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A study of spinal fluids : aqueous vanadium tetrachloride and its use in the diagnosis of syphilis

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Title:
A study of spinal fluids : aqueous vanadium tetrachloride and its use in the diagnosis of syphilis
Alternate title:
Spinal fluids; aqueous vanadium tetrachloride and its use in the diagnosis of syphilis
Creator:
Leopold, Robert Summers, 1915-
Place of Publication:
Gainesville, Fla
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University of Florida
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English
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111 leaves : ; 28 cm

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Subjects / Keywords:
Antibodies ( jstor )
Antigens ( jstor )
Fluids ( jstor )
Globulins ( jstor )
Incubation ( jstor )
Liquids ( jstor )
Precipitation ( jstor )
Syphilis ( jstor )
Vanadium ( jstor )
Water distillation ( jstor )
Syphilis -- Diagnosis ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Bibliography:
Includes bibliographical references (leaves 103-110).
General Note:
Manuscript copy.
General Note:
Vita.

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A STUDY OF SPINAL FLUIDS;

AQUEOUS VANADIUM TETRACHLORIDE

AND ITS USE IN THE DIAGNOSIS

OF SYPHILIS











By
ROBERT SUMMERS LEOPOLD











A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY











UNIVERSITY OF FLORIDA September, 1942










































147965















AC KOffI*


Th. author wishes to t his grtitae t
r. C. B. pollard without whose gui4d.e this invtilration would ha, been iosible. It wa. at the gostion of Dr, Pollard that this work was bg= a4d arriad on to completion.

Th- author would also lik. to give orodit to
Drs,. Therrell and eudiger of the Florida State fop$tal, Dr. Lonino of the Georria Stats Hospital ror the Insane. Dr. J. .. Patterson and the Kshn Staff of the Florida State Board of eAmIth, Drs. L,. Y. Dyronforth and U. Mar. shall Taylor of Jakemvill., and Dr. V . Do. T of the Lti e Tubaruloi3 aSanatorium, all for their splendid operation*

The author& a rutatfl thanks also Co to thc staff
of the Depwrhwnt of Chemistry of the Uniwrsi y. Their su estions on eLy.eat and methods hav proven invaluable.









TABLE OF CONTEM


Psag

Aekaowledu o............ .* . . .$...*..... o.*.oooso, V


Teots Proposed by Other A ho ,,. ..... .. .... I

The Inportano of Spinal fV1I4 TOst........,.........o 6

Sodi m and ColoIds..,.... ,,., .U.............. 2


The Preparation of a Sueitive Vanadium Solution far Syphilis Iteotion in Spina Fluid........................ 75
Remalte

Table It The Results of VC24 and r-& -93
Reaotimoe an Spinal Fluds..a......................... 77

Table n. Rovalts ot V34 ad KpTO'20I1IO
eaetions with Spinal Fluids Sh~vin& Agolzg Effet

Table ITU Results of VCIh " CroO7-,TMO1N0 Reatione
with Spinal ?luid.....,............................ 81

Table IV. Results of VClf, Reactiona with Spinal
Fluids, Showin p1V sed d iC ptIon of Preoipitate


Table V. Results of the VCkJ Rosetion with Spinal

Table VI* Results of VC1lJ Reations with Spinal
Fluids, showing Reproduibility of Solutioae......... 87
Table VIIo The VClh Rsetioa with Spinal Fluids
frM UnknOwn Dieea .....,....oooo......,.o,.oo 89

Table VIII�o.Suwy of Tabulated Results with

Table IX. Summay of Tabulated Resulta with
VCl!, Iesotons.................. . 91










T.ble of ContttS (Coninued) lage

Table X4 Bimwy of Tabulate Raeult* a the pH
of the W-.4Spinal Fluid Mxtm.. 98
Table XI* Results of Anlyes of the VsWous TCl4
8O~ut Ons...........~~o�. .. �..o... ***U* 95,

Disoueslon of Reaults i Conolui ,n 9,..5.............. 95

SUMa"y of Conluslis... . .. woe*..****a***so * s* we..t, 101











I TDa)1"fON


When it we proposed that the author undertake the development

of an original test for syphilis in the spiml fluid, the difficulties of supply were not fully realized. This obtacle-has been overcome to the best of our ability.

An investigation of this type is almost invariably carried out in a large hospital, or very near one. The clinical and laboratory ovidence of each ease is examined minutely. In this manner the true value of a reaction Is determined. The new reaction may be best under certain conditions which lead to failures with all previously known reactions. At any rate the value of a test should be determined by both the laboratory and wedie~l staffs.

In direct contrast 'o the normal state of affairs, we vere forced to use a source of spinal fluid samples some two hundred milo distant. The samples were shipped by express and their tine in transit ve a minimm of twenty-four hours. The data furnishod with each sample included only the result of the Kahn or Whsernamn test and the clinical presence or absence of syphilis.

The fact that Florida doe not have a medical college necessitated visiting other libraries for a study of the literature.

roiorto 106 (1), nfl but one of the leading syphilologists in thins country believed the appearance of seondary lesions to be the only means of diagnosing the disease of syphilis. In 1906, Waosieemzn,










Neisser and Bruck (2), made one of the two most important advances in the ultimate defeat of syphilis. The other was the later discovery of salvarsan by Ehrlich.

Although the theory that was proposed by Wassermann et al. was

not in accord with the modern work, the test itself has undergone only minor changes since its introduction. Despite the time required to make a Wassermann test, it is still one of the leading reactions for the diagnosis of syphilis.










TFWr P BY XFlMIT AUMOR
?OR TJ DIM3SS8 OF SYMMS

In the oplommnt fstion test, or Was ermn tet (2), tho
ueahmaim is postulated to be s folIes* 1) An antibody Is built up In s4 oonosztration In the ptinet's syete. 2) his anoibodo vill aeobine with and doestroy om speofie Vswt of the affect orgwaism whih i calle antion 3) In Order for the anutibody-.ntigen reation to tke plUe it is aeseoary that a reaction psmotr, or linking agmnti, bo preasntl this agnt to culled eoaplemut. 4) Zf the anti be addod to i aple of gmrv whieh has no ecmplm t, and then Just enough of this e sonpmbnt io addod for that antIns to react; than proviag tht presene of the umffteted ompleent would provo tho abseme of antibody. ) o pro. the presence or abence of this bridgi eatorial It is necesary that we have so otbhe raction which a1so rquirs complsmnt. uok - reaction Is the heuclysis of foreip blood eorpuseles by the soru of any corpumlo-mmum enlml. 6) To got ouch a syste, a rabbit is Injected with the red blood owpwcles of the sheep, so that his blood Il contain an antibody speoifis for the heolysis of shoep copu,les. 7) Complement Is thermolabilo; that is, heating it at 560C. will dstroy it; on the other han, aattbody Is neot o destroyed and is called thsoatabi*a.
with these taut in Mind It Is gey to vismaliss the smn
?est for syphilis. The first step is to bot the lptiouVo bleod sorm tho ells havig been removed. This heating o known as Imtivation










and 1s carried out in a 56�CC voter bath for 30 macute.. It destroys the patietv Cocplaent , The weond step is the addition of a knovi amoun of 00401406A. Tug cemplamet is $eed rpm the serum of a tids pig, and in reaity s Just the OWM of that animal, titated in h a ay asto kn how muh Oant n it wIll take per nit of volume. The third step Is the addition of the anUgen, The tube is then plae4 In a 3170. water bath for thirty minutes. At the sam time imativateiv c.rpuse*1u. rabbit serum and a suspension of sheep elUs is also put into a tube and Smubated. Xquvalent quntities ar* used in this system, as detersiged by titmation. After both tubes have been io tbe incubation bath for thjty minutes they are removed and the hm3.ytic system added to t3h mtigea. antibody tube. Incubation is again rqurod for a engt h of ti.. equal to the first period. At the end of this soond invubaton the tube is remove and the result read. If there in a eoeolet. lack of heolyds then the reaction is positive. On the other hand, a complete bewoysi Indicates a negative rationtext hvin been u oaffetod. In betwe these limits the amout of positiveness, i es timate by the operate.

It is obvious that several blank# mist be run on each serim of testo and that In order to be ceran of the result, a knao posit4ve nd negative serum met also be included. In addition to thee minor difficulties mast be stated the tact that the antigen hich is specific for sypblis baa never been found. Vassorman and his coe-wo used a saline extrat of the liver of sypbIlitic fetus, thinking that this did contain the actual antigen. It did not. Later It was found that antigen, substances of










equal merit were posseosed by the heart muscle of almot all umiale. None of these vs spesoift; Whether the aiml bad s hlis- or not as unimportant. Since the Um of the orJiLml test sovena -m fAto in anti gn have been the sole alterations. WAmSemw and PU4 (3) applied, U co lement fixation reaction t the spinal fluid with good resault, and this is the first test for syphilis, using pinal fluid.

Nioellis (4) 1n l907, anoumod the first precipitation testo, akin& use of the es antigen as the Waseruen reaction, the pateUs orum and malina. By ezporimt he found Ohat antigen an dnibody, In a salt, dilution of the, pr"or strength, Save a visablo precptate if allowd to Incubate for several hours. In an article (5) proceeding the awwane" nat of his test, ]ibaooll proved expertmenally that the Vasseruwon reaction probably used a non-specife antigna the reform, bis own test to bound by the sa limt, in spite of his claims for Its epsoficlty.
In the course of the work on antiens, Porge A Oeor (6), workift

under the diroetion of W seona, found that pure loelthin eould be used in plate of the antigen, and camplemant vould still be fied. Along the sawUm lne, vws the discovery of F3-iswuam (7) t)M oholsterol pav even otter results tan leithis. Varney (8) also made use of an Antlgonie substance in a precipitation reaction. He found that the addition of a few drops of V4 tau ie to I too of the patients earm gave a precipitate In the easo maimer as tOe Miobaelis test and did not require as long a tme of ineubation. mve, taud.a s nt effective enough to ever be of much ussl it led to too mma filse neptiv* reactions.











Forus, in 190MO proposed a test based on his laboratory$ 5 aperle s that a rphlltio serum uld osam" preoipitatior if added to a nomta swum. Plaut, Iiswk and Rossi (9) 1-,mbigats this, and toVnd that ay two, *or* regwdlss, or affetiozi by syphilis, might iw AiSO to a presipitae . The mehaniim of this reastion Is not olear. but it may have something to do with blood type. ulse it nay t M plas betines two noml sorow
The Abordetraldoa creation w applied to the diaos-s of syplis, Varney and Merst (30) olabod it wa speofio. ?hsy noted that the raoetion did not -pp bWatde the Waessmmm reastioe in useoftlnos This is a reastion depending on ais, or ferment aetion ?he srm or the affected person, mabd with a substrate of protein trm Spiroeheta pallida, should give rise to ftes msm aside by )b6roaytio satIou. brontenbremer (11) famd that a ruro liv osuld be used for substrft just a well as a proteins the proen being toted onma in the seram itsel. It m also tawA that alst saw ersoa would give rise to sa free ama soidso
Iawsnor (12, 13) sa is me to the rematio whieh takes pluee *s dilution of sera with distilled wtr* The syhilitis sera yield a pro oipitate, the noi-swypliti. feil to do so. It woo eadtto bowiw that the reaotia al sJo positive, 1th yp d m0, bpo and pnommula. a&mai usod the sea. raotion, bat uad the substution ot aloohol for distilled water. Both of those tests ae ba$od on the p10*01 cortent of the serm. X K e,, in 190v ws still ming u55 of sth










Dfauyi toot (14) In following tb. course of syphiliti treatment
A toeot of very o urs mchnaism was proposed by achwvan (15). Serum is diluted with 3-5 parts of physiologic salt soluioa and a drop of 3 p added, Now a "vy dilute slu ion of J Cd3 an phenol in added and the color rod. rom to blaek is a potstivs reaction, wie a green color is voM l. Thi, test Is not entioned pin ln he litemt , and must have ben gilty at too many false reaction*
Independently, aad using different inthode, sns (16) and4 Naguehl

(17) did all they could to got the Clobulin content of tho spinal fluid Into the diagnostic picture. They point out that tho spiml, fluid is not onwsr by the large onntration of otber mtel fou 1* the bloodftm mde zuse of the fact that globul4i towt eslub2* in a solution which to balf-.eaturated with (NH4)2 504. Negnohi clsia. that the addition of a W, solution of butyric aci4 showd the preeom. of oeess globulin much =ore clearly. Beltx (16) stat.. that a position, Nouns motiUoA (also called Ross-Jones reaction and (UM4)280,4 reaction) isa onfitaion of syphilitit diag is, but tbat a afgative reation is not conclusive. Als that tubwvuloim and tive affections of the meninges give positive resotions. Jo s (19) in a st4y of oneral paralysis (paresis) stated that ineroesd globulin Alust, iaral~y ocawurs that the Wasesormna antibo4y is associated with this globulil and that the e., globulin is the cause of the f em and et Soraade thse last two tests a the best sothod of sopartin general










*inrwT*s aid sypbi2tic affitos
Areigsr and Hoa (20) sowght to %Wfiy the WMeisonmaction b the us of KC103 as )oia. In a vaoM srtic# (21) they rke th statmnt that the active helytie agent it Minly in the COMUmeh and tb4 KO03 ae vatas it as well as the tu of opeeiri rabbit *or= Mille their eacton ch"ked Awth the wes*wr in 65 cases, it did not ratitvay favow, and Is not mentioned eata in the literate.

Jacoabstel (22#23) evolved an ultravderoopi test, based on his
obverat,one that. 1) eztrut of tk litia liver *how* .uats, oqualy sisod particles; 2) syphilitic sertin contains laes nuwrouss f�,a motile pnrticlos; 3) the addition or these two liquids gives rise to the toruslon of larg., Irrgurly shie Mleh, mkich are of fine Frtlcle, 4) on ives a top layer with no oolea mut binding ability, a better layr with vey strong oampewnt bindin ability 5) the fa ztlon of thes fUkes, ad the c bindinC- ability aft a Spseifie test for vyphils.

Jeobathal adited. that the por@Uta s of positive toots va not
as high by his ouwhot au b the Wassmrau, but state that it Mas 100% sure on negative results. .e als stated that hoa*r ausle and other artitfoal antigens ver of no use in this rssotloA. It v&# i4mted that the Wasvmnn is also a precipitation rvactioa.

Uruok azd Pidaka (24) node an intiatio of the tst propoted by Jaobothal, and in p a or cnfirue t. They also found 4 ws not toasant o to be a us. Rwaver, modifications mdo th pme ptat visible











to the riaked ee. Woling at 24CO., and livingg the remotion 94 hours for cmpletion, they found that oottr i fung gave a precipitat, of maroesople eIse This reaction oeouwred with fro 72 to 99% of the positive Wassenmm more, and frm 9% to 13% of the negative sof. The preolpitate, vas found to differ from that of Klmusnr (globulix) and that of Porges and Meier (leolthi). hil*e the globui Ise net empoeted in this resotion, thr weym most oertainly getting a lipoid, adterated with protein. The toot Is not use toduW, bemse of the log reaimia period and the nom-oonformity of results.
About this saw tim a reaction called the Rlloti. or Bo ,
ration appeared The reacion used as reagent a 1W% solution of NR05 with a slight exaes. of free Hg and a littl. 1". It mat proposed for an body fluid and Boveri (85) found that It gom negative rsults with am* 15 sem tfn afflicted patieote. tern (2) aleo oheeed this resotion and fouAd it had no spsoimioity with the urine.

Sabbtlidn, In 191UZ e.oted on the ometod of Rivalta, (27). " method Is to put equal volue of espinal fluid and wry dilute NW03 solution Into a tube and then pour down the side several 4MS of 9% aetie saoid. In I to 5 xmmtes the aeppLaaee of a Ibite i ohews a positive relation. Them are positive results In perelysis, tabos, eta., and the results are elamsed parallel to those with the *kam, Notoh, and Wa roae e. This roatiom y be s good as that of Somm and oguohb, but it would pr*mbly not parallel th Iseeu. It is










"ot "rswtda in5 th litrature.
M , a titrels invetigtor, next )ooke Into the possibility of an extract d" froe, a re culture of S. yalli (28). He. used thin Sn a #kin test, both with rabbits and san. A well tkad inflation developd hen the wet w used after the injection of the aninas 4th either a live or killed cultw.f. No reaction ims obtained in rabbits serig active orhit.i of cyTMIli origin, or in rabbits in which tle oonditicn had been cured four umnh previously. In wn it vas found that the reaction was positive in tertiary and congenital, but not in 1. or 2 sy ie. If there was any reacion at all in the p4. usry and secondary cases, it ve . Lus, and ri o (25) found that this ration vas a tUe 'onfirution# It positive, but when ngative had no special signfianco.
roporf (30) mda the obervation that the seru of an is hem io tovrd the red oorpuclea of the Cpise pi, and that inside oerta Units this property Is onsat Re studied a sries of dimsesas and found that the henolytic value of serum we altered ony in syp41i. Xn this "so the syphilis, by producing ree Upoids In the blood, apparently tied up a Vsat deal of ta coepluemet, and this, hindered the he lytie action. He found also that a great deal of the hoolytle value of a oa vas dependent on its content. Weilafka Is t1w mw given to a dianostc test dpnigon this loss of omltcPowe. Wil and XaM (31) ound that in the spinl fluid ther. vas alaot a total lose of cem duri syphilis, and a aernal level *as miatained in











soningitis. They only advisd the use of ouch a teot In the s-p l, aul4. Vwrtene (,32) fund tbat there Vas no paralle between thi test and the albu. s eotent, b he does n motion the saunt of globulin* the more r fasor. ThUs tost Is not mentioned ag in the literatre, bat it Is used as the basis of sev.ral ther work*1 notably the tOd test and its modtliationso

Lass' publis v4 the results of his work In 1912 (33), BY 4eductlv

ranenngho worked w~t ono of the mst important tests applied to

spIal fluid, ev tofy, It vas an entirey new roaction and cam as a result of his observil that all Is tests to that tim w based on cooid properties of liquids, Siso. one of the best methods for the Investi otn of ollo1doi solutions was the go)Al mbr of Z, ho applied this technique Th uthod Lang used was to muk dilutions of the rluld being investigted and add then to tubes a solution of aoUd-l gol. If the gold vas wiltered the reaction was nePtivo. Wen 10 dilutions wer. used son. would roact and som tal* Omphing these results gave o-rvea, and it ts In the intetation of these cures that the differently a of the varied i testations of neural syphilis lies. Kafka (34) coutlruet the work of las., and Krylo, Brand and mS (35) me this cement on its "The tost (Lance) runs parallel to the gloulin eonotnt, bu is ad u of it1. It appearn when other fail, and just an well my fall when others sippesro' Glaesoci and $or=m (36) did so=n work on the so4 Itself and fomd Oaf, the sensitvity decrased with inozeaiag VH and luoreased vIth the s~ss of+










the Partiles,

rZpe4nd u l =do ~ ow cocunM (31) on the theoy or syphils s p and in #of4Rt said that all $*t AM a fee4 On se1tw$ charge, ehages i surface teuson, adsrption and coagultin. They farthr a4d that the alteration of chemical structure of ressiu. substes~, or the tormtiie of new copud hardly eters Into the problou. Mother this en be acceped st Its face ,e--s or not in a mtter of debateo
RWkLU no roord of the origa work on the ?Inam oruts roaction is found in our literature, it is a reaction ung the moium salt of glycocholi acid with dde4 cholesterol as antigen. Z d (38) fte* this to be a eom-spacific reaction where there wre the follwin afftections prsoets pulmutr tueanmo-.i, oclania, eainow, typoi, sepsis A taimia, when tseting enum; and when testing the spil fluid u e and oclameia lut erferd. A large nu~or of controls ga' 94.4% positive Vkssermnn reactions ad 8V- positive Marman-?vuts rea ins. In connection with this test Y, Zfl*rnn (39) states that it is mt positive with tabo or prosis, bt oy with true syphis, Uq suc substaso" as the zlyoo)*.te, have been use"d as ant s, In pcipltaton reactions, but none of them hew been suces.
At this time an of the vaios toots and their iatie is published' b Wile and Stoke (40). Their dedution is that the coinneot indication of aoura ivlemnt isincreaed glabuia) and albucia, the position whoevana reaction And lyqgwaytosis, in the












Kolnr (41) Sgvoe us a record o the r tions of IA 4au. Tb*
first othod ft. to omay seru or fluid with Fraffin o1, contaning iodine, the second was to put tho todlim ir COC4 solution an run this mWor the lquid in qustin. Xt ws claim that in eithr c a svyi"t1o affection g~ve the liquid tho power to 4esolwMi th* lodin. It vas found by K#1mor that tbin ws a very poor nuthod# gi g far too way false positiv on. end Rodman (42) found this sav 30%1 false positives and only 6Z Positive reactions with syp itic eontrols.
*ordon (43) prpsda test using H8C2 in 'x diluteO slution (1,100). h se or fluids vere placed in tubes and a few dro of the reagert poured into thor. With a positive liquid a cloudiness appeard,, vit the iiorm a clear' solution re-oulted, Waichrd later modified this test and uns it for the detection of globulin increase. Krebs (44) found that the mamum Sl4u prc ipitation in this teat occum at an albumin percentage of 0.25# Thi. is nct a omo toot it this cunty, but soal.d be as good as the won or hi ethod.

Y., flnam (45) proposed a toot iewolvine the use or a etliioa or indigo, sulfuric &aid and Foling solution B in th. proper proportions1 Whn 44.4 to soVru et coupisto gal re-sulted if syp)hii wero prsent. The observation was aleo =do. that tuberculois of the boneosgb causo a false positiWe, as tis conition leads to increased proteins Just so syphilis doss.











Rcht (46) vwrbd mA an ant.in for u* in both a P'reoitation reaction and assorma reaction. When diluted for the precipittio reaction it vras turbid, but allowed the pneg. of light. Th asr= and omplemnt ware mixed before addition of the antigen and allowa4 to react for a t ae. After th a4diti*n of tee the tubes were iubatd for eight hours. A positive reaction ws shown by the forration of st heavy preeipitetej in a neotive the anutign swulsion rem 4 .
Ole tho nwo given a reaction first pb Qrku w.oil (47) in 1914. It wes a, hooyein reaction and vas maiftd byBro

(48) four yoarulitar. It isapplicable in abot 98g of th 1ptient teste4, the others not boring sufficient hs aloyti power in toir serm In the first paoe the hewolytio inez of tho ptients serm is foun , The units of the hemol..i% ine are volm of standard shop *11 esu pension henolysed per unit volum of *or=. )ow 1/2 this a of aheep cells is eded to a mix e of Mogushi antigen and erus The tube is incubate at 370C, ftr an hou. If the reaction is negative, hemolysis is usually complete at the end of thUis t1w. Tho tubes mro kept vernight in the icebox and read the follx m .

Emual (49), findin& the gold eol of Lmp hard to prsx In ai reproducible tuanr, proposed the use of a collidal solution of matic. Five tubs wer set up with the colloid and to the first four wer added graduate of epiml fluid, the fifth receiving only distilled ater. If the reaction vas positi the wastie in al five tubes precipitated* if negative only tha in the blank pmeip$tated,










In 1917# Cutting (50) noed, thmt the slt solu.tion (sp~mi~ flud) ina a negative reactlon did not a1lov the gm matio to precpitat. In order fr the blank to veuai* a .12*U solution, he found that the fie y of the tootwea not altoe d by theaddtio of I cc, of , .3 .lton to eaeh " to of the mti me up in al ool, The IgCO3 did, hovor, keep the blank fro precipitating,

In 193, Rnfel (521) proposed a o gi in thee atic test In order tha cuves slailar to the o.f the gold vol tost vould r sult. Pe prepared th coloid e st :ion from bufftted N&lI solution, ef 7.5s and an alooboU a beto sltion of mastjc. In ts manorn he co)lm to hAve a reprocble solutio and to get cur .e irh wore of wr. ueo, than those in the Lange test,* Brant (52) was still no~t satisfied with the sensitivity of the meo test. Ile found tht the speed of I nhad wre to do with sensivity a pr of t t t wa usually assumd. His test Is mmn in the fol.lowing- mnner. A series of tubes. containing the twos apouat of matis is set uap, and as rapidly as possible graduated munwts of vater ar* added. After stting for a while each tuibe is made to the ou. volume with moe mater. Owe tho particle size Is determined by the orlgial addition of vater, more vmor do oat anpit. In this vay a rowdable oll.oid of the sm son*m sitivity Is almayo obtairAblo. In a period of 1.4 year* some 70,000 toots were run 'by this method. Sines the normal and sypilitie cuwee both depend on the g3obuli/abdn ratio, there can rwvwr be a colloid retion which dttferentias syphilitic* from Uo-yhltic, if the gleWlS*, go"s










to thO Sam level. '1hre can not poseibly be a afenty one, as SM& nathors clZaia, but 'always a gadation~ from no-yhltic to *yyh41ti.
MSffw, Xober and Field (53) m an inyvstiption of the proteins in the ospial fluid# using nepholmotwy for their dstorx.ztiom. They fond tat if the norml value for eulobulin we taken no 0.004%, then fluids giving a positive %%aam test went over thLU value L* 14 out of 16 ass, and negjatva fluids e re under it in 33 out of 3 oaOss Tbhy also adnitted that in two of these disagmaing eanso their ra tn te were 4oubtful. It in concluded that th. e loblia eontent and the test are closely routed.

As a toeot for pathogesity, Hoftn and Uhmp the
use of the p te ber (54). Th5 is t me of cc. of OAN KI04 redued by co. ofthe opi flid. Coeme (55) found that this reaction -mo also capable of 'being eared out with thy)lne blue. No ov1erayed 1 ce. of t* fluid with I so, of reagent and the yellow color at the interface, follred by tLh ols liquids hea ye3lev after sAking, wa supposed to doente a pathologic fluid. ,overi, fter whom the prangate reatton is *d, found that this we A^ reality a tot for albuan (56), and that I co. of spinal fluid should not de-oloriSO I cc. Of li910000 l O4. Ouitain and Libert ( 7) also report that byperalbmimvie always gives a positive permangamtet,. Lowery (55) found that neither the pe mp ts nor the astic teot was of sufficieni value for the routine exw4mtion of spiml fluid~. His work w*a d prior to ll7, and while the perngamito test is not tsed, as ho advise , the metaotest is being used wre ad %or*,











ah an4 Oor4 (59) PMPOBe4 tbe tet bearing their ...s in

191e. ""hey mado use of an alcoholic extract of heart tissue to whi*)i was Added Choleterol. This antgen ag Mize with the serum (Ing.ti. v ted) n aloe4 to stw 18 t.o 24 hous, after a sort incubation at 37*O. Ylocouation ws a positive motion. ThiseI one.o the Most
U~r~tfoweunners of the uodera Kahn rotion,, and in fac~t the

faults in It are responsible for Xahn st--ring his researcb. In the eam "Aner, the Wer of Uonicke is .$It as rpnible.
Uoinieks (60,61) =oe ue of a phyeoia1 sheuical iss'tion ror the dianosis of syphilis. The reastioz was found to depend on the fa t)kat ak mixture of lipoSids (the antiffon) and syphilitic antibodies femn a precipitte, and that this prcolpitt. it Insoluble in salt solution. To 0.2 co. of the Inativated seumm is a4ded I cc. of the lipoldal antigen (made of hrw hoert extrt), the tube is then incubated for 20 hours at 3CC. After the incubation the precipitate in ehake and I cc. of 2, NCl solution Is added. If the pripitat, is insoluble, the reaction is positlv*. Yr. Reichert (62) found this reaction, after its third modifiotion, was still not applicable to the spiul fisd, the precipitated mterial, if any, not bin visible. The various modifications smde in this reaction by Usioks were not able to itrsaus its ussfulness, and in 1922 he a a =ea rction, the )bsnieOe turbidity reaction (.T.R. or Uoinieke 1). The antigen usod in this LT,R, (63) wms designed to mks the reaction clearer, and to do amfy with the addition of the salt solution at the end. To the I poAide










ztmrt, baisa of tolu was adde azid then th turbid smion placed in tubes with the ivtivs serm. If the section re positive, the turbidity inmcmsed, and mll flooulas appeared, Althou the K.T.R, only required ome eight hours to go to a, even Uiniske adnttad that It vas aot as godfor dnotcwrt'i rzm mth".
Still searehing for a better thod, ftirUk. (64) proposed the Iesnicsi Clearing Raaltion (E.K.R. from the 9s , or Msi~ek fl reation) in 1929. In this reaction he m ue of a beef heart, Upi atigan and added three tine, as nuch tolu balea e#in the .T.R. Juat prior to use, the extact of beef heart and tho baleam are heated to 451C. rnd I vlums of 3,A MCI solution at the ese teperature are added, rapid uixing being essont a. When the wixttur cools to 3C .., active seru is added, A positive issrus results in a pre4tation of the colloid, leavim a clear solulon. The cloi is not changed by a negative, non-rypilitiq "r=. This last test of Ubinike is appezr4n 11 his bet, but it Is not used in this country to sy eztont.

EZlinger (65*.6) tried a patented genmidal colloid of silver for studyizr the spinal fluid. The prepration, Collargol, is =d by a scret ptteub process but Aleksevsi (67) prepared a duplicate uatvrial. H{e wo as e 4In broak Orwi patents for the Rusesian govrad He suggests that the fiml solution Is not a colloid at all# but a mtzturt of compla9 jver salts of te anine acids in a disperod state. Rubelizg (68) in 1938, ten y-0 after E r, publseod Vnat he *ll~ d










a" m Totion for spiml fluid, usis CoUlerS*1 and4 ZHC1. ThO Met$.oa is Used on the fact theut the Pt,td silver Ch~ioOf ths, oallrg l is preipita4d by the ad& tion of epimi fluid ricb in gi.bulin. Rrrwa (6) states that the basis of the go4 sl ad col argol rction are different# end that for this reason differential diagPsis should Ue possible mktng use of Ioth reactions. Gince both aro deedent on the globulin content of the flid,, the truth of this statownt Is not appant.
In 1919 Stsinfeld (70) found that the action of spinl fluid on a suspemion of Pb02 dep on otherr it se pathologic, or not. lit round that norval fluid had no effect on the o, bt that fluid from a syphilitic eource ve varying ts of * utimstion and floeoulation. Io further report on this work io found.
Guillaia# Laroche wW1 *ahelle (71) introduced the use of bemoin as a colloid substitute fr gold in the LaMe test. Their test is sinluar to the first matic t st of . They mke the statemnt that the result are not In Aaeord vith the albuiun or call ount of tho fluid. Sinoe the globulin to the determining factor in this type of reaction, such a etatemnt is not too importentj howve, the cll count should be higher in patholoic c Lnditiono than In the. or , rig and Keraok (72) give ean exhaustive t e of the retion with beaoon, stroly advioin the use at'this coloi. iQurtl (73) in 1,2O, reports the use of gum zy'rh in spinal fluid testing, but doe" not give










x;q Oonsludons, as to the worth of the test. Xt in clear that wW eooloi my be used is thse mtion of spinal fuid, and that the renslts vill depend on the cs with w ich the technique is follmm4.
Vrsu (74,TS,7,7) bgan wr in 1917 on the diagnosis of syhils. At first he rodo use of a oit lil iron olutieon eontaig ?.(m%)rT,(C2H30)3. The soults wera not good * , so he md use of an antigen frota hores heart, which he cnlled thnol. Sinc thds CA a reaction viich he oould not see, he added a pig call hewlyio systea, for Indirect roadine. This test vwe si*1lwr to the Wsoernaf but not ae goe, so tht in 1925 he h lishe. his last sodlfication, a precipitation tst =kin& use of the perethynolp imative noerum an nstrument for reading the twrbiditr, Tkis i~nuinut is a, ypetotr with a apacial sonec, designed to show pathogenicity by tbos numricl rea4i. Cormll (78,79) discuss these tests in detal and preises the last modification highly for useo in the folUving of


D*1d (30) started putl4siabi n series of tests in 1921, but none of them was very suocosf The antigen he used wa similar to that of Sachs and Qeorgi and Minicke, and d4u d Ill with alt solution prior to use. The serum was then added to tYis opalscent solution a4d incbation carried out 'for two hours. at 370C. After another two hours at roca temperature the result va roa. It the solution ws still in Its original state, the result uns uegat$v) If turbdity developed* tbhn the rvntion was positive. 3trespel (el) found this retone less










somitivo tba either the 8owasorgi or the uesiks I, he fwad it Save falso negative results,
Dold (82) fond that formin er d tho reactivity, ef$"

especially syiitico and in 1922 mdo a sipiiainof Is crigift teUt# tinz fornhyde, *uatigen and serus. In order to read this test it was still necseaary for the incubation to go on two hours and the Wues were read after 2 sod 22 how* in the icebox. In line with this e the foraldhyde-el test proposed around this time by " and P . 'ohnston (83) =do an ivestigta.on of this test and found it i never to bo used with any urtvy. The tebhniqu is to add a drop of fomlin to I cc. of blood aerwa. The tubes are allowed to stand 48 hours and then examined for onu~lation. Isa 100 oases it vas positive in 23, the e s'mu a in . There was agroent in t of these, al the others onflicting. Considering this, peiAmpe the addition of forunlin to ths P.34 technique VMS a mistake, loading to erronou results,
)reyer and Vard, in 1921 (84), published a teot whioh is in uos J

En~land today, A choleatr~ii. antigen of beef heart extrat is prepare4 and two diluttior mde of it. Vine dilutions of the "ru ere then rode

tand five added to the stronger antigen dilutions 'four to the mkeoro
After * short incubation the turbidity in eah tube is read by wsa Of a special ealutinoope. The results ae xpressed In standseduad units. The neaesity of using a special Instrmeat keepa this reaction just as It does the Yernes reaction, In a 1ntid ield.
The IHewxhir reaction (C5, 84, 87), or prvctie tost, we firs










referred to as a *x toot. If a sin dose of is vas ainitrd ev. l mn =ds the caim that it had the *as ation as ttzuberc n, This was found to be a fae. assuiptio, and the rteatio is now defined as the t of ;thol pr fo antisyphilitic thomW. It is found, for o p tht od es wth a negative Vaoseren naeatioa my give a positive relation after a hevy does of .oars~em A., This does not oftom take plae, and i* is not a disnoti sn thod. It to theaptU shook ad to be avoi4*s aesor4i to Koor.
Tashiro and Lvisson r te he use o uIatur. of oul.osally11ic acid and I 2 in the diagnosis of syphili in the cerobrosirmal fluid. Kneohara (68) found this test positive with 11 forms of meingiti involvement.. No states it i never speocei*. It shouU be noted that ulfo-alioylio acid la used in the quantitative eimation of alba


In 17229 Kwhn started publishig the results of his roseAreoh, an his ethos (90). Nowver, thti toot was not fully ready until 192#, only the atige belnZ us.d before that tim. Wang (1) a test using alcoholic extract of human heart at the saw time. His results wars in igood t with the Wnseeonn test,. but the test never gaiwe any following. It had no apcial advatage over any of the other pro eipitation reactions of that tins.
Alwast as soon as Koh published directions for prajamtion of his antigen# 'Hrrold (92, 93) mde use of it8 first in a reation with *er*,










later in one wtth ay44sl fluid. Hrrold dilutod the atizen 12 and added eorum from a piatte so as to form two laye~r. A white ring in tho eru is a positive reaction. With spi Rl tluid., he tound it noeasary to Concent rte the globulin fraction, usint half Satuation with (MH4)204 After ream of the liqid, the glabulis w dissolved in a swU volume of physiologic saine ad them tested no abw. Uhilo the results were not too god, Kcn did not sucesully run a sQi2SX fluid until after he =do uas of Its cornnration by th4s matbo.
Bruck (94) in 1922, returned to proipitation tests. No mad. Us of, a w antigen# heavy concentration of salt, and c. antigen is na-olstria4.d e"tract of beef hart, Iraiti'vatod serms (0.2 cc.) is Ad to 0.8 c. or IV, ?k204 solution and then 0.2 ic. of antizen ewulsioo added. The tubes are entrifuged 20 minutes. An Ieo2bl precipitat is positive a native aer.o aleo gives a eipitat, but t0ia gaas back. Into solution on akiM. Tac n(95) notes that the reaction must be rum on positivo and noptive sera and gives the thod, Boker (96) tested 12,000 blood special with this ethod, and found almost perfect agraemat between It and the Waseenum tost.
Bchwartz and Orunemld (7) mad* use of colloidal silicon dioxide in the OxwAmhtiOn Of spinal fluid- A solution Of RiO2 (0.2%i) is used. Ton drop* of thin re mixed with I cc. or spinal fluid, and it is noted that luetic fluids floceulate in a few hour., or easi1 on heatin. A c method proposed i the dropis additionof spia fluid to 1 c. of the reagent. Fluids from p4asis, oerobral lues and tabes flocculate In










that order. This mtho4 ws foud by Kl) r to be 9q2 as offeati-fo that of Lange, and he reo ndo it forapi orientaton().

Rehm (99) foi4 that ;thologies spiml. flud, e.see%.ally luwtier

formo foeam wen mixed with an *"I volue of boxuono or tolwm. This is presented as evidence of the cfll4i4al change in the spial Mld duriog disease.
An i vestigation of the pass of Uanin (the sodium salt of florescein) into the spizal fluid, following its injection, or $inption, ws =do by Jervell (10). It vs found thalt the color of the spinal fluid was related in sow of the cases to te pAthology, in others there was no correlatiton,

In 1921 Eahn (101) published an article on the effect of heat on the roactivity of antibodies, At this tim he apwtly found that heat had a deleterious effect. At 56(C, he found a lees of Z in strong positive sera, and 5V0 in vZ poiativ,. lie also stated that in a great inny cases complete destruetion rsutd frow heating to 62�C, In his book (102) Kahn reports that heat has an enhancing value, helping to promote the reaction of *ar. fle explains all the reasoning that he and his co-vo*era have used in the developmnt of the Win tst,. In the first pyaes a concentrated atign is used to give bettr reactions with we �ly positive sort. t4eondly, this antigen is mde into an unstable emilsion, the we unstable this is, the faster it reacts. In the third place s of the antigen emlsion and the serum la Introduced, sinc, this also speed# the reaction. Palt soltion is addo be u e Kahn fouA the proper tion











of ThCl aided the reaetion. Cholesterol is used es a senitizing agent. Inactive serm Is used, since hentian the aerun increases Its reactivity#
Used on spimnl l d, the reaction is carried out as follows. The fluid is inactivated, and then mi=d with 2/3 its volu.s of atut 'ed (NH4)2s04 olution and itlloed to set I bour. T e tubes are then centritiged and the supernatant liquid poured off, leaving the globulin. To this globulin is added /0 the volum of the original fluid of phyio.logic salt solution. This cocentrated globulin solution is divided inte three parts, two of which are used with antigen the third, for a blank. Th antigen is added to physiologic salt solution, the tubes shaken and then the globulin solution in added. Shkin is continued for a U* and then the tubes are read. o itive tests are shon by the presence of r2o~culation, nogative by the fact that the tubes remin opalescent.

Epstein and Paul (103), as well as others, have investigated the precipitate, or flocculate, fored in precipitation rteat.ons, They find there io little evidence or any but included protein being present, and that thN in part is lipoid, Bruck, concernin: tho mee niom of then# tests, (104) stat es that they depend on the agglutination of finely divided lipoid partielos, this beint stimulated by soe constitute ts of the lustic aeru. Kahw, in a more modern work, (105), presents the view that a lipoid due to the Spirohete is responsible for the production of the globulin antibody, and that this globulin will than precipitate v like lipoid, no nattor what the source.

In 1926, several reaction* wero brought out. Kadich (104)










developed a rapid Massermn test, but this never Gained any favorable reation from ;ethorinit. Roenfeld (107) Mse a tudy of th itlu.ence or spinal fluid o e ctrolytio praoitetion of' positive ad ngatv* so, at a .init. H.Ho found tatif thispH of thespinal fluid vore ad1unted to $40, then the crail. flUdd would protect nieht'. blue against %2Go4 floculation. I. also found tVat if thor. ve )erl uanouis, the nightblu. reaction was nnsitized, and that in wnS itio the result was floceuation. tnhe othar hand, hyrblobuainosio had an opposite effect, the nightblue beine held In solution complatoly. Usia nighablo, "osenfeld was able to dtetinguish between meningitis and syphilis or other affections producing an exess of globulLn,

Sleeper and aonlker (108) investirated the method publbshe y Dolts. They proved tLat the use of aeetic uvhydrido or a malt of glyoma1 as54 with concentrated He4 Am' OPiMl fluid was mer"Y a test for tryPtophmnw tmt is wat an direct teat for incremsd protein, the amunt of regents bei such that the coor did not appoau in the normal and did appear at a value just pasu. norusl*
TacoO0e (109,110) advised the use of 5% K,*Cr207 with one drop of CC13-COCH per cc. He states that with seiml fluid this solution gives a white turbid ring a the intreoe, Wn that this ring is tor Intense and permnent with syphilitic than norwi fluid., no states that the dirfrenes in Intensity and structure of the turbidity allow a differ*AUXi diagnosed. In his second paper eaos inys that the 1hrit











reaction is the mofft s asiti'r. known; that it viis positive- with 92 of children having syphilis of the spirl chaord,, and that 45% o thes children had no apparent lorn of the nervous q ,tu. P4n11 had previous y found that CM l CCO- was a precipitation aent for aliain,









UPve a toot for nyphi .s Pruel foun it Indcaed a pahloi con,dition but not (11iy a3tils$a the it proby depended on elbT i m. e Au o nd 1ea r foun this Oml depIndre on ioin,) and v, of a In diag tl of hepatio Jh (s. It s no bettr than the gold test or other tests depending on globulin (114).

In 1926, Sachs and Witebeky (11.5) Mpoed the eitonhol reaction.
Sc nrated be heart antigen tbre time and then sholesteriaizend it. "he reaction is vwy rapid. They also imemtigated the possibilty of a Isutochol reaction, ulg lecthin in place of chologterol, but this VAG not as su.ceesb3.

Vion (1U.) mdO use of An antign omilvion which consioted of lseholie beef heart xtra tv, oblesterol, glycerol and NhC% solution. In t sting spinal fluid five uneate4, C-rauaed samee are used, It the reaction Is peitive there is a clseriog of the antigenic axulznlo and se0mwation. The reaction is earr aut at 37OC,, and if the











lath got* too hot, there wS41 boe leaning in all tub.., bW xo sedluan'.


Vuller propooed a couglatationo or cong rtJion reo4io in 1926, )ayser (117) found th was not as good as t e Ge or Mniiko tests. Ro applied it to 635 ewea. Troutor (11) also found tht this Vuller reaction gove for too w"V folso positive reotione to be of u"e unless choked by the Waesenazw vthod. In 1930 LluaIer brouCIt out a reaction knon o the U.LR. 11 or the ltaler &1IIW&15 IIktion (119), Ri. results on 937 spinal fluids givo agrooant in 883 positivee and negntiie) and 39 positive roations an WasmormM negative fluids, 15 netve reactions vith asserman positive fluids. The antigen Is a beef heart etract with a vry hmeavy cehol .ta tion. Mlaer and mndulma (120) explained that If to eooiant amunt of heart extract were added inoreasing amounts of sholestoroal, two sonme of optim precipitation resulted those baing separated by a sone of clear oleid solution. The b" reaction takes place In the sone with the large conoont ration of obol..terol, the ordinary tloculation reactions in the zone of low oholesterol concentrlion. TUna (121) reports that th. false Pocitive reaitions of the U#B.R. 11 are .aily distinguished from the trw positives# because tbisy are a course preipitate, ad not a tru tall. This bmIling is In reality an agglutinisation of the antiSen elsiono the stronger te rmoation the more C~pleteW it go.e Into one ball leaviMg a elear solution.

In 1929 and 1930 five reactons apri, the first ben the TOU"










utho the seond the reation of V4,eurl . bither of hsse we nev, or very ffective. Savor roports (122) on the Dme*Ube rmotion at this tim,. This action %eod a mxturo of' fonrlia# sodlt= oloate an r.l*14. This also vas nothing new ozeopt n the combination of reagents, and it wa rolt effoctive. Papi (123) explained the Beiarra reation,, which ise a Iaeosn teat with alcohol present. It has no paricular advent ge over the parent tet, Meleser and Dswoa developed a ztoro-mt te Uat, Uut RZiner (98) found it only Mob an effective as the Le tost#

archioni and Alloton noted (124,2), in 1932, Ust the diastaee content of the opi l fluid I irmolved in syhis. The naal of this mterial in the spinal flid Is 2"0 mg. percent, bu in erphilie It ia found to I* only 0*0-U4 x.%, This drop Is indoesdet of the sgrcontent, which reinss nrml. The over-lapping of the values does not prvet thi finan from bi of use in some ase diagnostically. The effect after tratm*n is not given, and should be eVOU more iuortart.
Xli4o (126) started puolieMan prsaxa t reports of a elide tot for syphilio in 1929. It usono ntll1932T.tht Uet, vw ready for ene,,a use with spinl fluids. At this ti a book we published by Kline. Kah had tbo idea that if all other fctors were equal, then the more unstable th e the =or* sensitive and rapid the test would be. Kline Psents the view t the physical state of the antigen e ison has moroe Jiortance than the stability. oth the










Kline and Kahn tests are the sam in that the antigen is concentrated to obtain diamino-phospbatidel mixed with as little Impurity as possible. This waxy material i' then mixed with water, cholesterol and physiologic salt solution. The mnner of mixing determines the state of the emulsion. In a reverse procedure to Kahn's, Kline adds the cholesterol to the water first, each particle being thrown out as a small plate. The antigen is added and forms a covering over each plate, giving great surface area. To this suspension is now added the saline, its purpose being the Beim as in all such reactions,. to maks the reaction more sharp.
Compared, the Kline and Kahn antigens are opposites of stability, the Kline not precipitating for hours, the Kahn giving precipitation as soon as it is mado. Two tests are proposed for spinal fluid syphilis detection with Kline antigen. The first is the exclusion test, the second the diagnostic. Again the reverse of the Kahn procedure is used, the antigen emulsion being concentrated in place of the spinal fluid. For both Kline tests 4 cc. of the antigen are heated 15 minutes; that for the exclusion test at 500C., the antigen for the diagnostic test at 350C. After the heating the tubes are centrifuged at 2#200 r.p.., for 15 minutes and then the liquid decanted. To each tube is now added 1 cc. of physiologic saline, pH 6. After centrifuging the cells out of the spinal fluid, it is tested with Bennedit' s solution for the presence of sugar. If sugar is present the fluid is good, if not it has been subJected to bacterial action and will not give a precipitation test of


lThe alcohol soluble, acetone insoluble fraction of beef muscle.










ww value. If the fluid is usable, 005 a*. of 2% acetic acid to placed on a slide and 0.25 o*. of fluid and 0.008 o. of antigen added. For either exclusion or diagnosis the quanoities ar the omne The slides ae rotwd for eamplete mixing and read under the mierosopo isdiatoly. If th exclusion test is negative, it is olaimed that there t no ebanoo of neural Involveamnt. The olunpig of matorial Is OL positive, teot.'
In Vie book Kline also reports preliminary work on a balling rotion. The antizen is strongly oholoestorinise, and when maleitied has the appearances of long needles of aholeoterol partially oovorod by ontigen. This has the suae appearance so the owlsic for the Mo.e. I* Since no further report of this tot has been made, ao udge it turned out no more osuawcsflly then the K.B.1R II.

nss (127) proposed a balling r esetion using a oephalin (phosphatid from brain) antigen with active sorwu. This tt did not get a very good reception. The statment is made by lieis that lecithin s good only when used with Inactive serl, oophalin only with active erue Reb has proven the fenwr asertion, but th. latter has yet to be proven.

$chubs wd Hair (328) proposed a colloidal cam tet. To the

spinal fluid is added distilled water and then a dilute solution to calie acid. After standing a time, diluted India ink is added. Preelpitation of the carbon is a positive result. Loon y (329) ad prevwuol tried to use this resotion with colloidal ourbon mA* by the, Br dig ae methd. but had been unsucoesefl. Seoubo (13O) explains the noehanim










of the remtions First onou& distilled wat4r is addd to render inert any protective eollold action by the proteins of the fluid. In the econd place oxa scid, whieh bears a poaitive large its added, and this �earp Is noutralsed by the tree neative c eargoe of the proteins in a normal fluid. Then the third step is the addition of the colloidal enrbon# which remains in ouspension. If thero is neurosyphlls, the oxalic noid is not neutralized, the free charpe of the proteins being less awasrous, and this unmutroliaed oalio acid causes flocculation of the o.rbon which bw a egative charge Deadma, Elliot and Mdth report (131) on this reactions "The result# uing the mtod of Sohub. and RaIns agme vith the Wassrmm and vther colloid tests," Although the reasoning on th mechanism is rather hard to undrstand, the toot undoubtedly works. $SwAo, Burack and Krislr (132) lAimw it Is specific for syphilis.
Fealep in 1932, proposed a new test (133). The antiwpn is mde of beef heart extracted ith ethanol and then treated with 0 vdxtur of equal p rts of corn germ sterol and cholesterol. This is eswUlifed in 40 NCI and ripered 48 how* before wsing. Inactive "os is used with an equal volume Of m4ulsion, the tubes shaken and incubated four hours at 370C. After the incubation the tubes are eentrifuged and 1.2$ NaC1 solution added. Strong alutimton is a positive teat, h geneity to a negative teat, all others are doubtftl. All doubtful terts are eentriftged again; if a vhito flake results in the bottm of the tube, the raction is positivel if not, negative* SiOOOn (134)











appli.4 th 091 toot to 171 Oyiral fluids, the results bethn ab* ;arallol to the Wa sermn roeton. This #..t runs about 20 awe sensitive tban either the Kahn or V'aps ma and give ftwr doubth1 results whe mod with "I**
ruskin bpu a *srie of papers in 193, entitled gPa

� klM id (1351,36). In the first he m4 use of the lose of color of oteh*10446.a NAA%., Oft it we added to spiral fluidl In the seonM be used glyooga.o-loi mixture. It vs caizad that the deolorimAtion was sog that a difformAtal dIgnosis became possible. On the basis of the results btained by FoUr, studyIr the Landau toot, this work as to have be" Ill fated before it was started (41), It my be that th* adsortIon kooe this a more successful reaction, but no further reports have boen published on It.
Koseasvwki (137) olabis tbat although mattio aci4 will serum
which contain increased globulin, this Is not true with syphilis. The p2 renotIon was seldom found with s.ph&34 This vas not publihed as a diagnostc procedure, bt meely to show that there appeared to be som differero in sypoiltle SlobulIn and other globulins.

The hwur (138) roaotioa# an VcCl2 flociulation of eeru or oplual fluid, is claim*e to be as d lt a teot fori raod glob l n as the Takta-Ara reaotlon. These tvt wers both proposed for syphilis, but proved to 6vl posAtive reat ote With any affectlon Inoressil the 810Win content of the spinal f d.

The ,abln tot (13W wa publsW in 195. Moore (140) found










It to be 09 in erid withthe n test and 2O with theW using Inaetivated Own is as only 10% in eror eared to the I" test. The reagent to a mixture ot hussein. ebmeaeefetl $* lot red aM pbyologleaal st solutions this s d44 to on extract of beet heart One 4 of the recent eaaleu is plsee a A slide and a drop or a"=i (or spinal flui) is a~0ed. The sppeaianee of red fess, seen ii indirect light Is a positive reation. Robinso a" Strod (11a) ra it 93% sffeetiw as against their Wasenmun and m7 SPIast their Koka. raieu (32) used it on 31000 ae and got go" resats. The longer it to used* th better this test It.

Vwwns OWL,) In 1936 stated the finding of a spoelt material in the spinl fluid was disgnowtie test. The xwtowlsa Is elled pallidin and is obtained by preelpitation. The spial fluid is preelo tated with CCl14 sd this prelpitate is e ,ith ether or seetone. The extroet yield a substane which gives a positive r ietion if added to a negative spina flid. Yam and Takagi fund the terial pallidi only in tbo later sta s of phis (1), sd that it is not dwoonstroble until soms ti after the secondary stage i oe.~
RytS "ported what he clle4 a rapid flaoilstIn re"toa in 1936 (145). hs am haray be sed rapid. To the etive spinal fluid i added MODUM sulfte Od act eolutions. An antigen 0ei1Is then ran owr this. The density is aw ech that the pratels in the tt ore forced ito the watigen a lelen n atitepgsuu After SWAttugin&o the solution Is node ligiter than the floeculate by the addition










of~ 4&Iut 1kC1 so)tlan. The tube* am~ centri%4 a omoosd tim and the solution poured off. The sedimat . is shakn wivitb hy ctologie selm.o, attia i. po ttv., a bas cSuepacon is tive and a e tion of the two extremes m12* 4oubtM. The eo pareom Of this test wth the Wsserman t not goodl but It is Um Sensitive, perhaps too voh so.
Usr (3.46) reports the Che dkahr, Dar r � ieak reaotio. The - ot the roation depsnde oa the owitry Iibi h the work Is done. One drop of 4.ZlbrUnt.4* dried blo d In wed. To the blood is ad4d 0*03$ a*, of a solution ontaix$.g 0,3%. hA2CO and 340 Y^Cl- A 1,10 dilutAoa of the Meuoke KUmge RAktion antigen w1th is o* solution o*f.WC03 and J Is pma.4 on a mWon *d., The awunt of satign diluton used to 0.03 sop The two .1do awe heated t 560C. and their contents znijed tbho slide ith the mixture to kopt at 37"C. in a wo$t cber for ft *iU an how# The rcetlon is rtod at the eid or thlo tim. The appearance of bsaek fc and olots to a psi-ie reaction, brown grnles is a negative reaotion. If a moifiod antipa MJ.*R. 11 (modified),Ia wwc4 the addition of the, W2O3 Vey be owtted in the prop tion Of the dilutint solution (147). fthuck (148) used op r* of blood, 0-3 ** of 343/O 1kC1 awe 0,,03 Co. Of tbo antIgen diutio. The *Vmao the rmation is the sen a. boforo. TM reanetto is st"M , romomiu its an additio l aid In d4icwo, by Ao, and 4. Britto (1At)h Althouh it bos received no mwAtiu in the bitod Statel, this toot ha Ve 6 a gmt auM of litomtwor











TOee nt rhe s.t of t wold.

ide snd 1de wen granteds a Femb patent for a d1ogptle reagont In 19M. The saget ts an extraost of bet heart musolo, to ubh is added oholesto-l mad crystal violet, or notlea blue (150). Van R ter4Iugen (151) found ts reaction less senitim o t the Uelanskoo Kohn and oltoohol reuntons, but more sonsitiv thas the Weseman ow ShOogi motom o# e states the speistfiit is The sae as that of the Wea5eroann reaeteno. It gives a gmret awbor of doubtftl rmoetiea, but sJlsity is its ,dntsep.

Rooaser, in 19"s, (1!9) proposed the uss of eentnt amount ot spinal fluid mixed with VU7iu amounts of osisium Shlorido. These mix. tures are then heated and the floosulation eurvs ued in diagnosis. This test has resolved no further mnAon.

In another of the ne and untried tests, Rrschteld ad lunger opos that the antibody be used to bilds tn ee If there I antibod present, then the th1in1okismoo will be unable to eoavplat plasms. xKahue)rov round tie to be an Inprsstieal test (1$3).
Mas" makes use or an antigen got by the euitiertiom of a mixture of en yolk and boeo heart muelo. It is claimed that the ogg yolk allows the mininal smoont of antipa to be used without loss in spooii city (194). Zn this r tion the eg yolk Is probably aeting in the manner as n addistion ot lecithin. This test is also in the trial stage.

u'hUloh (155), while ortioally studiUg the M.B,. I , hit upon an Idea of a new tpe ltes. Sine choleauster0 end antigen flooulate in










cartsAi dilutions, active, *Yphilitl ser=a ShMUld prevent this 1W binding the antigen. The t.eh4qu rsmeoded is to add a 3-5%
*C solution to the active soer contalned in eeral tubes. IergesIng amounts of heart lipaid ar added to the tubes. After a few hours incubation at 37�C. the ehobOe"Orol is add"d in the forM of 0.-1 Selution. To obtain optimal conditions a mixturo of dilute CI aM estowe Is than added. There is foeoulation Mly in the neGatve #Or&. ThS teot is merely proposed and no results gives as yet.
by no means all of the tests in the literature have been covered. When emitted a tesot did not embody any new or origial idea. As an example, Ve *LZ4 have considard the ue of sheIla., gta gwuie and soveral other substances capal of forming eoilodal oolations. To do this would be a waste of titm, one the astic reactions have been
*overwd. More recently ther have boon quit mammo publications on tests hich are all indstinguiehoble from the Dahr or Chedisk reaction.









mU XMWwSO 0 w5IR WID X


IU eact ONOStiona t th spu f1114 mst.eto *mo s o
O"OoS*4 Anhortie.* est and %'1at~ (196) WA~ VrIt (157) gjw W4epa"to M~ ft1 aR4voomt A* to the O*Iit"o f tieq4A


TM VOW~ f~t4 wise.4 0 ft 01 Pis of to bWain An

104S tft& te ves SUM"Of OW UvaI Tb*s= tm. he b
46" . To oupnets we not in mRy glvw ratio aopa 0t the blo4* ho*Wu.' This fot, aMW to te tet Vth ths erotatis presswo t aot pe* moo to aommi for thepesr of the f1xd4. IOWU to 0*e 6ea2IUto0 tbMt the 6611 In the dwrol4 ple =0s dis
*s# vol* Then to, not ftn ul1t afi1t*atI~ of U~e. ame* terb ateto the wa147 tww helu omo %W*Z t e pn In the blood a tie spinol fluid*
Proteins to~t to* 16-36 09 PoM of the norm Ir5n4a fluiMt Thse ofta are ixbt aqal divided betwe m bu~ .Xbumttk. The protei* do not apwto be ables to ps the anisiitle miter,, or,, it' *oy we able to pass ths bewrierv tho lzhm et esoooinv *law# as vI1I boooot amwamt 1tsr ZIsevs (156) fow two ptrotln In tho vpIWa tlv*. an* of which Uhave4 asa on ~ n the otbor being soW.Zb1 in 41st1111d ^Wt. Toe %ts lsAte pEotson would4 ba 1414 an a lbmls.
Per ftm reats tollovine Z ,svwtm' vok etthewittoe aeaioA that there, *a no )g.buliz In the spinal MIAt of * foww4 P~rsa. The %**to










used for globulin depend on the ratio of globulin to lbmmin, and wbat these mm have feand is that the globulin Ina noeual fluid is not d,tooted byr the method they used, The ratio of globulin to mabusn in a nomal fluid is sueh that there is no pr lpitation widw the .ooeAtim s of the test. The"efore when It Is stated that a troe of &lobulln is pathologie, the meaning lot the globullnalbtmin ratio for eaplete protection 1 passed and a part the Clebula thrown deva, the lek of protection belng pathologie. The oe modern authors reoog ntse this afat.
The nature of the globulin found in sypMh is amewhat different ftom that found in the normal ease. Raba gives a very eleoa plature of this fast (los), He first =plains that the antigmic substaee in syphllis Is met likely a lipeeld-protln ebin one The lipoid Is regarle s an inomplet antigen (alle hptm), *if it were possible to secure a pure protein erom the spirocheto, this wmld probably give moh morn satsfretory tests,

The antigen of yphlis Is not sapable of .usimag true autibedy fomatiom, but give, rise to two glebulim. The first of these Is insoluble and roum in the cllso, the second is soluble and U wshed into the blood oUom. ?he soluble globulin is the antibody whioh re- V with the hipoide in serologio toests. The origin of the globulin* ezoiting ipA ds is uiuertain They ma yme frm 4ther the spiroohote or the tissues or the host, In any ease it is neseesa y that th spirochet"e be present ftr these lpside to apper. XaM clams that










all1 tsstrsphi r %mood an th "II Sla a The fxwt Is Mti axsco n d stm s so= a t11 ealtn inn of qrpMu.
In~ Us -N v. Inah dorw 0l alot$ 41m.to *If 5*WL

=tbo5**, it to pabablo Vwt sy*I)4s; d" not prdw =0 "spW

glouli# 'at *thrs In &tot
13wfnt that tzwR antiO4*Re a"ntgnrtdinTroxaf
byv s~hili e ishv byt* swr of postive bloo rwnetos 4n


that true titibo45*s eaily pass tho mntabrirwdaefw Iniedi 1oly i the spinal fluid. A seam& point to owsi is the work of Isafeld (160) 0 *o pmved " th Pp ~aI fltu syto Is witbi. to vofture Its ova attb*o*
In as of 1.toypii tho g1.balin-Atibo4 so U ". te

Ini W spinal fluid syst, simn It is not alws tm4d in tho blood*

On the other ~ ~ ~ wi hnr* nUe isent in heoth $be spinal fivid um4 bo" stream.. We uay say that the .ptmAi fluid vA the blood oysto ar both ftpe of prfAL 1oalina m~he4 for sypbiste and that thase wssts work inMeea4mtly,
Agat andTreuea (161) state that "have PWW~f the to always no" atibody In tho ble4d stw'a tha In tM spitl fluid* Mei report to OvIEdi4)y mad anaInWV wrto Rev *s* MW we view their *a* in the light at t* mmy esat. in *$dh Moe to a gal











b1~d teatlan "A a povitive *Pima. fluid womtan?

)RA11#,* Uman WeSf (162) bave notod that the ant~i 1ipoiM, In procipitation test*, A"' ptroiptaWe b7 the globuXtla Idsi was -paiated out 1msg before by' Spts$in m aul (103). Wat ;a 1oy, Kemh and Wetall d1id find of ***Winterest was %bat the tloltwat wa n. a~aadin any vay If the globuin1 to Isolated t loot 1a XaM~ toot, the preet-pitate wany beame and tho globualin solu. t1OM still re'st* Its power U0 give W ~old #01 or Kast toSt.

The4. m of .esssA lip.14s Ina Mme sypil spinl flui& has 'beer, ntod by aare (163). Theso 11poi4. havoc son off eat on the col1*14. testoo an this was rrovm, by Irradiation, If I-he ignmu fluld to traW with Altrao-volst light eand then a gold toot vun those sum will bave Whit.4 to 'the Iloft. lri iation Is lkowi to affoct lipelds (1.64)#

A Vmft of tho iuperatlv* winson for OW",~ the aptiat- flu*id io ~mderttoo4 trozs tho pame Awe plainly fttat the west ot the ems

(S?). ow. MA the mmn working with him aith "Johng IiO*hUL Hospital hAlve tom that tie nervous myntsn t, not A$011'l inw1l*d in syphtlle. Tb* Immsi o~f tOle paf of the body la a later' otap %t the "swlogio tests ahov ths preasmee of this invuion l1pM Weore =ty Irqfptoma Vpm. This firmt sign of lzkvoa init Is the av esmo of


Ae~warnce of a Positive 310buin test. 4'astly there is a Poslitive


As theso a%,op go on the twesimnt mt be otM~ up# elns tOw










Owinge, flter* allow only a small1 part of the. towe Soucatiol I* PAs inbt e SP$Aa1 fluw6 It ts to '04 Aste4 taistw the poaa of %he mmttatlas * *eroSyjbIS OMe I& the M"04 as fthlr
Weams,,The oosle w globella 41*'po tilr, AM %wAW AnI complete v"umo b ssralogic mstion pwnIe*e. Under serolooe mwstlon In W~s ate is ItAw1Aod the cold -test This w.,)A twA to disra auy thorls a to the moa tmo the gol tes, bovvw, It iMW depend on *a ewtai& Part of the glowin easeU~b its oao.

aot, not all the clobull:.
Xshe, Xllii &M mwy other wokr* bwi fow that pms1pitaton

teets do not tewezee as rapidly as oo~1esmt fliton tests. Athv~h in this conotAm, asither do tb. vpitto tests beasos hstp as does tbo Wasrm -b 1AM Is wmat that no awt of treatmt v111 ftw*s a W5voat4 a n thog the diseave is elialoaly curdHt aiutboritle a"We that a prweapitatin testt a coplm flation toot and a clloW test shoul '1e m.4. with sine * Lwids for' 41w-ut. aad folloinw eatse
it smWNISind Wei ettion, w have the following 0.nldvatiam 1) ruw*1y to the splal fli4A test a prwa7 di setlo tst# If the ph)vrsicsi It Svez& a ppe ObWW to 'follow the 4seae.. Whe tri,~l flRd4 tot shew aevoriw ti InvIoinv % on before loslis arevi4mt axA shesl4 be prveial mtlanu a long as th 41*4s ezisto, 2) terow is no tro~ atilm4y fofimi bet a e4mi*.sltt* &Uo b'ilaL i ts plams T~he tosts depend an SlotaA Ua01p nonlsations 1a










00 VpeMe of oOVeuentt lipold4 floofeuaton b, the glloelin# an eo614 flooetlotlon by the globulia; 3) the euits tests for atoha are not In say vay ,eeif 1 tor the eex.l. glaeuln gonarato Wy eyphilto, bO ar aeely Ape"4nat on the total gloabulalblmu ratiol

a eoaton of tests Is most effeative in the spinal fluid. o test of awh type should b. uet. The pre4i4tateoa toot It more valuable thn a other trp for following tho pwogws of tietamtj 5) it Is eential that teeta of the apinal fluidbe etartot * soon se a positve U004 tot appwar, If the sppial fluid is positive by any teet the treatmt mot be f0r' nourtoypblls.

M eoeeoe glolaa In the pinAl flAd a be ewas by man y Othe patho1ogei emaitions in a4Ation t syphilis. Tuberclosis Ad aain.gitia (men - us) both lead to an oeoess protein, the former giving is, to globuin, the letter to albumSin (107), tiA praecox may give ris to hypertesio hglobdosie v-il hypewabminosts. Any or all of these eondttioaa may be foiam, or the spinal fluid ma be noral (165 # 166)#
We bay* also ea that soe SoUli *eats a# misleading na the preeeae of eel"ajwp , Meptlo eaoiwojo, m a, typhoid f~1 lupsi


In globui n preai tatift the pU at the t*i0 of preeipitation is of ia"Otwo~ This Aill bo AisoussoA in the vootion on

WA2I










SODIU YARAAA*O0OD


boyreu4 (167) mdo ma iaetigasla of mo tatioa or vmwwi asic and prtens R*2t 0 W9 i t)U tirot tie tho e tiei ot IOwh1140 DOI and Pwt*iU Mbs b40A t0*&. BifstA, wstie #ad gold solo r r 1 mt*o hydox14. (*11s%4) Is 1ybklI.. but has a 4lftwset apoaA #1ta 00. Th0 e rsao tbat Moylm4 o"sti vomiA4m 11#s to the faet that it to the on.ly on of the c e fs t ith form poaes. In an m sl w ution the *Met saW faeito RV6)60i7. The etat4est to osl that this em otbew ws, aw pol lt 4 positively It is so alslmA4 tht pwo1ae :% nW1vo 7 4rgit i neutral wA bsl sol* tio.a. =4 that In asiA solutas the. protlas bomsm positivWl


In the telrmm work the oomtwatio1 of Vs31Q4* on 981 atom per 3W ltto. YT$4os ad41tios wee wosdh by the aitin of "tet & It was t that slVbot in a solitioA of p lows th"
4 pralpitalSedsuglobU t o wSr aoi t P 5#2 &A gl .t Us in solnatips of pH lo thm 5 wo. wsd It&toL Te smmt of ouglobulia which a ivia .wt of albusin will pr.tmt Is isa*ms br lovering the p)~. No procipitati 1A. tWos p~tsocum~ no~lon ill41 t" P1A attk PH of nor* thmk

b UWM wa tvowsa s glbuin sm an sbm i sto s

bet" the In Vw Aume of W*av"s It tas to 15 of sash an Mw~ the p1 see mit In the v$intty of the olet

















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bttt Uwa the wo14l) remauls, *bore A I s a o f snsitiWty.
wI.a *Is* triod to dotomlie albmain end globulin in the 8we smple by o en Iag the pit of the vonde 6oidospiml! flid6 miXt"o. This Is aditted to be a fallur*.

The pR of the nomwi spinal fluid I 7.05 to1 This o hanw8 an et*ndIna to a value betwoon 8.0 end 8.1. The alkalinity ig gomoated by the large e rbonato reserve found In the aplual fluid (156k 157). In disease the saew pR values are fvomd with oe exoptiz meningocoacus m"IAtis. In m oms "eulagiti the pm1 is

7. 7* on withdramd anad the valu* I not thwngd on stooding (169). The Iso.leootrio point depends on the salt oomeatitton nd Ilies between ;a* 4. and p1* 5 for the moat part.














A M , fluid reaction of sy .lis. wag the a.b jeotivo ooleetevi In orer to be oossful this reuntion *Wmld shew a oler ttffrsntton t the eina fluids of eyIdllti pa. tients and those of non.,evpu1tI*** In order to saw time the labors. tory and literature work were begun sealtamnously,
The best starting point was to be sn eotnal o of

Pfoioter's werk (170). Ropots bad been hear of hi. dllasis by orystal patterns* Fortunately no eoy of ?Noiffert a book was available for s~o three onths. The present work sould not haes doveleped frm attopte to use the rather systie methods he rommads.
Having no idea what might result from attempting to orystallise metal malts in the presee of spinal fluid, sveral metal sat solutions wore mads up. Those, solutions were made from those salts which happened to be in the laboratory, no eonscious esleotion was oreeis . TMe solutions prepared moese 4?Ci2O7 (5. 2#5 & 1%). Co("K)g (lo,5 1%), v1e)%fO,() (104, 5', & 1%), CiUcl? (lot 5 & 1%)S WC1p1 (saturated &
hal..st~rst),A"p (saturated in 005N RCI), and Wi (saturated,


These solution were =ade ur with r,'rh weights of materials, It was re ognised that any solution found to be useotI would have to be analysed for metal oontent and purity,

4hile the organisation of materials in the laboratory was progress. Ing, n areromet was made with the Florida State Hospital for spiral











flvid ssplo, If we wold frniot shipg mater a exp m . they. would -urnish us wiih from 20--25 Oh tempe$ of spil fluid w*ly, as 1Wg as they wer* able. U boxze were b ilt for the shiping ov spinal iuld . 1spap.s Ssih box was bu1t to eawry 2 soft glana tst tubes with cork stoppere. As oig as th tubea were lid oloepd them in such a way that broealmg was Impo"ible unlost t'h whole box was broken, All shipping was by expros, the trip roqur-ing smo 02;-% hours.*

kior, use of soae aged spinal fluids remaining frm a finished rosoarh of Dr. Pollard'o, work vas tarted, Tho sults with Ker and Co(oP.) wmo of ro u80 In th diffeertation of syphilitic a none.syphlitlo spinal laids. Therefore a seie of seeoidary re.agts was also prepalr.. Thoe were mwdo with tw* ob-jects in Rind. The fi r t was to ahunre the solibility, th s"ocod to ohkn" th pU. For tho forer ethanol wtd aeoton wwre used, or the luttr purpos oneontratod and dilute (1,6 IVO , 323qO, LCI and NTJ4OC wero triodo All reagonts were put Into cm okmou glaes bottles with mdine r tops.~ Tho nerew tope we,,rs p aist"Lo#

After malig V~a aaeora roagonte, work w#r af-sin vtarte4 useing the di clwogat* sv.Uons. Thero "Or3 thrree ooluton ct Ypr %z ten secondary rgont. U1st ech cobinatin with both a syphl3,ti and nom-sypilitio spinal fluid, thOr ree 1tod 60 teats to be aftar zdxing ind then again after d ,ying With three eeeaPtion this work g av* o v si4ts worthy of motion.

It was, ound that a dr'op of syphiltie spiual Vluld tv whioh had











been added a drop Y , St regth X CrC gave S flOCOMtioU when a drop of 1#6 00O was also Aded. This roetion did not take plean it the spinal fluid a nomal (wn-msybilitio). Thro wus no difhrmtiatio with aiw of the other metal salt solutions and these partoular Spinal fluids, with hW eX0ption of the half saturated amndiun tetraohloride solution. While the floofulatma in this ease had a different appearane, the reaction appearod to be tho smo. this re stion of VC1k did not look as promising as that of the ftrn07? To aom-yphilitio eaoples flococulated inside of five mlimtes.
Duo to the age of the spinal fluid*, theoo r astions wars looked on with distrust. Any nmober of *os oould be Imagined to have taken ploo in the six sontho the fluids had boon in storao. As this testing was nearng ompltoti, two frtsh smsqles were reived. Those were pooled, that is, ech was a mixture of a large mmbor of empies. all giving the sae Wassemann resotion. One of these samples wa Wassemnn positive, the other nostivf. With both of these saupies the 40rr05O and Y014 reactions ooeurred. Wether the eplo, was positive or negative, a flooculation resulted In vvery trial.

It appeared that the most likely ohang in a fluid wuld be los in strength as it aged. For this rason thO reagents must have boom rade too stroug. It was reoailed that obsaging the oo m tion of the gCurgO flee-fold had had no effoo on the roastios so the 1#6 nitric aold m diluted. At a dilution of about 1,100 M" and aking Use or 2.5 or 1% ~rwc w. a elea differvnUtiaotla as ai S n obtained a











go" prt of the time.
This rauetion had ono &erious foult, In too *My trials the U,,i lg ws *at just rigt, and Instead of floonulaton tihe result was a h rd granular p e pitat.. Whilo the positive swple alwas g a tleeuoltioa or gramloo,, the negate, simple at timos gow, greaulos also, It was seen that repeated tests would be Aoesar for a alow differentiation when using the KfI2OrWO method*

AS seen as this reaction Ms , intOUSiV sarah at

the literature under potasalu ieh t was . Two refrne (109, 110) to the work et Theon were feu. Although the technique wax ditrent, the pwrioplo a the some. In ple of the twiehloro. aSetle eld, we wore making use of nilrio old, but the nation of thes two saide to o awh the same, Bth of these elds, have been used in the estimation of albabu. Prunell mentions tweh1oroioaceie (iii) and nitrie wa used by Douris (171), 1i6thew nothod is to be ro adad for the estiuation of aibusin but the albumin eos* is the Sense et the resation. In the light of these findings, it was decided that work on the V014 ohuld be the min objeet, and that the diohrsomt method be ued to deter ine whether its reults ee as reliable #0 those reprted by Tacomse

svmral dilutim were fndo m the half-saturated s lutio it vanmdiu tetraohlor4*e None of these was able to Ive a satisfotozy differentlatl of Syphilltictow nm-sphIlAtie spinal fluid* The solutions were allowed to stan an the desk me ight, and tried spin tho next d4, 0"a of these, No., gae Sa, a cler differentiation at











ayptll, hO 4alutODS. 96p~illy the Nam diZ~tv Onwss had *hanged o~lr in this length of ti.wo At the t. they vre 2ade Sh had a blue soler After ten hours th nwre dilute ones moe blue-gw the more the dilution, the, nwso ge andl Ues blue the solutions .p-eaed,
At this tiee fourwenu fresh amples of spinal fluid were roeed, Through i misuwderstanding tNo wor 11 Al po led. This wisundretsnd. ing was later cleared up, end individual sample. reevetd Sins 0 sensitive solution was at hand thes fourteen spIls were he hed. All rwe on# e a reasitio whioh was in etoord with its Wasserns reaction. Th. sensitive dilution -of VCl4 vas made by the addition of 20 drops of alf-satuOrte VClj olution to 12 ole of distilled water, The smmitisatiom was allowed to develop by letting the solution stand for W12 hours.

This shipont of fourteo samples was divided into three group#
or *Uas*** The torn alss is used by the hospital to denot he s oerlogic ree to end the pvewe or a*bees of syphilis. Thus elass I fluids are Wasseruaw or 1hn positive and yphilit3ie, Class II fluids are Wsormm or Zeu n tiwv and derived fto treated syphilitic patIents. Class III fulide are then from noinushilitia patients and are Kahn and iseemarm negtivo. The one fluid whish did not agree in its 2 reaction (positive) and Wassermmn section (negative) belougd to olass IT. A great deal of interest was taken in this fid ing, sinee it indicated that the V024 test might be a preipiation reation which did tot rover s soon as the W rm reactio











The faot that the wanadit totra*1.4de dilution had to ba m*o a day before use was a 41sturbing matter, This behavior m stoi*4 and found due to an oxidation of a part of the ,dim. That there sotually m not an oxidation of all the vanadi-m was mae o1ear by aqt os t to us* the solution two and thre das after ts proparation* go results obtained an either the mmond or t ird day had any comlow tion with either the Waomsum report, or th. sm dilution on the first day following pr patloa, 6h the e eond " thir d os the reaction with a olass III frin was as likely to be positive o that with a eless I fluid.

As stated befo*,. fmehly made VCl4 dilution to blue. Whther It is allowed to stand In the open. or Ia a tightly elodse vessel, the color at the aend of twelve hours Is a blue-green. After 36.4 hours the oolor Is greem-yellow, and after a eks tim th solution Is almos a pure yellow. Whn the y ellw olor ts roahed, no tho Mange, ooours. After a month and a haf an* or these solutions was found to still be the stwe yellow.

The oolors of tho vansAu Io s in the various *almos states are well know. Thee wo exporimerntolly produood to 4otemin. exastly what shmages wore taig pimme The lowest state of valeney vanadium asms is 2+ and the hidgbst i 6+, A defiite and dietinstly eelred solution egste correspondin to eeeh of the valmee states between two and six. Thus it us found that the colos and oorresponding mlonsies ares 24* laow der, blue-grem, 4+- # blue,











5+ yellow to .olorl#U, "d 6+ * red-br

ith the** soluiAos ase a pAde. an imol appeared to be takig place. The dilutions m#Ao wre originally blue, dsentiily totrflent. They then aquilre a bluo-.og color whioh matohee that of trivalent vawndiu, After a week that color has left, and the solution is a doftalie yellow coler, pntalent. ince blue and yellow will u~ke all eshes of blue-green, nreen-yellow and yellow, a trivalent solution was divided and me-half oxidiW slowly. At a walenee bvtwen four and five the exidis solu ti eculd not be distinguished fro the trivalent eontrol. lutions woe u *e of these more e oentratd W,4m * tion, and no nolor oh&o fowd. In ame eses the olor of an Lou in solution i* dtpen o the ooncentration, but this is not reportod or found true of vwdim, ith vanadium solutions the color depends upon the valence stat . not the cnentratiom,

To prov conclusively the valen atate of the somnittve dilution, it was oompured in two roactin with a known solution in whifh the valence of the vanadiUm ws a nidture of4+- and 5+, Both solutions are o pablo of forming a red oolor otely when a drop f 3% hydreCen peroxide to added. If *h. aount of I is snall enough both solutions will go only to a yt1low oolor on saying, Whon this test was repeated with a solution of Vl the result was a blue solution.

The second oxperimont to ehow the alence state we the addition of dilvte N&OH. It thore is p lont vandium present it will ooaglate to the extent of dkeing the s lutIon when it is nuutrol,











nd trn yellow when the solution Is basic# Wit the ba solution this emotion readily took plaeo. but the resulting solution was greent duo to the totroalet, vslux tons still preost* Th unktn, behavd In oxmatly the sa* innaw. Trivalent 'vanadium does aot ohWs". in basis #64utiozi. The ogulation and dakening of the **14.. tiozi at the neutral polat In due to the ormtatin of pelyusrs with the eperloal forWsU V".

The formaston or ., or its polYmWs, is Preventod in a soltion made fwo VClb* Viaoh a solution 1* aid due to bdrolysis =*d this leads to the formation o polyvanaio meld, probably 1V6Ol7. IU solubility of VC1t is not fouMd in the literature, only the feet that It deomposee In water, Thls deoompositlon is in %aoood with the following equsatlon

vcxh + , ++ + g + Wf*",

The oxidation of tb vamndyl io (Vo++) is Inhibited by th

preseno of hydrogen tIon To tebills eolutlons of VC111, ezooll SI io add It was, found in the, oouwe of our expeivnts that the more mtratd eolutions did not oxidie with nearly the speed of the dilute solutions. This Is probably due to a lower salubility of ozygn on4 a lowered nativity of vanadyl ions. The law of mes" mtion shm why hydroea 'n is effective in prrmflion of oxidation in the following wq!matI

4V4+ +o2+ 4( )" + 11 a O + 41t

heo acldity of a solution made from a aluitw of VOCIO and VClp,











to such that oxidation Is rapid In dilute solution,, A stabilized saU tIn was diluted to tho sOA mItWAn Oontnt showed no oxidatiqu after three *Avths.
After finding the mixture of valOW S to be the sensltising prinelplo, thor was no hoed of zwking a dilution ton hours before use, The old. overly oxidised solution was save and added book to the frosh solution to pro&** the mixture at the time it was needed.

The fourteen pooled Spinal fluids in the first shipment were tested with the diobrmtse and ,ltrie aod ethod. Class I fluids were all positive In their creation. CUSS II fluids were all posttive or doubtful in their roaation. Class II, whlh should have all been negative, gave all negative results, with one exception, The false, positive at this point, coupled with the toot that Tooo neve got ony reognitioan for the dithrate, roaetln, all but led to the stopping of fmeatI oI this mothod. It was deolded that a total of 100 tests would be uade with this teohnique, and the resu)*e oheaoked to a" whether it should b1 oontinued#

on roooipt of a seind box of fourteen pooled spinal fluids, the asuplis were oheoked with a Sensitive dilution of VCl. The dilution was prep d in the following new' 1) two oI*" I and two olaos III fluids were seloteld at randess :) to 12 m#. of distilled water w e added 20 drop. of half-saturated VC14 solutions 3) an old dilution of the s se strength ws added five drops at a tne to the fresh dilutionj 4) after sach addition .t five drops of old solution a test was ru on











all ftur fluide sleatods

The four seleted tluds Sav the proper reaction arter a total of 40 drops of old dilution had bom added to the fresh dilution, sensitivity was shown after the .k4fition of 50 drops, "d at 60 drop* the Wassomm negative fluids gave posl tive reaction, When th solution was at a fairly sensitive point (after the addition of 45 drops or old dilution) all the omple. in the box were te ted. All

*lass I and TI1 fluids Agreed with their Waoesevm reports. Of the four *less 1 fluids In the .hi pmt, two were positive and two egatiVe to V(l1t.

These same fluids were thon toted with the dlohvmate-l"

motthd. All *laos I fluids 4heaked. The o1^ I1 fluids gave two negative results, on positive and on doubtful from the four fluids* Class -jI av* one doubtful result an the remeindor gative. The presonoe or 11 Allthee*oubtal ratione may be the degree of poitIwv* ity to whiab ?aeooo refor*, but earoetl oainatilon under the mioros"p failed to reveal any differ ce Ia structure of the turbidity. At any rat., these reaoti o appear to be graded frM positive to aegtive, by the .iut of protein presoAt,

Up to this tU.* all fluidI tested a been pooled somplos. Tho* isa action on the addition of oertmin spinal flui d to others, Plat, Bauch and Rosi found this roesfon unrodiotable, end net related to syphilis (9). -obile this fmat wa3 not kwon at the tie the fluids were known to b. altered In sc om rg they were not the,












eharaeteristio wate white, but a turbid white,.

The third ohipent of spinal fluids reiV4d contained aieht

fluids in ach olass, ll InAvidual emples. The shipmnt was do livered late in the aternoo, and wa kept on Joe overnight. In the moming an attempt wne ade to prepare a sensitive dilution of 14. The eso vvthad ais used before was used. with the exeption that only six *l of 120 and 10 dros of balf-,aturat. VC1, were tak*n to aks the fresh dilution. both aleo* IN semples taken for testing the solu. tion gave a groat deal of trouble. A 84esItve dilution vas not sue. oesstully prepard. Vie fluids wore left on the desk unti late in. the atornpou,

At this time, four othar nonplus were seleeted for testing the sensitive dilution. The afternon trial at ensitization of a V14 dilution *As �UoKtlI11o trkiuble was enooutered to ua wa. Aftor the so4nitivqe dilution woe obtainq4 all tho fluids were tested. All class I fluids agreed. Class Il fluids gave five positive and thres negative re~ations. Cl* III fluids gave smn negative and mw posi tive teaotlonl, This positive was onie of the rluids used in the morning attempt to sonslti e VC14 aMt for part of the tmble, at least, It roeelve treatment In, the seotion on Disoussion of RIteegt

By the dichronte ,amthod all elass I and II fluids were positive, as ,ell as V1e fle positive whieh .me out of el. 11 with VT14* All other close IUI fluids were negative#

Faith fresh fluids on hand it was decide to check all the metal











solutions previously pro-ared plus few more whieh it w theurht ,iht be e~gativo, Cobaltous nitrate (10%) failed to give a reaction with either ols I or IlI fluid. Saturated arsenic trioxide also was without affett

Cuprie ohlor1do gave a reaction with both class I and III down to 0.1% dilution, and then failed to vivo a r*e*tioi with either sypili-tic or non-ayphlitia fluids. Cuprous chloride (saturated) gives a reaction with either class I or III fluid, and when made half.sturatod reacts with neither class I nor III. between those orcentrutiono it is possible thrat Cuop will differentiate syphilitie from nonsyphilitic fluid, but the Instability i ouch that further work was not indicated at the ,ime.

1ismuth triehloride in dilute I mipht be used to good advantage, but an reaction of the metal is masked by tho PiiCl precipitated. The amount or HCI needed te clear this precipitate Is efficient to floouw late the protein of a naaosyphilitl fluid. Ferric smwulaw oalate Is not a stable ocnpound in solution, and it presents the eame difficulty as HICL. heon added to the spinal fluid the reaction is masked by the Fe(OH)g precipitated. Vernes (74) used oolloidal F*(0H)3 in hi* first test and this proved unsucoosafl.

Volybdie acid gives a proeipitate with negative spinal fluids, even at trace conentrations, It is clear that a great majority of those metal i#ns ore able to precipitate albumn, as well as glot ln. ,ilver nitrate is capable of giving a precipitate with either class I











or III flulds. Aj# t strength of the solutioA is out down, ,the pwooipitation stop gmd a Vdlky Aggb~~itn taks its Pl66, APPairently protection of the silver is neededs Collargol. contabig protected siler, may be of sam se (68).

The next fluids, re ived were f ro . Luko, aospital, JaCkSonville. While thee were all s -hat cloudy. they locked with the vanadiia chloride. Oie of these vs marked only syphilitis, gold aol# positive. This also gave a positive reation with VClI3, The weekly box of fluids twim the tate Haipital gave good clear cheeks with claso I and TIt fluids# As usual, olaS 11 was divided, the eight samples givint 4+,3.. and I t results. Fro the thirty-five sexploeram colved in this week, only fourteen were checked with diehromate and nitrie scd. These were in line with the Aesseraann report*, with the zoeption of those l elss 1# all at which wore positive.

The fifth box of spinal tlUd samples from the tatse HospiW ow. taned osnales which were, for the moat part, q(uto eloudy. In spite of this. the VC34 SOlittiono proere sd susitisaed In the usual MsMs was effective. Class 11T gave eilht negative reactions, class U, four positive and four negative, *"&us 1, siz positive and two weh wrt recorded o doubtful. These doubtful fluids eontained bafterial oo tmirination, bewyd a doubts The first of then gave two eek psitiv reactions and orie very weak positive reaction, The second questionable sample gave one negative and two positive results, the positive reactions being week, Siznoo sAyn other tests exhaustively,, It isap~w











thot this whole box of fluid* should bay* been d sarded due to ban. terial oontsmination. however# try wro tested, and thm results an not so for out of line as to varait striking them tram the w.ord.

This *one series of asaples wa tested with dicro te and H0 The eight positive sy1ii o and the eight negativ nonyphilitio fluids gave perfect rsulto. A deviation from previous e enee was found in the *lass It smplos. Three of tho eight were ne*Xatji, four were doubtful md only ono was a true positive. Th t.,ree negadves and one doubtful oofmspond d to the four negative results obtained with W14.

At this tUim an 1tept was soe to prepare. sensitive dilution or VC41 from the originta "ftirted solution, go re-so was sn for not using this, mixed with wj, equal vvluao of wutor, sineo the half. sattrated solution in use had boon mad from it in the sae maner. The sensitive dilution wa being xu*e new in raller mou-s, the usual teohnique being to toke 6 al, of distilld water, add to it 10 drops of oRe-half 8a ' 1I4d ,"o* 4 nd then sonsitize by the addition of an old (oxddisod) sensitiv dilution. Trial testing still ws us to find the apeflo mMat of old dilution to be added.

To ake use or the eaturated solution of VC111, 5 drops ewsh of the solution and distlled water were adde to 6 %l, distilled water, FOila the ft timhat drops am not &omuoe for measurement was rocopnsed, it was supposed that this would be seourate mugh tor the purpose. although several attats were made to sionsitize solutim











maao in thi maner, the 't of the lot goe oe " ls poitiv with the zC,&tiw samples and tie negative results on Wasserx positive, syphiliteio saesl. ?hInldng tbtt 'this asm not "aurat* enough work, 20 ml* of exactly hif-zaturatd solution was "d* This WUS placed In the eae t bottle as the other haIsarate4 sol . tion and mark~sed 0l/A So'td ?V No. It* ?he *e. dropping top m& then used tor making a dilutim from ts no# S2 soliution as was us" oa the orl.ginal hvl f-saturut4 solution* The rsults in this as" wre no better or wore, than thoe got by use or the saturvted solu.. tion the first plaog, ?ho foot that &100W takes som t1 to ix wit water wid form a solution which mW be dupleat*4 has been known to the liquor trade for $*ars, Howowro no record of this is fovad oooorning mel salt* hat some partial oxidation Is ubtle to take plae in tho =or dilute solution is possible, but It woul hv no offeot on the rults, As long as an oxidied solution Is bein added for sensitization, -* only result would be th n essity of using more of this oxidisod solutions whep propari- se siti i l. tion frm the saturated soluftwu*

It was found thot with the next hipmnt of sinal fluids, either the go* 1 or No* 2 half-saturatod solution of VC111 go" the s se su ts. Ola I wnd II wore In sOeowd with their a~svera rportal elase I1 was equally di vded, four positive and four neatvo re*ttaS it vast be that ak definite period or agin I neooeossary &Mtr the dilution of saturoted vaadivA teteshlori4*, To aooek this. the











saturate solution w.e ae~an use to mika a dilution, and again it w ia",aible to seltise it. It me discount the fact that a defltoe mzing time is required, the only altrnativ, is partiole sise. This could not enter IA, tre there la no polyvanadi. aid domed until after oxidation to the pentavs3rlo State has taken place. Moto if indeod not all, of the pentavulent vwiadium is added ir the sensitisati0no and this has bef the .ein eaeh oese.

Several questions as to tohnique and acncentration were taken

up. The use of droppers for sun was decidedly a thing to be avoided, so 25 pipette., 1 ml., grduatod in 0,1 and 0.01 mri. were then employed. Approximtoly equal volumes of spinal fluid and sensdtive dilution of VC11. had been used in all testing, and it was found that exactly 0.1 ml. of oach Cave a taest which was easy to road anc handle. 'Tis volume relation was used Uhroughout the reminder of the work. A separate, clean pipette ws usd for ch sample# Ater uszz eaeh pipette was cleamd with cleaning solution and rinsed six times with distilled water. After rinsing, the pipettes were dried by drawing warm air thniuf,h them with a water pnp&

tn order to save tia, th ratio of oxidized (old) solution

needed to senitlse a giv-n volume of ftesl solution was dearmned. Uip to this tim sonuitixAation haad required upwads art an hour osmh UAW tests were to be run. Trial tstig was used to determine tho ratio ot old to fresh solution, sod It was found to be 1 5. This ratio of 1 al* of old dilution to 5 Ml. of the freshly mde was used with success for











a period of two moath&.

The ocnooetration of vanadiums a. vamAdia tetrachloride, was detorsined in the asitife 4lutlon. After a oonsi~draiton of ths many mothoad for vnsadum analysis the oolorlotrio mathod we* ehoase. in the first plate a vary dilute solution was at had, and this would require the use of miorooequipment for gravinetrio or volumaio analysis, Thit equipment was not available. In the second place, a pure ample of ammonium wrta-veanduat was on hand for preparation of known olorimetri rsa dard*. A direct omparator with split field viewing was used for this detoxezition. An Heli ; type instrument would have been much more efficott, but none was evailable.

A known weight of" th* fV5 wa" made to volume in a volumetrie flask. Into one cell of the comparator was placed I %I. of the sensitive dilution, 0.5 ml. of lz6 1 O), and I oml of 37 hydrogen peroxide. Into the seond cell wore placed equal amount* of the H2SO4 and p, followed by a deflulto volume of the known solution# made to I al, with distilled water. ahon the approzAste streogth of the solution had boon round a now unknom was used and the standard mixed ixi the asse order as the unknown. Wbhen a aolor matoh was at last found, the deternluwtion was seen to be aturate to 3-% oInoo it wms reproduolble to 0,01 ale of the standard* Tho eoneratia of VC1h was 144 0 Cros per liter, _+ Later analyses verified this result.

Tho reasou for soas, early difficulties was aexidotally disovered











during. the ourese of routine tostizig,* nid. were beint usod which ha jut been remov4d frou the lce-*box, and the sonsitive dilution, Aade by a dernte volue ralutioashIp gae threat false positive roactions In auo0e ieo, In the routine work, three or four slides were lined up on the desk. spinal fluid from an equal number of zapli. pi ptted onto the left hand sid. of the slide and dilution pipette4 onto the ight hand s.ide. he two liquids were thi. drawn together with a amIl glas stirring rod. stirred and th slides rotated by hand fQor tVirty seondse As eah slid* wa vixed, it "s replaced of the desk and allowed to remain there a few minutes before readingC In a majority of the positiv roatlons the floeulation is easily seem in a Indirect ligit with the naked aye as soon as mixing is oomplate.

In the Instance referred to abov throe Kahn negative s.aploe

had been so treated, " l gave a .visible flocculation. The work was being done under a desk imp, tilted 01oso to the top of the desk to Provide indirect light, oile starting to make up a second dilution, b.evliing an error had been made, these slides were left under t s li1ht. hile making a nw ansitIve dilution, it was Toted that the f Ulso positives had apparently alearoe4 kz~crosoopio exmtnation ton.,firwd thin ftaet

In order to bo satiefed en this butter, alide, were made with spinal fluid at IO , and 25. The rootiea of flcoulation took place li positie and some negative fluids alike at wOol, Only Kama or Waserwam fluids Caw this reaction at 250C. It ws found that











It OWN false oO144d-tood rations Von llw4 to sa to roft t vatun there wIIJd be a re-dissol.in o the flemlstlo. It was aleo fund that true plsitiv reactions were not re-di*elvi b oooling or hestia g, but refined =uAtl the water had omletely mporatdo The evn ertion o a nixtua vo seaitivo dilution and %#.g.& tivo *Pima tuld at ro temperature did nt result In floealstion at a time In its .ours..
to this tib. the f p a the Inetigation had boew Istentionally put aside. The ature at the flometoats, the pH at whieh it aoswrrd, and slmiar qastioms now tquired maws, A total of one hundred and sixttmtw spinla fluid amplo. had been "4heeed by the V014 dilution. In these tests on Wasseran neative flud had given a positive rwotionO and two waeraMa posItive flulds had given doubtful wea'*ts. Prm those empls 100 had been run by the diahoaate-aitrie acid vmtb*d ?his latbor Method had been tapered oft for oeral, rmeaon. in the ftrt plao it gave rise to too mey false, ra tlms, sd too ma 4cubtful naeft ns. In tUs soond plaoe the solutions won, not ooetsst. tcrgO, whish is not suppoed to aheae with ago, gots mote ed wmo en altl., so that a frsh solution mast be made up oac veek# Sitrio sold to also able to chase on storing, both through xitrIte foUao ed &WvporisatloC.

The nature of the spsil fiud Is .h that the vVlpitate obtaied ti livIted in Its possIblo 00oo dtion. it It ir l s abu"ant, It w4ght be Mny of a wokber at things. The mount is sU4s











that it is moroseole in divanalons, wtd to obtoidud tram Ol al. oft th* spile rium. Clearly it aust be emp oe ot' either tho potois or vanade aold, or saw 00*101atoR of tboe materials. To prots ham beaw disood, w, their sipifloan"e in pillss pointed ft, In ordwv to dotomine whether the prolpitato was orgewe or laoe' gauLo, a oerios of amia Oopio tosts wore run.
with smpls hayl a Volume of 268 *1. or Mre, it ws Possible to doteMie the "&tue ot tho ppewepitato and fin the pU of thte solu. tim froM whih it It . .1 3e m P mat er wa aaloysd dor these dstceul~tiona. For its, operation wo"."e awde 35 *1* of solatiot ia addition to 2 m. for preliminary rIing of the ele uotpoes ad & oll, Kqa volows of spis fld and seniti,, dilution were mixed In a oenwtifug tube, and if flomstio ssutoeod It was ooatrifutd et at 3,000 rpn. ?h pH, e h Aom d on the uper t liquid WAn-the prolpitat e"in" epartol, Def'o"e using "Opus in tais mar, mioroaeopio teaos wore n- in deteamin their VOtOU .
A toIt of 70 Yf dotamimattsw mea"*. =w 24 o tese fluids gaoe ps"U"v resatios both aioft.sad sae"os tialy. go sipilfi. oust differs in the pit of a podtv moou san ne4atwvo reAtlonists. ithor VMAI Sw s lavrae tPH of ,9.
After . trif ot Ote OWNrlpitabt. Its Solv ai s maw deotftsr
mine.ft no ease "as the sa ilivt test poaitliv Is distilla **ter*' wad in every eaethem' was solubili In diUte "M#d bae or salt.










This is ehsnoateruatio of gisbullm. Ybw o th, preelpLtatir wen remoe4 to eOft bler and fused. The ohavrte ritie e or burning protein Wu vey strag, ad a very s1iht residue of yellow powde reMIA4J. The residue wan soluble, in Ields nd prtod to be The prOAeIpiUte was thus found to sonsist of globulin end v.anodia


To detovmlns Whether es V, or ydrated vanadlo "Iid was tied into the prespitsat in a GmpMPn, or just earuied don by the floooclatel four of the p*06iptateWeve washed with distilled Iter repeatedly. After the third washing no test for vanaium was otAned in the wash water, end the prolpitate had ohanoged tm a Sry eolor to a POW whto. fAuion of one of these samples of Slobultn ptM so r1esidu. To hook the 0miowt St vnadium being asorbed by the globuia. V*0m uM detem ion were made an the supeuta liquid 3* two ases. The liquid wed was ftat from the pH motor ello ate ~ had been read. The liquid should have ooutaad 0.7205 go.., sa" was foqnd to onain 0.750 g./%- of Wl . ?bs JMauN t to a l0ss of about 6e $in* tho onslysis is ely ascurate to 3%. at best# t my be stated that the asetu of VaUSm adsorbed Is not the snmt In the pmaAmlent state. This is at lest 16.67%x the souuta for sonsitisatio.
While wo*lng sionwooplolly with some of the negative flui, I this serLos, a pocalar oourreaso was neted. Za a tow eases a fow.1m nation took pise, *ut a os oeMtiagn the uaosepie test of the sin











flutd, no prolpiute'" toud. Uovertless, 0 slid. teet wae showing ase t ieW wsestion. It. this wM an ests s1bumint it should be soluble in water, For ome tine It had been kaM that water did not obange the aspect a 'a tuo Positive roseton# ense it had tans p%o Tha a sodifioat.s was =e* in the prooodure. To 0.1 ml. of the spnal fluid W added 01 ml, of the sensitive dGin. tion In the *e inner as before. If there wa then flestIees, this ss allowd to set for a Idauhe aM 0.2 all or distilled wer added. After stirring w3 aillovwig amother mIM~te to pes, the sido was omimed, Flooeulatio was persistent only In the positive luid.e All fluids from umbe *ere nm niO seolly by this method.

One fluid, marked amosyphilitio, *sssmasm n*sgaiW, gave a

true positive reaction, with vanedium tetroohloride., The fact that the proipitate ma a mixture ot glabulla sad VpQ5 ms verified. While the ounx t of globlin got by eutrifing wee quite aml, Its identity mae quetionably prOws Th esuse of this r uic is diseusse"d later.

Although this phase at the AVestigatica Oeevers s0 92 sPIa flutds. only 70 of these we ot Suffieleat Volume to alow PH determiations. The daem was made at the eutet that So 'spea equwipmeunt be pureed for VS work* Xt io possible to toke a pU rea. Ing frsoh eao lide with the proper eoudas t After the p*r44be error of the sen had been elomlated it Was see that the mthod 4e hAd been satisfactory In all detail. ihe lar. ..d.vation botmue











inmluakal s84w to not hoed to umderstzd. Sesh fluid h" a difforent alkai rser and onriiml Whil there, Is little vwae tion at Uhe tiU the fluids ore 4 m, standng sOuses *a to bome alkalao. This takes plow sp otameomslyo but depends m the tightness or th stopp , heat and the mature of the ivdividma fluld. $ino. all work of this tWe iS otaUgtUi a large, dwustime are nmt


Fr the prepwatim of the originma saturted solution ot IC , a amplo whioh had been o head at *Uis laboratory for ye"s was used. This was the so sailesld**i salt. V014 is in reality a rod liquid, highly deliquoseent. No liteatue reforome could be fomad to the material sold under thes oswe Van*Aius Tetrachloride. C*V.(** The original ample of salt wma taken by a amber or t. inargani. prepaUos elass for purifiattion. Ts senplo was a a blue-geo nass of non-eoystlline xteriel. It looked b)eal 1Spurs mAd tMs student had the dea that It soid be purified. At Uat tm the use of this salt had Just been beom# etd there wa so rfson to wersat such a proposed madevtKai, Zn the sere of the puritflation ell t" material was rimed. This wrowamaeo oe0urreme 0eft the laborato7 with Id ml. of the saturated solution end a Itke mount .f halt aturated solution. Over 250 fluids were em ed before fet, V% was ob"Lned, aW ev then the,. was saffiasat solution on head to run about 1O0 mpee.
Simae the solid mal# V1t. w&s listed In tbe ewtalop*u of tha






68


Vnadtua Cororation of Ameria, so tbouCg4 was givm to setrift a soond "tple uil mar the ed at the .iparbutal work. At the tLjO samples in the eleveatk sb wamt wor being eomiodo, the que** tiou was raised as te whother the solution was just a ehos results made from impure .atorla, It a matte of shame pteroO the ue)4ag of the solution, then a very difficult nal4uis, o2 almat a micro oale, would be required to duplieto the oolutlm* . AmItatio of the saturated solution gave hope that this quantitative analysis would not be naoesery for anythig but vanadium. On evaporatioa of the sluticn the only residue we V2, The solution gw a sliht fime test for A end nothing else.

An order for solid VC24 wa se t the Vanadimu Corporstim and their reply was quite d4ishartonimgo Sine there was no sale for solid YC14, they had disomiiAed the preparation and had no Ida tbAt there was any available In the eommtry. Euquiries were then sent several hous dealing in rare 0hemialos, befro, attmpti to prepay ay of the materials A mall supply was iamlly located at the A. P. MbskeW Companym Ri York* An am"e of their V02J4 (so1i4) 1"s purdts, This was found to be pwtleally ivsoluble In W r and the mtall mount that went int. solution gave the grona-yoll" o0leor of a mixture of pentaval et md tetravalet venadium.
A *all sMpe of the material "M placed in a anieble mad igited. At a very lo twe4m turo there was a sliht sew o material velatilised." The Lvolatile- parbipt* ft" in tUe air. aul











totwashlovide Is volatile st low tomperstros md vry easil b drely . , The onaOlueslao vw rod that the trial pirohasd did have a very low owatent of VC14
on mGtimwd host"g a oson portian of the moel volatillld. fthe took plae just as a *all rod heat was, rosohe Depsited sn a Gold wt oh lJasso this bad the. properties or 41t s oWNsub lds (vanadyl ohloride, VC1)o Wm tis *mat*l Se disolvd in heloW s e sitso saod the prom of vadim end hiorld isa wer esLly demastratod.
After the aublimatin of VOCI2 had been camplete4, there me no ehmang in the rsidual material. evm at a bright zed olorI ColIo l the oruolbl to t s, there remained an owsao-y.llevw ado~otalline materil., This was proven to be voodim pentidde

?v") he soli0 C*?. V024~ was therefore a =Ixtur. WWii '~1 vocl a V2o5. In tion to e three .oaounds, proven ps*% a wmail perootag of vQcle is Probabl pmos5*.
Or the various Omedo it i possible to us. fo the pseparete of VCI4, the on. ised to make Us smple puwobssed as tbs, pas*Wg ef a sts of -bleriva ga ovw heated TiMs would yl4 all the pro~aebs fam, s"d that v4speodob It was rosalled Ahat the oraiul sml of saturstod I1 m mode frm a materil iich furs sol partially oolublo* No 4oat 45t* that It io this xixtaW of 5 salts which besre 1oe tmae w m4I ~.w Ovd.w,
Tn this wstur.o ra " dium smd the only soluble sslto ame










the atid and ., ?he VPC1IO is a ery rre sonaid swd so umstoble that ly trues an, liu to be preoet As tar aso edld be d t 46 t im., )ne*er, ho saw eoleoom U owuU be smd. frft such a sIturj, s"d not ftm puws Vh a addition to greortt ON* or puP ons"tu, ths also sllwumd for the ditfforea in smidift whioh would realt fram solution or a mixtu at VC and Vlh *n soutUon f pure VC). t is n tm a e very aid $olu4ou of VC made at this larmt" several oru% ao do mot wderp a oidation when diluted ad left eiposed to the airs Tds Is not a dosirabi effect for the prossut work. As ftr " Is raosled by the diresotr ot this eearlie work, iqVdd VC14 slow ws used for t preparation of thee soamlos fho early work wa net eor emmp3et4 . wri tea up, so so abso*tAsowlse et, th*e elutin referred to d4 be go%
A simple *" t" U eed fr the OMei at the Old *AteuIal on hand bs t@o sitreaoil aif son. VC14 eentinA 14OAf the ,app&VUO oon44stod of' a obldei. gesnmtor, C,1e drying tube, tube o .ntsimig the smple, a sA tue sad an oeit tube spaSg om a " solutifto Mf W . For the $esnemtion of Olt# e ed 01 aw hypoohlorfte wem Usd, The saple tube was so arroage that 1ib Gauld be bested.
Abm 2 groS of the s i r uO played in the apparats sad
hlori e pasd o~or it t a ta s a , wit a stead trom or Cl Sein th.OUh the apparats., boat Was applied to the "icro tabt











Por this puin*e a imovig Room lme,. 1/t inch lx height, was Used After the beating ba eem ocmtIm4 tfr ninety mlt** the apparatus W&# tare down and the sample allied to ooel,, Duriug the owe of the heating a al queaztity of red ulqd distilled fim the easUim imbe to -the safety tuhe.' Thi ws romd to be VCl2. After oo u the Smple me reo'ved end bftkme up.
It VMe deolded that no attempt would be madei to protdoo a fpli. se atu rated solutim, therefore the ample was trSktd In Oemttfwe tubes with an exeew water. A deep blue color developed in the soluton. The solid r due, was sestrifuged out and the solution ti)terad, usirn very fine filter' papor This solution was lbelled oem. oentratod VYlh and put on the shelf,. he nut at would be Ihe "uair. e*. of this and then its dilution o a 'vmodim eonte t the a" as 1/t~ saftrated V~, Yanedtz aalyses were made Pt the. nowly prop"r. solution and of' the hatfa/ttiratd VClj1 solution. Th m thod was the ane as that preIously outlined. After "loulations had been *. pled the mow solutim w"a diluted so that its venAdim eontmt WAS equal to that of the o gu m al1-a0 turated solutiOU This new bat. saturated solution wa ilalw"d to age fr a week ad then used Ax pavllel test# with old V1j solution.
So diff ioulty arose in runnAS the lost 70 asapes with somitiw diluftiMs prepard ft= both of the halWf_=uted solutions oab ftd* Noth enditiv d13uttater for mvaA ton a to hav I.410 crom of Vloiter. The aerae so of the smsi, v -











dilut on propared from the o14 solutioa differed only 0.08 pff muit from that of Ihe xew sonsitive dilafteta. esidering erro" *ad* In dalutions the smth itsift of the BD m pH aetr 0md all tasters invoveds the two solutions mre judged to be eIq4ilmt,
I arder that *A little yv mdiv solutiom U wasted m .c414e.'O the utiive dilutions woro prepaed, by the addition of 0.35 al. of hal.emituratd V01 to 3 ml. e ,120. The WWtmmm smeItivty ot this solution MW got by the addition ot 06 ml. e yellow, idiact 5lu*. tLon. Wil* the eoemtratim o the am a always, %he raoto or am to old wa ohmaged by this uor fro 5.1 to 5s0*9 This tnge may be due to the use or a wes oxidiaed solutiom or hals-aturstsd iClIh then originally umo.. This slightly lowered pHIx the older solmm ion would iwiloste this tet. Sims * pure yellow solution was used throughout the wrk,, ome the ftie was etablished, there i not sue ohnse, that it would be more esidise one t tn anther.
Thi b ofttle rat r smnaigation toek plsee over a priod of thee monthos. It movmt to 6%. If the seme tmistan rate wre detrmined each mouth there would be a* diffieulty rising twos th gra ohmos, As Wuch ma on might perohmg do* at effn e the "mdItwIvi to 0 grant #xteato but on m omefw tluids, Iees twom 10g the ratio mUs be asomrste to�g or th roultift rmati Will be weak enough to omma, dmbt

Daiiu this InvostsiSoti tho question of ts ffToet ot sensitieo
4 dilutint m spinl fluids ti. patients aufete Sm oewtal disesim ther than. *yVlie has bees emoeidee










obfthqpl4"e team* wo ft sedat " at, , tth ea noe - seek


amaple .lsdil, L, otate wu den 'un stqo aWPq~t ti mmudn .t Va to


be v mo ewsG fs . ., am











liar the woirntbim of wait tw rqai mba to ae!, I e eoui. edo ssur * eemtdo of ii.gf wpiites lasi �144.. ?m Stt 3v ot 804Alt Labiratw anti 4*r sa~~ slWh t ftat mW #aeb fblut med be Naa ost t art*"* 20 ft""d WOve not sdppd for tor 0011t. O nb at$n - o
Atiipatis* fti tot wk to be dAme at Josmil*e
~emstuomn " tat tw '4 oUfma Im Ut 1bmtNyo. A eawk pwIoPitwb. -I" taswd to be ft"" i f ha bos duo to uh oat, or ea 14t it the ftbber ft tho etsopp "im S"U. tics 1." bows let ftn V~th pos0140 ad be" bor voytatod ***I* ditalyat Saw tiase I& the AWr* IS p~optt a Intarod ..t ot am at the savutams ot UANlt-e15meA "34 a" tim slst


Whorsas the SOUM4ir 1*d WWigimlY 00*tindW 0004 W14 pus
litor, the omustm me was s ol ad to be IN"5 g. Pot' liter. * order to amaesuitioate oao *on Atlti of to Ossti ildv.1Ufat (14440 so per, iter), 0.s 0094 ot* sthin *&sVLutM 0"1 Nw*d up- to ri* soluiom saumAlod to uudavo t osaueusasa. ems s tbo wook. to be dame latero










Al.weub the State Boa*d of NHalth a week in vhbq to saw splw, a tLp mss mads to their labortovy to tost them On arriwi It was towd that soevoutoe pml n *I'ut )ad boom sw, ?bwe wae nmua'.qlitie omVoss ui had bon set in for �abun smz. q"antita" i Proti and lbalaux daetbrmatem.

A positivee dilution ot vamsdiam me pa'qvSei. AI - v.1
of *0 solution proparsd a Ieek PvViously to SostbsOe ve **l. of the trehy mAe dilutim. In order to obe* t, ar Iutfo tr' solzlvtyo thro ef t positlve ,nd throw Uh neptivo mtples were tested* All sm po~ oonfivmto7- re~tlns
The oevatoem ps.al maple wave tbm v and oesh gave a

neativo ve"Us ten. Fer at thes, fluid had OXN"e globuta somtest nporbd to the plWSlelOn WktMdng the o*#
At tha point laboratoy work m* stoped. It mse felt that the teot was ree*d for hosital work of a routine ma--r. Qly by smio work as the tw wasth of mW tet be tsbliMsd.











IS MWARATION OF A B TIM MUM ROLWXQU
VfOR 51BP1 =Z0 SYU R W OM

t4itm "1ods,, C?.,* 00ld to flirt tms4d with dry ahlorio "s end mild hoat tin e tow htw hr e .lw d pu t wa .ttig t lo od with watsr to yld a blmo saltj. t I ao UeAd au rtwt ast al, 50 ml. ot o io u.ofilet tor leaams . The oolutim is aml)sd tor VmO ta seontet sad dilute to gie a emvattti na or OsVAdI oorspeda to 1x3*.1 awa at C14 e ltbw U t hi* solutan is allomd to t o am we*.
Mtsi' 44gisg tb. soilUlios, it sowIu3'alydtr
wuuadiwaas VO14. P"pw- 5-6 ml. sr a dilutim ae uiug 14Jl+ % r sim of VC1 pv liter and alow- ths ti ao pnow a yeoW au


my be oomd.
Prop"r 10 *1* or 911e4h dilaties oataiM&Ue I.44 gis ot ~g
per ' liter. soue *""%I wfakly M"b psibisp s ui Ids am Xa" megatiwv Opisl fluio. DXvA t1h t wh dilution Uto 5 equal "ats* m to esA pat dd a eknow vui of twh Yo*lw as" Ot lutils whioh hoshe * eYa madie otomat. 1 T WIRO Od" "0sld J$" VOUN* m. gtai of ea i)* 21M ha ft dilat"~ or 0*b9, ,,,


Fa or. the$ sea WWre tosoted *Ih the spinal ftulds I the o llug amer. PUe 001 ,L a tfe spinal flu"d a a mis s11d, sd bealde it, but Mnt I%' eetaft" wih it pl#oet go am e ulaw










of the istur. The two selufit swe Ied with a M li glass "A and obseved, the low powe of the Smlresopve.
To eaoh slide *a atbioh is d" ted a loamlaet presipitat" I* added 02 al. of distilled Watr. Ak the end oSt a mx1aU *a& of Ith"O slid#$ is 6aai bow d*
Or the mixtures or fresh and old vmsan a 61,utlaw. one *ald give a ver seitive. taetion differntiatinS the UKhn posative spia ftluids b flmulatim tron the hah negative spinal fluids whisk do not flootlaf.

The pH of the emmtive nsztre 4hold be 2A.6 7, Tke ratto of dilutimas *sfth Is mst sensitive mq be used for at l..m a amth wi being re.-w rmied. The more oonmtmted solution (13-14 g. k per liter) shmud be stable for there to six aethe If kept tightly stppered and in a al plmes. Rubber is net v i nwadsd for stpertia vmadium solutims it has saew atlyfLt properties for their odation a e .








TABI,4U I


THE ASSM1


OF VC14 AND ON f3?INAL


I (pooled) + +
2 (pooled) 4


I,


~LL4a~1


��


+


4 4.
4
+


at, = l blao 6,wr w Positive

zMAX1t1pe tests# not a amo trn t o a*tI4 ,
*T# roa 4 sb~i.lI~pWo"fm"SUO


FLMDS


PUMIM A, (makAl








*.+ , , . I S I


54,
I! mil


II

U


# + #*t tI #+ t ++ + ~#, + + + *,*


I, S S Sf4


f+ 14* - 4G40


,2


t #


I
If


ft
a










a'C















I






I
*4


4i
# # *t~ .5++..+4


if


+ ++


4. +4.4.4.4.4.4.


+ 4.


f t 4 . 4


4.
















et WA Ki &a - i g�M190,Y~l MEW. p.0LMA/





94 4 - " 05 - -


Qnot

100 T+ + 101
1020
104 + 106 +



by04 + + :+ 104. - 4 4 110 + . + +1 + 11 +. 4 + 4 +IR + + 1$+ + + +

frm 3 *b Luks

1141~ + .4.4















l t I � 1 I S I t I I S t S I







* * *9*


+*+++++1+54 I


,4 4


i








TOL III (COMXUD)


148 T
149 T + � m5 T

I6S 7

184 7 151

4 4,
155 + 1850 + 159 +
160 + + S
-6 +
14 W awl*






83



?L S -01, RCO WI SMAL -LIID 8pH All) COIUPQOITZO0? OFP!ZITATR ZP FRE






164
168 - - 8.


170 5*7
171
17P - 540 171 T " 176 T 5,0

174 T - *17*1 T -5*7 178 T - I77 a , 178 T - 3,9 a
77 a 5,9

+ + &1bi1+ 181 , +
m +
+ + - ob1I

a0
188 - 0.,O 107 6*7 .
188 - 0 3,89 - - 0,0 a, 1 )0 - a 5 a 191 - a 5,9o a
193 509
194 6,0 19- a0*25 40

mFluid In von bad hpe on reevl. v









TABU IV (CONTIMMD)


610bu1is. + 81obulin + globz1ai.n + n~ot vwm


196 197

POO v08




P08

209 $4-O.0


229 V14 ~17 19


globulin + V globultn + V slouln + V clobulln. + slobulin +. V

Globuin +


1%o flomoul. of~ *ant1fuS *


eould be So


out for tetn by same


�


9.0
5,85 680 85


Met._ 10. T
T
T
T
T

T


83 6*1
6.0
U..


5lobtg$J + globulin~ + V


0*


-


$00 6,06






5,9 602
4
Z47
58

6,2

.0


5 5,9 5+,9


7FTiia'?TM - *-779m- - - -- ---
0 tion rp-4.









TAM IV (CoNTIMM)


men,



01# g1oba4�n +

34 509
* *




T+

1242 T +0.8 globulla +

r'4 T + .

4 46,+ +
247 +4 b* bu1Ii + V AS 4 8
GO9 8 S0 *It1an + 250 4+ 604 slouiln +
:1 64 g1bultn +
tALI 339 top'41 40##
+~~~A 4- 0~ N,*#~s
















2M 1 5 4 .......'L ....





284W
88 -

2 -







6 T

270 T "'75
274 4 + 44


S =,p1e of tth*s s wer r a# u o












8 OF VC14 IONS 1 w IHAL FLUIDS*


~vo1~
0 _____________ ______


77 4"8-

281 Tvze T 0- A
6T -"








4" 40
3T -0 4 0T +


44- +





40 4 +O - -


saless of this mu sn as unkwww,
















+# # 5+# i t I 4


iIiiJII


t *I S fI I #


t * I # I t IA- 4 4 o 4 4 ejL


I Ni


A


4,


0 blo*ol








I'RZ

MOTON M PINTMUD POM


4 #4 ZV 4


4
333 40 330 64

3m4* 4* 340*



3"%



umS3I44i me a* 04t
tetsmiivt o 04al4t* oao r4*o a u r OtM










TABLE VI U









A ntlgozdtc n.Cativas , treated oyhjt*.#$**t IL 27� AvOrthqresltswiI Pezkeematag.4i,84 SA




440r-00 - rosult witb Z.Ze~. - ~*









T'ABL\ I




Total secn nd "iiq posi1tive, syrhllltic arinl Miud sr$ pes........ L00 A al as.-3eermarm cM abn Tw neatiVe, r~ns~~hilt n piral fluld otmploso,.. ... 145

trea!~ o eym lI�le spinal fluid sesmples. ,.i C Tota number of samples...


Peents dioa~e e ......... Absolute = 4%


vcl4 resultsi withi 14- I lItVe 1.4'
VC14 resbAlte ith B.0........,..0, -boo -,0 V
aanta~g dlsagreeaent ....... , - Absolute 1.4


VGI re It ..%h .,,,, . ,....,..,B1 ,
eren e i eeen.,..... ....,About =-


:- tor mpresaent.











or A? fiIUL 'CD'O T"i pH OF
ME VC4w-SKNAt, YLIZ KMIWJR.




'oal V& dttiot......
AV rS de ato Of asinge mamwoent ,.,, Frobab oftvr of a O..V . u... llrbeate 'a, or the n, ....,.....









Additional pr peirtateq o foe Iezoipltasto toi to oOni tn Vmsaditm
.b


69





units O4

9


-~---~- -~-


l�o Qi-matrie qailtativ mot4 used.











A- 10$ JV"I"5 Vol*

Tho *oQorl.-otvle mtW-A -s7ng ase of nulftrie sol .ma tlwooe 7pollet hr drogen ,,oroxide vitooused The oolor
sta~drd ~r ~~efron. q -uo wnn of* R~mwnt

- Ina senaltiVe 9*1ution.., (1) 1,"1410 Vc. .A* ~(4) 1#4410 sVCl /1#


aonula. of the mntalysi or, alution ftor tho comPlro ')&L" tets"I a r~a~a Q. oe IzrecipittQ by the




_,'_,me . ., (1) O,00..o0!7 VCI./l.
(2) 0.67r.50 vd
y taflht 2.1uviton theo ,nlutionra chiuld hav e
Vwcdw~ ozteft Of C0,Q& gra Vol* per liter. "Ibe 44tru foronoe i dus o the soztlon by the f1oou1dts


Rosults of the vc stra onslysis of the n.w solution prepaad#e wl of half-saturated Qa,1


1 -etuatedVCI.......,..(I)1398 gyn VCI/#




1e0Oilte of the vanan 173s of senWItve Lulution rrep od from nm va old Ibt-erutedur to sto oluionn't o no nalutlon (X).......,,(1) 1.4410 grams V(14/1 lo=old solvuon ( ..,..,1 4410 jvw VCI*/I




Full Text
kl
bean added a drop of any strength EgCrgO? gave a flocculation when a
drop of 1*6 HHOj? was also added* This reaction did not take place If
the spinal fluid was itomal (non-syphilitie). There was no differen
tiation with any of the other metal salt solutions, and these particu
lar spinal fluids, with the exception of the half saturated vanadium
tetrachloride solution* While the flocculation in this ease had a
different appearance, the reaction appeared to be the saae* This
reaction of VCl^ did not look as promising as that of the KgCrgCty* The
non-syphilitic samples flocculated inside of five dimites.
Que to the age of the spinal fluids, these reactions were looked
on with distrust* Any number of changes could be imaginad to have
token place in the six months the fluids had been in storage* As this
testing was nearing completion, two fresh samples were received* These
were pooled, that Is, each was a mixture of a large number of saaples,
all giving the same asseraann reaction. One of these saaples was
Wassemann positive, the other negative* With both of these samples
the KgCrpO^-HKOj and ?Q1| reactions occurred. Whether the sample was
positive or negative, a flocculation resulted In every trial*
It appeared that the most likely change in a fluid would be loss
in strength ae it aged* For this reason the reagents must have been
made too strong* It was recalled that changing the concentration of
the KgCrgOp five-fold had had no effeet on the reaction, eo ths 1*6
nitric acid was diluted* At a dilution of about 1*100 HHO5 and making
use of 2*5 or 1% KgOr^, a olear differentiation was again obtained a


89
TABLE TO
TUB TO* HBAOTIOH WITH SPIRAL FLUIDS FROM
mrnrnm diseases
BCTT
number
Kahn
TO*
tw
524*

*
*
32S*
4

ses*
4

8
**
327*
*
40
3E8*
6*
m
...*
389*
OS
m
*#*6
330
4
m
m
331
'**
m

532
m
m
m
333
m
SO
334
m
OS
m
333
*
*>
4
336
m
4

337
m

m
338
m>
*
m
m
m
340
m
4
341
m
0

342
4*
40
4
343
m
m
so
344
m
m '

343
m
so
<*
346
m
P
#>
*3yphUitie m& aon^sypkUitie series used to
test sensitivity of TO* dilution*
*Hot reported at tine of testing*


5
Baranyi test (14) in following the course of syphilitic treatment.
A tost of very obscur mechanists was proposed by Sehuraan (15)*
Sonsa is diluted with 3-5 parts of physiologic salt solution and a
drop of 30£ hydrogen peroxide added* How a very dilute solution of
FeCl^ and phenol is added and the color read. Brown to black is a
positive reaction* while a green color is normal. This test is not
mentioned again ia the literature, and must have been guilty of too
many false reactions.
Independently* and using different nothods, Monne (16) and Noguchi
(17) did all they could to get the globulin contest of the spinal fluid
into the diagnostic picture. They point cut that the spinal fluid is
not encumbered by the large concentration of other materials found ia
the blood. Nona made use of the fact that globulin is not soluble in
a solution which is half-saturated with (NH4)2 SO^. Noguchi claimed that
the addition of a 10^ solution of butyric acid showed the presence of
excess globulin much more clearly. Belts (IS) otates that a positive
Bonne reaction (also called Roso-Jones reaction and (IS^JjSO^ reaction)
is a confirmation of syphilitic diagnosis, but that a negative reaction
le not conclusive* Also that tuberculous and suppurative affections of
the meninges give positive reactions. Jonas (1$) in a study of general
paralysis (paresis) stated that increased globulin almost invariably
occurs} that the iVaasermann antibody is associated with this globulin}
and that the excess globulin is the cause of the Bonne and Noguchi teste.
He regarded these last two tests as the best method of separating general


fluid ampios. If wa would furni ah shipping material and expense, they
wotild furnish us with from 20-25 freah samples of pinol fluid weekly,
a* long te they were able, Two boxes wore built for the shipping of
spinel fluid sample*. Baoh box was built to carry Sh oft gises test
tubos with eark stoppers. As long as ths tubes were stoppered, the
lid clamped them in such a way that breakage was impossible uniese the
whole box wee broken. All shipping wse by express, the trip requi ring
am 2h-?6 hours.
Peking use of sosas aged spinel fluids roaaining fro s finished
research of ihr* Pollard* s, work wee started* fhs results with KgOpO?
and Co(M0g)g were of no use in the differentiation of syphilitic and
non-syphilitic spinal fluids. Therefore a series of secondary reagents
was also prepared. Thee were made with two objects in mind. The first
was to ehanpo the solubility, tho second to change the pH, For the
former ethanol and acetone ware used, for the latter purpose concen
trated end dilute (1*6) HSO^, HgS0|tt hCl and SE^cH were tried. All
reagent* were put into ene oimoe gises bottles with medicine droppor
tops. The screw tops were plstic.
After making the secondary reagents, work was strain started using
the dichromate Solutions. There wars three aolutioaa cf KpCrpOy, and
ton secondary reagents* Os t ag each combination with both a syphilitic
and non-syphilitio spina) fluid, there remtltad 60 test* to bo es*iidned
after mixing said than again after drying. With throe exceptions this
work gawe no results worthy of mention.
It was found that a drop of syphilitio spinal fluid to which had


107
92. Herrold, J.A.H.A., 22, 957 (1922)
93. Herrold, ibid,, 8, 203 (1923)
94. C* Bmck, Munch, saed. Woehachr., 69,, 569 (1922)
95. Teichnann, J.A.K.A., JO, 286 (1923)
96. Becker, J.A.K.A., §g, 1525 (19*4)
97. Schwarz and Orunewald, Klin. Wochschr., 3. 1026 (1924)
98. Gustav Kleiner, Klin. Wochschr., 1, 811 (1932)
99. 0, Beba, Hunch. mad, Wochschr,, 21# 1793 (1924)
100. Jervell, Korsk Magazin Laegavidonskaben, 86, 757 (1925)| Abat,*
Che. Abete., go, 949 (1926)
101. Kahn, Abstracta of Bact., 17 (1921)
102. Kfthn>t |erg^i^^^|^l^e. & William* A
103. Epstein and Bawl, Beat, md, ochschr., 48, 89 (1922)
104. C, Brack, Klin, Wochschr., 1, 1656 (1922)
1Q5. Kahn, Tissue Xnaunlty. Chas, C, Thoms Co,, Baltisora (1936)
106. Kadlach, Med, Klin., 1649 (1926)
107. Bosenfeld, Blochs. 35., 16£, 343 (1926)
108. Walker and Sleeper, J. Lab, Clin. Hod., 1£, 1048 (1926-27)
109. Taceone, Podiatrist Beviata, 131-41 (1926)
110. Taecone, ibid., 3£, 1097 (1927)
111. Prune11, Prensa Med. Argentina, 232 (1919)
112. Prunell, Compt. rend. aoc. blol,, 2l, 1315 (1928)
113. Takata and Ara, A, Trap. Med. Tr. sixth congress, I, 667 (1925)
114. Statib and dealer, Klin, Woehschr., 14, 1638 (1935)


4
Format, la 1907 proposed o tost b&sod on his laboratory*s ex
perience that a syphilitic scrum would cause precipitation If added to
a normal scrum* Plant, Houck and Fosal (9) investigated this, and found
that any two sera, regardless of affection by syphilis, might give rise
to a precipitate. The mechanism of this reaction is not olsar, but it
may have something to do with blood types, sins# it may taka place be*
tween two normal aera*
The Aborderholden reaction was applied to the diagnosis of syphilis*
Varney and Morse (10) claimed it was specific* They noted that the
reaction did not approximate the Wassexmana reaction in usefulness. This
is a reaction depending on ensyme, or foment action* The serum of the
affected person, mixed with a substrate of protein from Spirochete
pallida, Should give rise to free amino acids by hydrolytic action*
Bronfenbromer (11) found that a pure lipoid could be used for substrate
just as well as a protein* the protein being acted on was In the sern
itself* It was also found that almost any scrum would give rise to coas
free amino acids*
Klauaner (12, 13) gave his name to the reaction which takes place on
dilution of sera with distilled water* The syphilitic sera yield a pro*
olpltate, the non-syphllitio fail to do so* It was admitted, however,
that the reaction was also positive with typhoid fever, lupus, and
pneumonia* Daranyi used the same reaction, but made the substitution of
alcohol for distilled water* Both of these tests are based on the protein
content of the serum* Krameke, In 1923 was still making use of the


105
46. Kgo Heeht, 2. Iswmitate., 4, 258-66 (19X5)
4?. Gradwohl <&, 240 (1914)
48. W J, Bruce, . Lab. Clin, liad., i, 2X5 (1918-19)
49. Sijaaanuei, BerX. fclin. Tochachr. 0, no. 30 (19X5)
50. J. A. Cutting, J.A.U.A., 1810 (1917)
51. Hoeonfeld, Ked, Kliaik, 26, 553-5 (1930)
52. . Brandt, J. Lab. Clin, Ued., 5, 1077-84 (1940)
53. Pfeiffer, Kofeer and Field, Pros. Soc. Ixp. Biol. Sed. 1, 153-7 (1915)
*
54. Hoffwm and Schwarts, Arch, Int, fcfed., , 293-302 (1916)
55* 0* Genoese, Pollelin, Rom, 26, 97-100 (1919)
56. Beverl, Folielinlco, Jg|, 450 (1921)
57. Guillain and Ubert, Ann. de Med., 771 (1921)
58. L, Lowery, Boston Ited, Surg. J 121, U5-7 (1917)
59. Sache and Georgl, fed* Xlinik, 805 (1918)
60. E. Meinioko, Z. ImamlUt*., 350-63 (1918)
61. B* Uolnlcko, ibid., page 51>7
62* Fr. Reichert, Cent, Bakt. Bamelteak., I. Abt. 90. 328 (1923)
63. E. Uainicke, Deut. md. Wochachr., 48, 384-5 (1922)
64. B. feinlcke, Klin. Wochachr., 8, 112-3 (1929)
65* ?. Klliagar, Berl. kilo, Vochschr., 8, 1001-3 (1918)
66. P. Ellinger, 2. Phyeiol. Cheau, 16. 245-65 (1921)
67. Alekeeovokli, ?* Rueo, Fhye, Cheta. Soe## 53 1. 331 (1922)
68. Rubellng, Klii, Wecfeechr,, 12, 501-4 (1938)
69. Hermann, Kolleid 2., 8$, 297-301 (1939)


INTRODUCTION
When It was proposed that the author undertake the development
of an original test for syphilis in the spinal fluid the difficulties
of supply were not fully realized. This obstacle has been overese to
the best of our ability.
An investigation of this type ia almost invariably carried out in
a large hospital, or very near one. The clinical and laboratory evi
dence of each ease is examined minutely. In this manner the true
value of a reaction is determined. The new reaction my be best under
certain conditions which lead to failures with all previously known
reactions. At any rate the value of a teat should be determined by
both the laboratory and medicll staffs.
In direct contrast to the norm! state of affairs, wo were forced
to use a source of spinal fluid samples ccmo two hundred miles distant.
The samples were shipped by express and their time in transit was a
minimum of twenty-four hours. The data furnished with each sample
included only the result of the Kahn or lassenaann test and the clini
cal presence or absence of syphilis.
The fact that Florida does not have a medical college necessitated
visiting other libraries for a study of the literature.
Prior to 1906 (1), all but one of the leading syphilologists in
this country believed the appearance of secondary lesions to be the
only means of diagnosing the disease of syphilis. In I906, Waasers&nn,


70
the VC If, end VOClp, The VOGlg ie a very rare oompound and *o
unstable that only traces are likely to be present* as far as could
be predicted at the tine, however, any new solutions should be nade
from such a mixture, and not from pure VClj,, in addition to greater
ease of preparation, this also allowed for the difference In acidity
which would result from solution of a mixture of VOClp and VC If, and
solution of pure VClf,. It Is known that the very acid solutions of
Wlfj made at this laboratory several years ago do not undergo any
oxidation whoa diluted and left exposed to the air* this Is not a de
sirable effect for the present work. As far as is recalled by the
director of this earlier work, liquid YClft alone was used for the prep
aration of these samples* The early work was not ever completed, &/
written up, so no absolute knowledge of the solution referred to could
be got.
A simple apparatus was used for the conversion of the old material
on hand back to a mixture actually having some VC If, content. In order,
the apparatus consisted of a chlorine generator, CaGlg drying tube,
tube containing the a ample, a safety tube and an exit tube passing over
a ysf* solution of HaOK. For the generation of Clg, concentrated SCI
and hypochlorite were used* The sample tube was so arranged that it
oould be heated*
About 2 grams of the mixture was placed in the apparatus and
chlorine passed over it for a few minutes. With a steady stream of
Clp going through the apparatus, heat was applied to the sample tube*


108
115. Sacho and Witebsky, Klin. Wochechr., £, 1233 (1928)
116. Hinton, J. Lab. CUn. Bad., 4, 621 (1928-29)
117. Ifeyser, Klin* Wochsehr., £, 792 (1928)
118. Treuter, Boot, sod* Wochochr., 54, 994 (1928)
119. H* Bailor, Klin. Woeheehr., % 1405 (1930)
120. Buller and Kandula, £, Irsaunltate., §g, 183*92 (1934)
121. Flinn, J, Ub. CUs, Bed., 682-6 (1930)
122* Sauer, I* lenonitato, £g, 51*2 (1930)
123. L* Fnpi. Urol. Catan. Rev,, XI* 188-94 (1931)
124. J&rehioni and Alinatela, Klin, Wochochr,, 3, 1345 (1932)
125. Marchioni and Allenstein, Ibid,, pages 1424-6
126 B, S. KUne, %icrqacpj>ifi Sllge Lrecls&fifelSg Teeta £or ho Diftgnc-
ia m gxclugjon S Williana and Wilkins Co
Baltimore, lid., (1932)
127. Kiss, 2. Iraaunitots., XL* 195-241 (1932)
128. Sehttbe and Harrsa, J. I*b. Clin. Bed,, 8, 65 (1932-33)
129. Looney, J, Biol. Che., Ji, 63 (1927)
130. Schube, J. Lab. CUn. Med., 2£, 314 (1934)
131. Deadcen, KlUot and Sadth, Ass. J. Clin. Path., 2, 246-50 (1937)
132. Osante, Buraok and Kreisler, T, Lab. Clin. Bed., 26, 1349 (1940-41)
133* agle, J. Lab. CUn. Bed., 1,8, 684 (1932-33)
134. Siiapaon, Lab. J* Australasia, 2, 18 (1940)
135. Oruskin, Ara. J. CUn, Rath., 2, 441-7 (1931)
136. flruskin, ibid., 5, 162-4 (1935)
137* Kopaeseeeki, Coapt. rend, soc, biol., 11,6. 932 (1934)


BIOGRAPHY
Robert Somera Leopold es born In Dayton, Ohio,
the 21st* of June, 1915* He attended granger and
high schools in several sections of the country,
finally graduating from high school in take Charles,
Louisiana, In 1932.
Ur, Leopold graduated frota Mississippi Stats
College with a Batchelor of Science degree in Zoology in
193?. The year following graduation ho assisted in
chemistry at his eisaa enter.
In 193, Hr* Leopold entered the graduate school
of th University of Berth Carolina, and graduated from
this institution with the degree of Master of Arts in
Chemistry in 1940,
Since the fall of 1940, Mr* Leopold has been
enrolled in the gradante school of the University of
Florida* During the second semester of the 1940-41
school year he held a Graduate Council Scholarship and
continued on this scholarship the following year.
Hr, Leopold is a member of the teoricen Chemical
Society and the Bets Beta Beta and Kappa Hu Epsilon
national fraternities.
i
\


table n
KSSQVSS OF VCl* KSACJXOHS OT SFXSAL ifcSISS,
i'IW0 HEPROOTCXBILITt OF SOLtmCHTS
SpXal fluM
W
JL
£70
m
£77
m
<*
£79
m
@00
m
£01
m
£0£
£03
4
£04
V
£83
%'
£06
f
£87
T
£08
T
£89
f
m
T
£91
T
£0£
4
.£83
4
004
. 4
£93 *
4
893. ,
£97
. V
4
£98
*
£99
4
iMzmmt is
300
m
301
m>
302
m>
303
m
304
m
303
m
4
4
4
4
4
4
4
4
, :y
*Samples of this group m sub unkrouns*
44444444


1+8
good part of the tine*
This reaction had one serious fault* In too many trials the mix
ing was not just right, and Instead of floeoulatlon the result was a
hard granular precipitate* hilo the positive sample always gave
flooeulatloB or granules, the negative sample at tinea gave granules
also* It was seen that repeated tests would be necessary for a clear
differentiation when using the KgCrgOyHKO^ method*
At soon as this reaction was discovered, sn intensive search of
the literature under potassium dlchromato was conducted* Two references
(109* HO) to the work of Taoeone were found* Although the technique
was different, the principle was the same* In placa of the trichloro
acetic sold, we were staking use of nltrlo aoid, but the notion of theae
two acids la vary touch the aaste* Both of theee acida have been used in
the estimation of albumin* Brunei1 mentions triohloro-acotio (111) and
nitric was used by Dourls (171)* neither method is to be recommended
for the estimation of albumin, but the albumin excess is the cause of
the reaction* In the light of these findings, it was decided that
work on the ?C1|, should be the min object, end that the dichromate
method be used to determine whether Its results were as reliable as
those reported by Taoeone*
Several dilutions were made from the half-saturated solution of
vanadium tetrachloride* hone of theee wae able to give a satisfactory
differentiation of syphilitic from non-syphilltio spinal fluid. The
solutions were allowed to stand on the desk overnight, and tried again
the next day* One of these, ho* 11, gave a olear differentiation of


Neisser and Bruck (2), made one of the two most important advances
in the ultimate defeat of syphilis. The other was the later discovery
of salvarsan by Ehrlich.
Although the theory that was proposed by Wassermann et al. was
not in accord with the modern work, the test itself has undergone only
minor changes since its introduction. Despite the time required to
make a Wassermann test, it is still one of the leading reactions
for the diagnosis of syphilis.
vi


table iv (Gomimrn)
Ipinci Huid
seraalt numbs*1
. ..reaction
reaction
pi
Precipitate
196
Mpaa?- M L%>tttlnu041
** #
m
197
T
e*
* *
m
im
f
*
5*85
*+
im
Â¥
m
6*0
m
POO
f
£
3*3
£01
f
m
5*9
nm
4
4
6*3
globulin 4
£03
4
4
6.1
globulin 4
5504
4
4
6*0
^.obulin 4
£05
4 4
Shimant 11
* *
not run
£06
*
m
6.0
ee
£07
m
6*1
-
£08
**
s*
5*6
m
£09
**
5.95
m
£10
#
m
5*85
*P
£11
M
m

£12
4*
m
*#
*
£13
*>
*
5*8
*
£14
T
4
5.7
globulin 4
£15
T
m
5*9
m
£16
Â¥
m
6*8

517
Â¥
m
5*9
m
£18
T
as
5*7

£19
T
m
5*8

£20
T
4
6*8
globulin 4 V
£21
JP
4
6*0
globulin 4 V
£28
4
4
5*3
globulin 4 V
: £3
4
4
6*3
globulin 4 V
£24
4
4
5*9
globulin 4 V
£25
4
4
6.8
globulin 4 V
&£
4
4
,6.6
globulin 4 V
a 7
4
4
5*9
globulin 4 V
£88
4
4
*
£29
4
4-
5*7
' globulin 4
llo floeeulat could be get out for touting by aeons
of centrifuging*


56
solutions previously prepared plus a fon moro which it wo* thought
might ho effective. CobIt mis nitrato 110%) foiled to give a reaction
with either class l or HI fluid. Saturated arsenic trioxide also was
without effect.
Cupric chloride gave a reaction with both class I and III down to
C.1? dilution, and then failed to give a reaction with either syphili
tio or non-syphilitio fluids. Cuprous chloride (saturated; gives a
reaction with either class I or III fluid, and when made half-saturated
reacts with neither class I nor III. Between these concentrations it
is possible that CupClp will differentiate syphilitic from non
syphilitic fluid, but the instability is eueh thst further work was not
indiosted at the time,
Bismuth trichloride in dilute BCl might be used to good advantage,
but ary reaction of the metal is masked by the FloCi precipitated, fhe
amount of HCJ needed te clear this precipitate is sufficient to floccu
late the protein of a non-syphi 11 tic fluid. Ferric ammonium oxalate is
not a stable compound in solution, and it presents tho sane difficulty
as #ici^. When added to the spinal fluid the reaotion is masked by
the Fe(oH)^ precipitated. Vemos (?U) used colloidal Fe(OH)a¡ in his
first test and this proved unsuccessful.
Molybdic acid gives a precipitate with negative spinal fluids,
even at trace concentrations. It is dear that a great majority of
these metal ions are able to precipitate albumin, as well as globulin.
Silver nitrate is capable of giving s precipitate with either class I


72
dilution prepared from the old solution differed only 0.03 pH units
from that of idle new sensitive dilution. Considering errors nade in
dilution, the sensitivity of the Beokaann pH meter and ell faetors
involved, the two solutions were judged to be equivalent.
In order that ae little vanadium solution be wasted as possible,
the sensitive dilutions were prepared by the addition of 0*35 ml* of
half-saturated VCl[t to 3 ml* of HpO. The maximum sensitivity of this
solution was got by the addition of 0*62 ml. of yellow, oxidised solu
tion. While the oonoentratlon is the same ss always, the ratio of
new to old was changed by this work from 5*1 to 5>0*9U This changa
may be due to the use of a more oxidised solution of half-saturated
VClj, than originally used. The slightly lowered pH in the older solu
tion would indicate this feet. Bines a pura yellow solution was used
throughout the work, once the ratio was established, there is not much
oh anco that it would be more oxidised one time than another*
This change of ratio for sensitisation took place over a period
of three months. It amounts to 6$* If the sensitisation ratio wars
determined each month there would be no difficulty arising from the
gradual chango. Ae much as an eight percent change does not offset
the sensitivity to a groat extent, but on some few fluids, lass than
105$, the ratio arnst be accurate to25$ or the resulting reaction will
be weak enough to cause doubt*
During this investigation the question of the offset of sensitive
VClj^ dilution on spinal fluids from patients suffering from certain
diseases other than syphilis has been considered*


31
applied the Eagle teat to 171 epinal fluids, the resulta being about
parallel to the laeseraatm reaction. This test runa about 20?* store
sensitive than either the Kahn or tfaesenaann, and gives fever doubtful
resulte when used with **ra'
Grushin began a series of papera in 1931 entitled Studies in
Cerebrospinal Fluid (135*136). In the first he nado use of the lose of
color of starch-iodine mixture, when it was added to spinal fluidj in
the second he used glycogen-iodine mixture. It vaa claimed that the
decolorieatlo was so graded that a differential diagnosis became possible.
On the basis of the results obtained by Kolaer, studying the landau test,
this work seems to have boon ill fated before it as started (41). It
nay be that the adsorption makes this a more successful reaction, but no
further reporte have been published on it.
Kopaezevakl (137) claims that although lactic acid will gel serums
which contain increased globulin, this is not true with syphilis. The gel
reaction was soldo found with syphilis. This was not published as a
diagnostic procedure, but merely to show that there appeared to be some
difference in syphilitic globulin and other globulins.
The Bauer (136) reaction, an KgClg flocculation of serum or spinal
fluid, is claimed to be as delicate a test for Increased globulin as the
Takate-Am reaction. These tests were both proposed for syphilis, but
proved to give positive reactions with any affection increasing the glo
bulin content of the epiml fluid.
The Laughlon test (139) was published in 1935* Moore (140) found


7
to the naked oye* Working at 24+C*, and allowing the reaction 2k
hour* for completion, they found that centrifuging gave a precipitate
of maeroeoopio also* This reaction occurred with from 72 to 99?* of the
positive ifuaenum asm, and from 5/£ to 13% of the negative sera* The
preolpitate was found to differ from that of Kleusner (globulin) and
that of Forges and Mador (lecithin). While the globulin is not expected
in this reaotlon, they were most certainly getting a lipoid, adulterated
with protein. The test Is not used today, because of the long reaction
period and the non-confonaity of results.
About this samo time a reaction called the Bsllostl, or Butenko,
reaction appeared. The reaction used as reagent a 10% solution of HgHOj
with a slight excess of free Hg end a little HKOq. It was proposed
for any body fluid and Boveri (25) found that it gave negative remite
with some 15 sera from afflicted patients. Stern (26) aleo checked
this reaction and found it hod no specificity with the urine.
abb&tini, in 1911. reported on the method of Rivalta (27)* The
method le to put equal volumes of spinal fluid and very dilute HagCO^
solution into a tube and then pour down the tide several drops of 2%
acotio aoid. In 1 to 5 minutes the appearance of a white ring shows a
positive reaction. There are positive results in paralysis, tabes, eto.,
and the results are claimed parallel to those with the Sonne, Noguchi,
and Wassomann reactions. This reaotlon may be as good as that of Hoaao
and Noguohi, but it would probably not parallel the Hfassenoann. It is


8
ro reported again in tho literature.
Hoguohi, a tireless Investigator* next looked into the possibility
of an extract made frost a pure culture of 2. pallida (28). He used this
in a skin test, both with rabbits and sen. A well narked inflammation
developed when the extract vas used after the Injection of the ansale
with either a live or killed culture. i?o reaction was obtained in
rabbits suffering active orchitis of syphilitic origin* or in rabbits in
which this condition had been cured four months previously. In man it
was found that ike reaction was positive in tertiary and congenital* but
not in lc or 2C syphilis. If there as any reaction at all in the pri
mary and secondary caaes* it was very weak. ifenu-Suscel* Alexandreecu
and Friedmann (29) found that this reaction was a true confirmation, if
positive* but when negative had no special significance.
Pcpcff (30) made the observation that the senas of man is hemolytic
toward the red corpuscles of the guinea pig, and that inside certain
limits tliis property is constant. He studied a series of diseases, and
found that the hemolytic value of serum was altered only in syphilis. In
this case the syphilis, by producing freo lipoids in the blood, apparently
tied up a great deal of the complement, and this hindered the homolytic
action. Ho found also that a great deal of the hemolytic value of a serum
vas dependent on its complement content. fell-Kafka is the name given to
a diagnostic test depending on this loss of hemolytic power, ^eil and
Kafka (31} found that in the apinal fluid there was almost a total loss
of complement during syphilis, and a normal level was maintained in


18
any conclusions as to the worth of the teat. It is clear that any
colloid my be uad In the oxaniontion of cpiml fluid and tiiat the
resulta ill depend on the care with which the technique la followed.
Vemos (74*75*76*77) began work in 1917 on the diagnosis of syphilis.
At first he raid use of a colloidal iron solution containing Fe(CH)y
FetCjHjOg)^. The results were net good enough, so he made use of an
antigen front horse heart, which he called perethynol. Since this gave
a reaction which he could not see, he added a pig sarun-shesp coll
hemolytic ayate, for indirect reading. This test wee similar to the
Wassermnn, hurt not as good, so that in 1925 he published his last modi
fication, a precipitation test making use of the perethynol, inactive
aerura and an instmsent for reading the turbidity. This instrument ie a
photometer with a special seal designed to show pathogenicity by the
numerical reading. Cornwall (70,79) discusses these tests in detail
and praises the last modification highly for use in the following of
treatment*
hold (BO) started publishing a series of tests in 1921, but none of
them was very successful The antigen he used was similar to that of
Sachs and Osorgl and hsinicke, and diluted It 10 with salt solution prior
to use. The serum was then added to this opalescent solution and incu
bation carried out for two hours at 37C. After another two hour at
room temperature the result was road. If the solution was still in its
original state, the result was negative! if turbidity developed, then
the reaction was positive. Qtreaspel (81) found this reaction lees


KXPEHIMKNm.
4 aingle-ooapound, spinal fluid reaction of syphilis was the ob
jective elected* In order to be successful this reaction should show
a clear differentiation between the spinal fluids of syphilitic pa
tients and those of non-syphilitic*. In order to save tie the labora
tory and literature work were begun simultaneously*
the best starting point was suggested to be an extension of
Pfeiffer* work (170)* Reports had been heard of his diagnosis by
crystal patterns* Fortunately no copy of Pfeiffer* s book was avallabia
for sons three months* The present work eould net have developed from
attempts to use the rather ayatie methods he recomends.
Having no idea what might result from attempting to crystallise
metal salts in the presence of spinal fluid, several metal salt solu
tions wore made up* These solutions were made from those alts which
happened to be in the laboratory, no conscious selection was exercised.
The solutions prepared worst KgCrgOy (5, 0*5 & 1j£), Co(HO^)g (10, 5 A
1%), FdHHl^CpOfi) (10, 5, & 1%), CuCLg (10, 5 lj£). VC% (saturated &
half-saturated), AspOg (saturated in 05K SCI), and Bicljj (saturated,
clear solution in 0*5S HCl)
Those solutions were made up wit!) rough weights of materials* It
was recognised that any solution found to be useful would have to be
analysed for metal content and purity*
hile the organisation of materials in the laboratory was progress
ing, an agreement was made with the Florida State Hospital for spiral


that it is macroscopic in dimensions, and is obtained from 0.1 ml. of
the spinal fluid* Clearly it must be oapossd of either the proteins
or vanadio asid, or sorae combination of those materials* The proteins
have boon dismissed, and their significance in syphilis pointed out*
In order to determine whether the preoipitate was organ!o or inor
ganic, a series of maerosoopio teats were run*
Kith samples having a volume of 2*3 ml* or more, It was possible
to determine the nature of the preoipitate and find the pH of the solu
tion from which it oame* A Beckmann pH meter was employed for those
determinations* for its operation were needed 3*5 ml* of solution in
addition to 2 ml* for preliminary rinsing of the electrodes and cell*
Equal volumes of spinal fluid and sensitive dilution were mixed in a
centrifugo tube, and If flooculetion resulted, it was centrifuged out
at 3,000 r.p.m. The pH was then determined on the supernatant liquid
and the preoipitate examined separately* Before using samples in this
manner, microscopic tests were run to determine their reaction,
A total of 70 pH determinations were made, and 2 of these fluids
gave positive reactions both micro and raaoroseoploally. No signifi
cant difference in the pH of a positive reaction end a negative
reaction exists* Either reaction gives an average pH of 3*9*
After eentrifuging out the precipitate, its solubility was deter
mined, In no case was the solubility tost positive in distilled water*
and In every ease there was solubility in dilute sold, base or salt*


11
order given.
Kolmer (41) gives us n record of the reactions of Landau. The
first method was to overlay scrura or fluid tilth paraffin oil, contain
ing iodine, the second ana to put the iodine ir. CCl^ solution and run
this under the liquid in question. It was claimed that in either case
a syphilitic affection gave the liquid the power to decolorise the
iodine. It was found by Koltaor that this was a very poor method,
giving far too many false positive reactions. Bronfenbrenner aw! Rock-
man (42) found this gave 30,1 false positives and only fi$)t positive
reactions with syphilitic controls,
Gordon (43) proposed a test using %C12 in a dilute solution (1*100).
Tho sera or fluids vare placed In tubes and a few drops of the reagent
poured into them. With a positive liquid a cloudiness appeared, with
the normal a clear solution resulted. Woichbrodt later modified this
test and used it for the detection of globulin increase. Krebs (44)
found that the aaaiaum globulin precipitation in this tsat occurred at
an albumin percentage of C.25. This is not a common test in this country,
but should bo as good as tho Nonas or Noguchi method.
v, Dungern (45) proposed a tact involving the use of a solution of
indigo, sulfuric acid and Foiling solution B in the proper proportions.
When added to serum a completo gel resulted if syphilis were present.
The observation me aleo made that tuberculosis of t5j bone might cause
a false positive, as this condition leads to Increased protein, just as
syphilis does.


57
or III fluid*. A* the strength of the solution is out down, the pro*
cipitation stops and a milky agglutination takes Its place. Apparently
protection of the silver is nsedsdy Collargol, containing protected
silver, may be of scam use (69).
Tho next fluids received were tram St. Mikes Hospital, Jackson
ville, /hilo these were all sots whet cloudy, they checked with the
vanadium chloride. lino of these was marked only syphilitic, gold sol,
positive. This also gave a positiva reaction with VCl[t. The weekly
box of fluids from tho State Hospital gave good clear cheeks with class
X and III fluids. As usual, class XI was divided, the eight samples
giving h+, 3 and 1 results. Fro the thirty-five samples re
ceived in this week, only fourteen were checked with dl chromate and
nitric acid. Those were in line with the ttaeseraans reports, with the
exception of those in class IX, all of which were positive.
The fifth box of spinal fluid samples from the State Hospital con
tained ample* which were, for the most pert, quite cloudy. In spite
of this, tho VCljj, solution, prepared end sensitised in the usual manner
was effective. Class XXX gave eight negative reactions, class XX, four
positive and four negative, class I, six positive and two which were
recorded as doubtful. These doubtful fluids contained bacterial con
tamination, beyond a doubt. The first of them gave two weak positive
reactions and one very weak positive reaction* The second questionable
sample gave one negative and two positive results, the positive reactions
being weak. Since studying other tests exhaustively, It is apparent


9 NS *03 fO
*3 *3 *#**§ *§
+ + + + i
86$

II
8£8*P8S8SggS kpj
I t t t t t 1 I t I t
03
O


ACKSffLKIXMZKTB
The author wishes to exproa* hie gratitud* to
Dr. C. 0. Pollard, without whoso guidance thi* invest!-
got ion would have boon impossible, It was at the sug
gestion of Dr* Pollard that this work was begun and
carried on to completion*
Th author would also like to give credit to
Dr8. Therrel1 and Heudlger of the Florida State Hospital,
Dr. Lcmglno of the Georgia State Hospital for the Insane,
Dr* J. 0. Patterson and the Kahn Staff of the Florida
State Board of Health, Drs. L, Y. Dyronforth and H, Mar
shall Taylor of Jacksonville, and Dr* K. D* Thompson of
the State Tuberculosis Sanatorium, all for their
splendid cooperation,
The author's grateful thanks also go to the staff
of the Department of Chemistry of the University, Their
suggestions on equipment and methods have proven
invaluable.
11


75
THE PKEPAiUTION OP A SSWSITIVK VAHAJDIUM SOLUTION
POR SYPHILIS DETECTION IN SPINAL FLUIDS
Vanadium tetrachloride, C*P*, solid is first troatod with dry
chlorine gas and mild heat for one to two hours* The solid material
resulting is leashed with water to yisld a blue solution* If 2 grams
are used as starting material* 50 ml* of water is sufficient for
leaching* The solution is analysed for vanadium content and diluted
to give a concentration of vanadium corresponding to 13-H* grass of
VCl^ per liter* This solution is allowed to age for one week*
After aging the solution* it is oolorimetrleally analysed for
vanadium as VCljj. Prepare 5-6 ml* of a dilution containing 1*14*10
+ 3/S grams of VCl|, per liter and allow this to age until a yellow solu
tion results* To hasten this reaotion of oxidation one drop of HpOg
may be used*
Prepare 10 ml* of fresh dilution containing 1*14*10 grams of VCl^
per liter* Secure several weakly Kahn positive spinal fluids and Kahn
negative spinal fluids. Divide the fresh dilution into 5 equal parts*
and to eaoh part add a known volume of the yellow* aged solution
which has the seme vanadium content. The amounts added should give
volume ratios of fresh dilution to old dilution of 5/3*9* 5/3*99
9/U 5A*5 ad 5/M*
Each of these mixtures is tested with the spinal fluids in the
following manner* Place 0*1 ml* of the spinal fluid on a microscope
slide and beside it* but not in contact with it* place an equal volume


point* it toko six times the numbs)r of hydrogen ion* to satee tills
change with an alborada no with a global In. For this reason th albumin
is meh harder to precipitate by neutralisation. fhero la no known
roaaoa why these effects nr* not additive in the protection reactions*
fhsy nr# not found to bo of such a nature* boylaad proposed that it
sight Ho in a compound formation V tho aglobo!in and salt* this eom-
pouad hawing mm specific reaction with tho poly-van&die add*
Precipitation Is cannot! by the neutralisation of tho charges of
tho protein with an excess of vanadio add. It then Is not an orneas
of the acid, bat an roots of tho protoin, then tho poly-acids should
precipitate vhm they are neutralised, the fact that this does not
happen it probably doe to the outlier partidlo siso, as because the
vanadio add particles only weigh about one fiftieth as such*
Seri (l68) attempted to apply the work of 3eyland directly to the
spinal fluid. He nado use of a solution of sodios vanadate end acetic
acid* His test does act parallel the Waseoraana reaction very well,
only fifty fluids were rm la caparison, twenty-one being basseraann
positive. Of the twenty-one positive fluids, the teet ef eri may be
judged positive with IT Of the twenty-nine %#seraann negative fluids
there it a positive reaction with 10,
fhc method of Start consista of adding spinel fluid Aropwise to
exactly 5 al* of the vanadio add solution* If lose than 10 drape pro
duce flocculation, the test is probably positive* $h pH of the vanadio
acid solution used mat be 3,94, Although the results with paresis arc


30
of the reaction* First enough distilled voter is added to render
inert any protective colloid action by the proteins of the fluid.
In the second place oxalic acid, which hears a positive charge, is
added, and this charge is neutralised hy the free negative charges of
the proteins in a normal fluid. Then the third step is the addition
of the colloidal carbon, which remains in suspension. If there Is
neuresyphilia, the oxalic acid is not neutralised, the free chargee
of the proteins being less maw roue, at! this unneutrallied oxalic acid
causes flocculation of the carbon which has a negative charge, Boadrnn,
Elliot and Smith report (131) on this reaction* "The results using
the method of Schubo and Harms agree with the Wassensann and other
colloid tests," Although the reasoning on the mechanism is rather hard
to understand, the teat undoubtedly works, asunto, Burack and Kreisler
(132) claim it is specific for syphilis,
Ingle, in 1932, proposed a new test (133). The antigen is made of
beef heart extracted with ethanol and then treated with § mixturo of
equal parts of corn gera sterol and cholesterol. This is emulsified
In 4? f'feiCl and ripened 46 hours before using. Inactive serum is used
with an equal volume Cf emulsion, the tubes shaken and incubated four
heure at 37C* After the incubation the tubes are centrifuged and 1,2$
itoCl solution added. Strong agglutination is a positive test, homo
geneity is a negative teat, all others are doubtful. All doubtful tests
aro centrifuged again) if a white flake results in the bottom of the
tube, the reaction is positive) if not, negative, Simpson (134)


50
The foot that the vanadium tetrachloride dilution had to bo nade
a day before use was a disturbing natter* This bahart or was studied
and found due to an oxidation of a part of the vanadium. That there
actually was not an oxidation of all this vanadium was made olear by
attempts to uae the solution two and three days after its preparation*
So results obtained on either the second or third day had any crrela-
r
tion with either the %ssenaann report* or the same dilution on the
first day following preparation* On the second and third days the
reaction with a olass X1Z fluid was as likely to be positive as that
with a olass X fluid*
As stated before, freshly made VC1J dilution is blue* Whether it
is allowed to stand in tit# open, or in a tightly elosed vessel, the
color at the end of twelve hours is a blue-green. After $6*1$ hours
the color is green-yellow, and after a weeks time the solution is
almost a pure yellow* When the yellow color is reached, no other change
occurs. After a month and a half one of these solutions was found to
still be the setae yellow*
The colors of the vanadium ions in the various vlenos states
are well known. These were experimentally produoed to determine
exactly what changes wore taking placo. The lowest state of valency
vanadium assumes Is 2+ and the highest is 6+ A definite and dis
tinctly colored solution exists corresponding to each of the valonee
states between two and six* Thus it was found that the colors and
corresponding valneles are* 2+ lavender, 3+ r blue-green, 1++ * blue.


65
This Is eharaeterlstlQ of globulin* throe of the precipitates were
restored to crucibles and fused, The characteristic odor of burning
protein was very strong, and a very slight residue of yellow powder
remained. The residue was soluble in acid, and proved to be VgOg*
the precipitate was thus found to consist of globulin and vanadium
ponteadde.
To determine whether the VpO*; or hydrated vanadio acid was tied
into the precipitate in a compound, or just carried down by the
floooulate, four of the precipitates were washed with distilled water
repeatedly. After the third washing no test for vanadium was obtained
in the wash water, and the precipitate had changed from a grey color
to a pure white. Fusion of one of these samples of globulin gave no
residue* To check the amount of vanadium being adsorbed by the globu
lin, vanadium determinations were made on the supernatant liquid ir>
two oases* The liquid used was that from the pH meter cell, after pH
had been read* The liquid should have contained 0.7205 g/l*# end
was found to contain 0*6750 g/l* of VClj^. This amounts to a loss
of about 6/ Since the analysis is only accurate to et best, it
may bo stated that the amount of vanadium adsorbed is not the amount
in the pentavalent state. This is at least 16.675?, the amount added
for sensitisation.
While working microscopically with some of the negative fluids in
lis series, a peculiar occurrence was noted* In a few eases a floccu
lation took place, but on centrifuging the macrosoopie test of ths seme


106
70. SteiafioU, J. Leb, Clin. lied., & 445 (1919)
71. Oulllaln, Laroche and Lechelle, Bull. neo. One. S3ed. Bop. Baris
M W99 (1920)
72. tt. Wright and Koraack, Bloch. J.# 1 635 (1923)
73. 0. Quart!, Boll. soc. ital. Biol. Spar., 1145 (1926)
74. 4, Vernee, Compt, rend. Acad, da Sc* 165. 769 (1917)
75. 4. femes, ibid., 66, 575 (1918)
76. A. Vemos, Ibid., jLJ, 500 (1918)
77* A. Verms, Compt* read. sec. Hoi*# 23, 1425 (1925)
78. Cornwall and Aronson J.A.M.!,, 25 1697 (1920)
79. Cornwall, J. Icb. Clin, Sed. 0* 831-52 (1924-25)
80. H. Bold, Had. Klin., 2, 940 (1921)
81. Strenpel, Munch, sed. Wochschr,, 2, 120 (1922)
82. H* Bold, Beut. sed* Woehachr. jt|, 247-8 (1922)
83. D. A* Johnston, J. Lab. Clin. Med £, 277 (1923)
84. Dreyer and Ward, Lancet, 1921 I. 956
85- Spsroni, J.A.&.A., 22, 1056 (1921)
86. Houghton, J*AB,88 719 (1927)
87. J. B* Moore, The Mft4sr.S %£*&%£ £ .MilU Charles C. Thorns,
BoltlaoreTl933 5
66. Kasahora, Intern. Med. News, (Jap.), no. 987 (1921)| Abst.i Che.
Abete,, M. 438 (1922)
89. Ayer and Foster, Repert phar., 24, 114 (1922)
90. Kahn, Arch. Bormt. A Syph,, 5, 570 (1922)
91. C* Y* Wang, Lancet, 1922 I. 274-6


6
nervous and syphilitic affliction.
Brelger and Hen (20) sought to tacdlfy the assonannn reaction by
th, . ot KClOj . Mm.ly.tn. In a .end articl. (21) tMy mk, th.
statement that the active hemolytic agent is mainly in the ccopienat
and the KClOj aetivntoo it as ell as the use of specific rabbit serum.
While their reaction checked with the Bhoaermrm in 65 caeos, it did
not receive any favor, and is not mentioned again in the literature.
Jacobsthal (22,23) evolved an ultramicroscopio test, based on his
observations that 1) extract of syphilitic livor shows minute, equally
sized particles; 2) syphilitic senes contains less numerous, fine,
motile partidos; 3) the addition of these two liquids give rise to
the formation of large, irregularly shaped flakes, which are composed
of fino particles 4) centrifugation gives a top layer with no comple
ment binding ability, a bottom layer with very strong complement bind
ing ability; 5) the formation of these flakes, and the coiaplenent bind
ing ability are a specific test for syphilis.
Jncobsthal adratted that the percentage of positive tests was not
as high by his method as by the Wassetmna, but stated that it was 100/'
sure on negative results. Be also stated that heart muscle and ether
artificial antigens were of no use in this reaction. It ma indicated
that the Maaanmrm la also 9 precipitation reaction.
Bruck and Hidaka (24) mda an investigation of the test proposed by
Jacobotbel, and in part confirmed it. They also found it was not constant
enough to be of use. However, modifications made the precipitate visible


10
the particle*
Epeteln and Paul randa sotso comments (37) on the theory of eyphille
aorodiagnoeia, and in effect aid that all tete are baaed on electric
charge change in surface tension adsorption and coagulation. They
further add that the alteration of chemical structure of raacion sub
stances* or the formation of new compounds hardly entere into t
problem. Whether thle can be accepted at its face value or not is a
natter of debate.
While no record of the original work on the Hermann-Perots reaction
is found is our literature, it le a reaction using the sodium salt of
glycocholie acid with added cholesterol ae antigen. Zadek (38} found
this to be a non-specific reaction where there were the following affec
tions present* pulmonary tuberculosis, eclampsia, carcinoma, typhoid,
sepsis and uremia, when testing o enea 5 and when testing the spinal fluid
urania and aclamis interf erred. A large number of controls gave 74.4*
positive Wfissenannn reactions and 8positive Henmnn-Peruts reactions.
In connection with this test v. ^Hermann (39) states that it is not
positive with tabes or paresis, but only with true syphilis. V&ny such
substances as the glycocholatcs have been used as antigens in precipita
tion reactions, but none of them have been successful.
At this timo a comparison of the various teats and their indications
was published by Wile and Stokes (40). Their deduction is that the
c aso nest indication of neural involvement is increased globulin and
albumin, the positive Wassenoinn reaction and lymphocytosis, in the


69
totraohlorido le veletile at low temperature end very easily hy
drolysed, the conclusion was reached that the material purchased did
have a very low content of VCl^.
On continued heating a second portion of the sample volatilised
This took place just as a dull red heat eras reached. Deposited on a
cold watch glass, thla had the properties of vanadium oxychloride
(vanadyl chloride, VOClsj), When this material was dissolved in halogen
free nitric aeid the presence of vanadium and chloride ion were easily
demonstrated.
After the sublimation of VOCI3 k*n completed, there was no
change in the residual material, oven at a bright red color* Cooling
the crucible to roost temperature, there retrained an orange-yellow,
miorocryatal lino material* This was proven to be vanadium pent oxide
(VpOtj), the solid, C*F* VCl^ was therefore a mixture containing VCty,,
VCCln and VpO^, In addition to the three compounds proven present, a
nail percentage of VoClg is probably present.
Of the various methods it is possible to use for the preparation
of VCljj, the one used to make the sample purchased was ths passage of
a stream of chlorine gas over heated V#Ot*, This would yield all the
produets found, and that suspected* It was recalled that the original
sample of saturated VClj, was made from a material which was only
partially soluble* Bo doubt exists that It is till 9 mixture of several
salts which bears the trade name Vanadium tetrachloride, C.F
In this mixture of vanadium compounds, the only soluble salts art


7k
Allowing the State Board of Health a week in which to save seat
plea, a trip was made to their laboratory to tost thorn. On arrival
it taras found that seventeen spinal fluids had been saved. These
were non-syphilitic steeples which had been sent in for Ashn. Eagle,
quantitative protein and globulin determinations.
A sensitive dilution of vanadium was prepared, using one ml.
of the solution prepared a week previously to sensitise five ml. of
the freshly made dilution* Is order to check this solution for sensi
tivity, three Kahn positive and three Kahn negative samples were
tested. All gave perfect confirmatory reactions.
The seventeen special samples were them run and each gave a
negative vanadium test, four of these fluids had exosss globulin con
tent reported to the physician attending the ease.
At this point laboratory work was stopped. It wss felt that the
test was ready for hospital work of a routine nature. Only by such
work can the true worth of ary test be established.


34
treatment in the rest of the world*
Ide end Ido were granted a French patent for a diagnostic reagent
in 1937' The reagent ia an extract of beef heart atusle, to which is
added cholesterol and crystal violet, or methylene blue (150). Van
Sverdingen (131) found this reaction lose sensitive than the Molnioke,
Kahn and eltoohoi reactions, but more sensitive then the Wessermann or
Saehe-Georgi reaction, lie states the specificity is the Mae as thet
of the ifaasertaann reaction* It gives a great number of doubtful reac
tions, but simplicity la its advantage*
Rosegger, in 1938, (138) proposed the use of a constant amount of
spinal fluid nixed with varying amounts of calclua chloride* These mix
tures are then heated and the flocculation curves used in diagnosis*
This test has received no further mention*
In another of the new and untried teats, Hlrsohfeld and Klinger
propose that the antibody be used to bind thrembokinaee* If there is
antibody present, then the thronbokin&ae will be unable to coagulate
plasm* Kaehueharov found this to be an impractical test (153)*
Hastial makes use of an antigen got by the extraction of a mixture
of egg yolk and beef heart muscle* It is claimed that the egg yolk
allows the minimal amount of antigen to be used without loss in speoifi-
clty (13^)* In this roaction the egg yolk is probably acting in the
manner as an addition of lecithin* This test is also in the trial stage*
Prohlioh (155) while critically studying the H*B.R, II, hit upon
an idea of a new type test* Since cholesterol and antigen flocculate in


25
reaction is the most sensitive knctm; that it positive with 92^
of children having syphilis of th spinal chord, and that 45/' of these
children had no apparent lesione of the nervous system. Prunall had
provimmly found that CClj-CO* o a pr.clplt.tton <** for lWdn.
(111).
later Prunell (112) investigated the reaction of ?akata-£ra (113)*
This reaction is carried out with 1 cc. of spinal fluid, 1 drop of &2
CO^ (10/Q and 0.3 cc. of a very dilute solution of JigCl2 (G,2$f,) and
diamond fuchsln (0.0l£). It was claimed that the color nial flocculation
gave a toot for syphilis. Prunell found it indicated a pathologic con
dition hut not espocailly syphilis, that it probably depended on
albwiin. Staub end Jealer found this Takata-Ara reaction depended on
globulin, and was of use in diagnosis of hepatic cirrhosis. It la no
better than the gold ieet or ether tests depending on globulin (114).
In 1926, Sachs and litebsky (115) proposed the citochol reaction.
They concentrated beef heart antigen three times and then choleeterinised
it, Th reaction is very rapid. They also investigated the possibility
of a lentochol reaction, using lecithin in place of cholesterol, but
this was not as successful*
Hinton (116) made use of an antigen emulsion which consisted of
alcoholic beef heart extract, cholesterol, glycerol and HaCl solution*
la testing spinal fluid five unheated, graduated sables are used. If
the reaction is positive there Is a clearing of the antigenic emulsion
and sedimentation. The reaction is carried out at 3?C., and if the


la
the presence of a apiernen t, lipoid flocculation by the globulin, and
colloid flocculation by the globulin) 3) the existing teats for globu
lin are not in any way specific for the especial globulin generated by
syphilis, but are merely dependent on the total globulin-albumin ratio)
4) a combination of tests is most effective in the spinal fluid. One
test of each type should be used, -ha precipitation test is more
valuable than any other type for following the progress of treatment)
3) it is essential that testing of the spinal fluid be started as soon
as a positive blood test appears. If the spinal fluid is positive by
any test the treatment must be for neurosyphilis.
In excess globulin in the spinal fluid may be caused by many other
pathologic conditions in addition to syphilis, tuberculosis and menin
gitis (meningococcus) both lead to an excess protein, the former giving
riee to globulin, the latter to albumin (I07), dementia praecox may
give rise to hypertension, hyperglobuLlnosia and hyperalbsminosis.
Any or all of these conditions may be found, or the spinal fluid may be
normal (165, 166)#
We have also seen that some globulin tests are misleading in the
presence of eclampsia, hepatic cirrhosis, uremia, typhoid fever, lupus
and pneumonia (13# 3* 113)
In globulin precipitation the pH at the time of precipitation ie
of importance# This will be discussed in the section on Sodium,,Vanadate


TESTS PROPOSED BY DIFFSREKT AUTHORS
FOR THE DIAGNOSIS OF SYPHILIS
In the coapleiaont fixation teat, or Whsaenrann test (2), the
mechanism ie postulated to be me follows 1) An antibody is built
up in some concentration in the patient's system. 2} Tills antibody
sill combine with and destroy some specific part of the affecting
organism* which is called antigen. 3) In order for the antibody-anti
gen reaction to take place it is necessary that a reaction promoter, or
linking agent, bo present this agent is called complement. 4) If the
antigen be added to a sample of serum which has no complement* and then
just enough of this complement is added for that antigen to react then
proving the presence of the unaffected complement would prove the
absence of antibody. 5) To prove the presence or absence of this
bridging material it is necessary that we have a cm other reaction
which aleo requires complement. Such a reaction ie the hemolysis of
foreign blood corpuscles by the sertas of any corpuacle-tesunc animal.
6) To get ouch a system, a rabbit ie injected with the red blood cor
puscles of the sheep, so that his blood will contain an antibody specif ie
for the hemolysis of sheep corpuscles. ?) Complement is the molabile;
that is, heating it at 56C. will destroy it| on the other hand, anti
body is not so destroyed and ie called thonaoatabil.
With these facts in mind, it ie easy to visualise the Wesesraann
Test for syphilis. Hie first step is to heat the patient's blood serum,
the cells having been removed. This heating is known as inactivation


TABUS I (OHTISBD)
79
plual' jSbT"
mifeuatesaL
ItaEh'
TSXT
Xoaotlon reaction.
werr***
K0r07
potion.
63
64
T
as
a
66
T
f
66
T
a
67
T

4
08
T
4
60
T
4a
4
70
T

71


7£
4
+
4
76


74
4

76
4
4
4
76
4
4-
77
4
4
78

4
79
Sklrsaeni S
striSr^spitai
raECtion
** a*
4
80
a
m
a*
81
a
m
a*
88
gold sol a
4
4
83
+
4
4
84
4
4
85

4
86

> '
*. 4
87
4
4
86
+
4
4
88

4


32
it to bo 18$ In error with the Kuhn toot and 20$ with the saseeraann.
Using inactivated sema It was only 10$ in error compared to the Kahn
test. The reagent is a mixture of bensoin, cholesterol, scarlet red
and physiological salt solution, this is added to an extract of beef
heart. One drop of the reagent emulsion is placed on a slide and a
drop of serum (or spinal fluid) is added. The appearance of red floes,
soon in indirect light, is a positive reaction. Robinson and Stroud
(lip.) found it 93$ effective as against their ffassermann and 97$ against
their Kahn. Thcmaasen (11*2) used it on 3,000 sera end got good results.
The lunger it is used, tho better this test is.
Vernes (li;j), in 1936, stated the finding of a spool fio material
in the spinal fluid was a diagnostic test. The material is oalled
pellidin and is obtained by precipitation* The spinal fluid is precipi
tated with CgCljj and this precipitate is extracted with ether or ace
tone. The extract yields a substance which gives a positive reaction
if added to a negative spinal fluid. Yasitsa and TakagL found the ma
terial pallldin only In the later stages of syphilis (Il4t), and that it
is not demonstrable until some time after the secondary stage is over.
fiyt* reported what he oalled a rapid floooulation reaction in 1936
(ll*5) This can hardly be oalled rapid. To the active spinal fluid is
added ammonium sulfate and NaCl solutions. An antigen emulsion is then
run over this. The density is now such that the proteins In the fluid
are forced into the antigen emulsin on centrifugation. After centri
fuging, the solution is made lighter than the flooculate by the addition


l4
to the asm level. There can not poaaibly he a safety sons, as seas
authors oala, but always a gradation fresa non-ayphilitie to syphilitic.
Pfeiffer, Kober and Field (53) sede an investigation of th# pro
teins in the spinal fluid, using nepholosaatry for their determinations.
They found that if the normal value for euglobulin as token as 0.004J,
then fluids giving a positive Ifesaermrm test -sent over this value in
14 out of 16 casca, and negative fluids were under it in 33 out of 35
cases. They also admitted that in two of these disagreeing eases their
measurements were doubtful. It is concluded that the euglobulin con
tent and the Wasseraann test are closely related.
As a test for pathogenicity, Hofftean and Schwarts proposed the
ue of the permanganate number (54). This is the number of cc. of 0.1*1
KHn04 reduced by 1 cc. of the spinal fluid. Genoese (55) found that
this reaction was also capable of being carried out with methylene blue,
ke everlayed 1 cc, of the fluid with 1 cc. of reagent and the yellow
color at the Interface, followed by the whole liquide becoming yellow
after shaking, was supposed to denote a pathologic fluid. Bovori,
after who th permanganate reaction is named, found that this was in
reality a test for albumin (56), and that 1 cc. of spinal fluid should
not decolorise 1 cc# of ltlO,COO KKnO^, Gulllain and Libert (57) also
report that hyperalbusainoois always gives a positive permanganate test.
Lowery (56) found that neither tho permanganate nor the mastic teet was
of sufficient value for the routine examination of spinal fluid. His
work was dono prior to 1$1?# and while tfc permanganate test is not used,
as ho advisee, the mastic test is being used acre and more.


52
end turn yellow when the solution is basic* With the known solution
this raaction readily took place, but the resulting solution was
groen, duo to the totravelont vanadium ions still present* the un
known behaved in exactly the same manner* Trival ant vanadium does not
change in basic solution* the coagulation and darkening of the sola*
tion at the neutral point is due to the formation of polymers with
the eaparioal formula VjgOj;.
The formation of VgO5, or its polymers, is prevented in a solu
tion made from VCl^* Such a solution is aeid due to hydrolysis and
this leads to the formation of polyvanadio aeid, probably i|tV^0i7*
solubility of VCl|t is not found in the literature, only the fact that
it decomposes in water* This decomposition is in accord with the
following equation
Wl|,+ HgO s V0++ + 2H+ + liCl" .
The oxidation of the vanadyl ion (V0++) is Inhibited by the
presence of hydrogen ion* To stabilise solutions of VCljt, excel 1 HC1
is added. It was found in the course of our experiments that the atore
concentrated solutions did not oxidise with nearly the speed of the
dilute solutions* This is probably due to a lower solubility of
oxygen and a lowered activity of vanadyl ions* The law of mase
action shows why hydrogen ion is effective in prevention of oxidation
in tiie following equation:
4V0++ + Og + li(H)~ + 0HpO s kHVOg + |*B+
The aeidlty of a solution made from a mixture of VOCl^ and VC1|(


103
BIBLIOGRAPHY
1. 0. Morgan Moran, J.A.K.A., 40, 937 (1903)
2. Bbssarmaan, Malaaar and Brook, Boot. aed. Wochsebr., 2, no, 1$ (1906)
3. Wasoenaann and Plaut, Bant. taod. ^eehaehr., 31* n0* 44 (1906)
4. L* fftefcaelle, Borl. kUn, Woefeaehr., 44, 1477 (1907)
5. L* Mlciaaolio, Ibid, pagan 1103-7
6. Saaaamwm, Bari* kiln. iSochaehr., <4, 1599 (190?)
7., Flaiaotaaann, Bari* kiln* Itocbachr., 4£9 490 (1908)
8* Varney, (discussion), J.A.li.A,, 1, I994 (1908)
9. Plant, Keuck and Roaai, J.A.M.A., &, 13 (1908)
10* Vsrnoy and Morae, J. Cut* Die., 3g, 624-9
11, J. Bronfenbrenraor, Blochea. Boll., 87-9 (1915)
12* E, Klsuener, tain, klin. aochsehr,, 21, 213-46 (I908)
13* E, Elausner, ibid*, pages 345-78
14. Krcweke, Red. Kllnik, BarUn, 7&t 210 (1923)
15* w* Schuraan, Bout. mi. Wochachr., 35, 6l6 (1909)
16. Rax Honra, J.A.R.A., 33, 289-96 (1909)
17. Roguehl, (editorial), J.A.K.A,, 33, 1931-2 (1909)
18* L. Beltz, Dautsch Z Korvenhoilk, 43, 63-80
19. E. Jones, Rev, ileur. & Faychiat*, 2, 379-91 (1908)
20* Breiger and Hans, Dent, mi. Wochechr,, 33, 2203 (W)
21* Broiger and Bans, ibid*, 36 78 (1910)
22* K* Jacobathal, Biol. Abt*, Artxl* Vereina, Hanburg, 3, Id (1909)


91
TABLE XX
SOWAR* OF TAmAT'E) RE30LTS ITH VC1* HHACflOKS
Total Pfassemcnn end Kahn positivo,
syphilitic spinal fluid samples 100 A
Tot al Aassermarm and Eaten negative,
non-syphilitic spinal fluid samples **..,.* 145 B
Total Wasseiutfm end Kahn negative,
treaood syphilitic spinal fluid samples. ....... 101 C
Total number of samples .oieT
vCl* results Tilth A.$ +, ^
Percentage disagree:¡ont Absolute
Hoi stive *s
VCl* results with B...................... 143 -, £ 4
Percentage disagreement ....... Absolute 1.4$
Relative 1 4£>
VCI4 results with £ ea 35 , 5+
Percentage disagreement*......... ... Absolute 38.9#
Relative 39*9$
1Bacterio present.


19
sensitive than either the flachs-Georgi or the Ifeinieke III) he found it
gave false negative results*
Bold (82) found that formlin enhanced the reactivity of sema
especially syphilitic and in 1922 made a simplification of his original
test, using formaldehyde, antigen and serum. In order to read this test
it was still necessary for the incubation to go on two hours and the tubes
wore read after 2 and 22 hours in the icebox. In line with this was the
formaldehyde-gel test proposed around this timo by Cate and Papaccsta.
Johnston (83) made an investigation of this test and found it mas never
to bo used with any surety. The technique is to add a drop of formlin
to 1 cc, of blood serum. The tubes are allowed to stand 48 hours and then
examined for coagulation. In 100 cases it as positive in 23, the Wasser-
raann in 27. There was agreeaent in 11 of these, all the others conflict
ing. Considering thio, perhaps the addition of formalin to the Bold
technique was a mistake, loading to erroneous results,
Dreyer and ard, in 1921 (84), published a test which is in use in
England today. A eholeeterinised antigen of beef heart extract ia prepared
and two dilutions ade of it. iline dilutions of the oerus are then rand
up and five added to the stronger antigen dilution, four to the weaker.
After a short incubation the turbidity in each tube is read by moans of a
special agglutinoscope. The results are expressed in standardized Sita
units. The necessity of using a special instrument keeps this reaction,
just as It does the Vernos reaction, in a limited field.
The Berxheimr reaction (85, 86, 87), or provocative teat, was first


38
all lasts for syphilis aro based on the soluble globulin. The foot
that this soluble globulin disappear as sow as the exalting influence
is geno plains why serologic loots sometime* fail I the latent stages
of syphilis.
la the same way In which certain diseases generala non-specific
antibodies, it is probable that syphilis dose not produce one specific
globulin* but others In addition*
The fact that true antibodies are act generated in persons affected
by syphilis is shown by the occurrence of positive blood reactions end
simultaneous negative spinal fluid reactions, flam (139) demonstrated
that true antibodies easily pass the meningitic barrier and are found
immediately in the spinal fluid A second point to consider Is the
work of Hoof old (l6o), who proved that the spinal fluid system is unable
to manufacture its o antibodies.
In eases of aeurosyphilis the globulin-antibody amt be generated
in the spinal fluid system, since it is net always found in the blood
On the other hand* true antibodies muid always be present in both the
spinal fluid and blood streams We may say that the spinal fluid and
the blood systems are both capable of producing globulln-satibodies for
syphilis and that these systems work independently.
Almost and Tarbouroich (l&L) state that they have proven there is
always more antibody in the blood stream than in the pial fluid This
report is evidently made tas an incomplete work hew else may we view
their work in the light of the many eases In which there is a negative


100
the relativo agreement of the venadltsa reaction with the Kahn
reaction (based en olese I aat III fluids onjy) is 99*2^. The abso
lute agreement is lie propose that this is a sensitive test
for certain globulins found In syphilitic spinal fluids*
With regard to other conditions producing excess globulin in
the spinal fluid, little has been determined. The work of Borland
(167) shows that vua&dio acid ie more or less specif!e for certain
globulins* the work of eri (268) goes even further In showing there
is a tendency for -vanadio old to be specific for syphilis globulins,
but without aay sensitivity whatsoever*
We can stake definitely that every ease which sheers globulin in
excess of the normal will not give a positive vanadium reaction* It
is Impossible for us to state tbs offset of various diseases on the
test* The samples examined at the State Board of Health have no hist
ory attached* The attending physician rarely inferas ti laboratory
of the diagnosis*
Koore* Eagle and Mohr (172) point out clearly the facts on falsa
diagnosis of syphilis Very little critical work has been done in the
field and a wide opening for further research exists*


83
TABLE IV
RESULTS OF VOX* REACTIONS WITH SPIRAL FLUIDS* SHOWISO
pH ASD COMPOSITION OF PRECIPITATE IF FORMED
Spinal finid
NKtita. nwabar...
Ha
mr
resetIon
asr**-
pa
Preciptate
163
afea..?
** m
6*7
164
m :
*
5*0

165
vs
m
6*0
as
166
> S..
vs
6*0
m
167
m
V
5,7

168

**
3.9
vs
169
4 m

5,9
*
170
', J
vs
5*7

171
-
*
#**

172
-
m
6*0
-
173
T
X
**#
4*
174
T
m
* *
175
T
m
5,7
as
176
T
m
* **
41*
177
T
m
5.7
m
178
T
as
3,9
m
179
T
V*
3.9
VS
180
+
' 4-
# ,
globulin +
181
4
*
#
globulin 4
18£
4
4-
, *
4
183
+
4
***
globulin 4
184
** *a
6,1
m
183
6*0
m
186
V*
*
6*0
as
187
' a*
a#
3*7
as
188
-

6,0
as
189
*
vs
6*0
as
190
*
4#
5*9
m
191
vs
m
5*95
v*
192
Â¥*
** ,
vs
193
vs
4V
3,8
4
194

V*
8.0
4,
19
V
**
6*15
VS
^Fluids la very bel shape on roeoipt.


t
71
For this purpose & moving Punten fimo* 1/2 inda in height* me used*
After the hooting bed been continued for ninety minutes the apparatus
ms taken down and the sample allowed to cool* During the course of
the heating a mall quantity of red liquid distilled from the reaction
tube to Hie safety tube* this was found to be VClj^, After cooling*
the sample ms removed end broken up*
It ms decided that no attempt would be made to produce a dupli
cate saturated solution, therefore the sample was treated in centri
fuge tubes with an excess water* A deep blue color developed in Hie
solution* The solid residue ms centrifuged out and the solution fil
tered, using very fine filter paper* This solution was labelled con
centrated VCl^ and put on the shelf* The next step would be the analy
sis of this and then its dilution to a vanadium content the same as
1/2 saturated Wl^. Vanadium analyses were made of the newly prepared
solution and of the half-saturated VCljt solution* Tho method ms the
sane as that previously outlined. After calculation had boon com
pleted the new solution was diluted so that its vanadium content was
equal to that of tho original half-saturated solution* This new half-
saturated solution was allowed to age for a week and then used in
parallel tests with old VClj^ solution*
Ho difficulty arose in running the last 70 semipis with sensitive
dilutions prepared from both of the half-saturated solutions on hand#
Both sensitive dilutions were analysed for vanadium content and found
to have l*iiUlO gmas of tClj^/liter* The average pH of the sensitive


80
TABLE II
RESULTS OF VG1 AS ^Of*Or*BSi0* REACTI0H3 XT8 SFXJtAL
fluids shoots aosisg effect o 1/2 saturated vex*
Spinal' f^uidt Weasemann
Vifr* reaction ktOPOr'^,
JKi
JMt.
jMftiL,jamaiaeL
90
**
m
m
we
91
m
*
98
ee
-
4#
*
93

m
4
94
*
4
m m <|#
P
96
*
m

a#
96
a*
m
ep
m
97

r
1*
m
98
' T
not
i .'?*- >
99
T
run
* i -
H*
100
T

4
4
101
T

p
HP
108
T
4
4
103
T .
*#
#
104
T
#
4
£
105
T

4
£
106
Kahn
reaction

4
4
4
107
4
4
4
4
108
4
4
4
4
109
4
m
4 4 44
4
no
4
m
** *
4
in
4
4
4
4
118
4
4
4
4
ns
4
4
4
4
114
Single fluid
from St# Luke#
laaeermsm
4
4
4
4
1Humbers 109 and 1X0 would bo reportad as doubtful#
the reset Iona being so uncertain and weak#


59
made i this uanmrt tha best of the lot gave ene falta positiva
with tito sagatlva samples and two negativa results an Wessons am
positiva, syphilitic ampias. Thinking that this was not accurate
enough work, 20 ml, of exactly half satura tad solution was mads. This
was placed in tha ttse type bottle as the other halfsaturated sola*
tion and marked "1/2 SaVd VCl^ Ho. 2." The earn# dropping top was
then usad for making a dilution from this Ho, 2 solution as ms usad
on the original h lfsaturatad solution, Tha results in this casa
ware no bettor or worse 'than those got by use of the saturated solu
tion in the first place, Tbo fact that alcohol takas some tine to
mix with water and form a solution which may be duplicated has been
known to the liquor trade for years* However, no record of this it
found concerning wetal salts, That soma partial oxidation is able to
taka place in tho more dilute solution is possible, but it would have
no effeot on the results, As long as an oxidised solution is being
added for sonsitication, the only result would be the necessity of
using moro of this oxidised solution, whan preparing a sensitive dilu
tion from the saturated solution.
It was found that with tha next shipment of spinal fluids, either
tha He* 1 or Me* 2 half-saturated solution of ?Cl}j gave the s me re
sults, Close I and XII ware in accord with their Waesefwfe&n reporte*
olasa II wae equally divided, four positive and four negative reactions.
It must be that a definite period of aging is necessary after the
dilution of saturated vanadium tetrachloride. To cheek this, the


73
Enquiries were sent the State Board of Health mod tho State Tuber
culosis Sanatorium regarding tho furnishing of sample# from diseases
other than syphilis. It was found that at tho time there wore no suoh
samples available* laboratory work was stopped at tho end of May, to
bo resumed In July,
On the resumption of work moro enquiries wore made as to a possi
ble source of diseased, non-syphilitic spinal fluids* Tho State Board
of Health laboratory gave the only enoouraging reply. They stated
that any suoh fluido would be raved for our testing* The fluids wore
not hipped for fear of eontsmlsatioi,
Anticipating the test work to be done at Jacksonville, an
examination was node of the VCl}t solutions in the laboratory* A black
precipitate was found to have formed in each one* This say have been
due to the heat, or eoatnot with the rubber In the topper* The solu
tions hod been left In an upright position and had been overturned acci
dentally at some time in the muster* The precipitate was filtered out
of one of the solutions of half-saturated TGI}, and the solution
analysed for vanadium content.
Whereas the solution had originally contained 13*9U g* VCl}, per
liter, the aoneentratian was now found to b# 15*25 R* Pr liter. In
order to make a solution the concentration of the sensitive dilution
(lJ&lO g# per liter), 0*& ml# of this solution were made up to 5 ml.
This solution was allowed to oxidise for sensitisation use in the work
to be done later.


no
159 flama, last. Path. Lund*, Acta Bath, et Microbiol, Stand*, 5,
16-24 (1928)
160. Heufeld, Krankheitsfrsch,, g, 63-9 (1925)
161. August and Tarboureieh, Bull* 00c franc* dernatol. syphilog*, 45.
368-72 (1938)
162. Malloy, Kahn and Westell, J, Infectious Diseases, *8, 203-11 (1931)
163. F.ntmer, Momtaachr, Kinderbeilk, 1, 387-93 (1932)
164. Warren, J. Lab, Clin. Med., &, 1146-50 (1936)
165. Victor Meyer, J. Biol. Chora, j>, 115-31
166. Havout and Laignel-Lavanatine, Bull, era. see. raed* hop., 3* H51
(192C)
167. Boy land, Moche, 2., 26, 1383-90 (1932)
168* A* Keri, Rnssongn di studi psichiatriei, 21 639-5 (2uly and August,
1935)
169. Levinson, Ass, J* Bis. Qilldren, 1§, 568-76 (1919)
170. k. Pfeiffer, i^indUcJa* gia 22S
l2£MSMm£tSB 4s Mm* desden (1938)
171. Bouris, Corapt. rend, 00c. biol, gi, 106-8 (1927)
172. Moore, Eegle and Mohr, J.A.M.A., ll£, 1602 (1940)


92
TJSLS X
mmmt of taslas results oh the ps of
m voi4-spxjal flci mm¡m&
Total pS doteroinAtiona...... ........ 70
Arithmetic 6*9 pH
units
Average deviation of & single measurement....... 0,15 pH
imita
Probable error of a single measurement ..... 0.1S pH
units
Probable error of the mean....... 0*016 pE
units
.reolpttttta
' v>- 'y " i V ,
Total precipitates oarained 64
Precipitates behaving as globulin (solubility} 64
Precipitates examined for vanadium*............. 9
Precipitates found to contain vanadium...** 9
Additional precipitates examined for
vanadium by fusion*....*..*.*.,...*........., 6
Precipitates found to contain vanadium
by fusion*.. 6
^Colorimetric qualitative method used.


29
any value. If the fluid is usable. 0*03 oc. of 1% acetic acid Is
pl&oed on a slide and 0.25 so. of fluid and 0.008 oc. of antigen added.
For either exclusion or diagnosis the quantities are the seme. The
slides are rotated for completo mixing and read under the microscope
iranodi&tely. If the exclusion test is negative, it is claimed that
there is no ehanee of neural involvement. The dumping of material is
a positive test.
In the book Kline also reports preliminary work on a balling reac
tion. The antigen Is strongly cholostorinised, and when emulsified has
the appearance of long needles of cholesterol partially cove rod by anti
gen. This has the same appearance as the emulsion for the l.B.h. II.
Since no further report of this tost has bean made, one may judge it
turned out no more successfully than the &.B.R. II,
Kiss (127) proposed a balling reaction using s oephalin (phosphatid
from brain) antigen with active sonsa. This test did not get a very good
reception. The statement Is made by Kiss that lecithin is good only
when used with inactive serum, oephalin only with aotive serum. Kahn
has proven the former assertion, but the latter has yet to be proven.
Sohube and Hams (123) proposed a colloidal carbon test. To the
spinal fluid is edited distilled water and then a dilute solution of oxa
lic add. After standing a time, diluted India ink is added. Precipi
tation of the carbon is a positive result. Looney (129) had previously
tried to use this reaction with colloidal carbon made by the Bredig are
method, but had been unsuccessful. Sohube (130) explains the mechanism


101
SUiaiARY OF CQSCLUSIOHS
1 The reaction of potass!xm dichromate and nitric acid solu
tions on syphilitic and non-syphilitic spinal fluids has boon investi
gated* It 1b judged that this reaction is too unpredictable to be of
use in tbs diagnosis of syphilis or tbs following of syphilitic
treatment*
2* The reaction of an aqueous solution of vanadium tetra
chloride with syphilitic and non-syphilitic spinal fluids has bom
investigated* It has shown definite value for the following of
syphilitic treatment* The action of vanadium solutions prepared is
the manner given is specific in a great degree for the globulins of
syphilis* tittle Is known, or can be found out with regard to the
effect of other diseases on the vanadium test* It is reasonable to
assume that any condition causing a great exeess of globulin would yield
a positive reaction* This would occur with almost any metal salt one
happened to choose*
3f* The reaction Is that of a reproducible solution and is ready
for evaluation In actual hospital practice* It may be a much surer
test for syphllltio globulin than any proposed to date*
4. We may say that this is not intended for a diagnostic test*
but that if the blood test is positive in the secondary stages* this
will surely shew neurosyphilis as scon as the Kahn test* Perhaps the
VCljft reaction will show turnroayphi 11s sooner* since non-specific


85
table iv (oomimm)
~rar~

eso
SkffttjRfwrfe It?
5.7
4*

Mr
£81
#
+
5.7
globulin 4
2S2

m
5.8
48
ess
8
m
5*8
M> '
£84
*
4
M
M
ess

ft
3,0
*
236
**
8
5*05
#
257
8
Mi
5*8
*
£38
T
4
3.95
M
£39
T

5*6
not osntrifugod
£40
T

* *
£41
T
Mr
* * *
*
£42
T
3*8
globulin 4
243
T
fr
5.3
globulin
£44
T
1
.
*
£45
f

* *
£40
+
* -*
*
247
*

5.7
globulin 7
£48
3.1
4*
£49
*
+
6,0
globulin
250

+
@.l
globulin
£51


6,1
globulin
252
4*

6.9
globulin
£53
*
+
.***.
.*****


97
the wait for partial oxidation to talco place.
This sensitive dilution contains polyvaaedio aoids of the type
%V617* hydrogen ions, chloride ions, and a vanadyl ion (V0++). This
latter ion is not named in the literature. All VO ions, regardless
of charge, are called vanadyl, ~ous being added to ahow monovalence
and le being added to ahow trivalonee. The divalent form which
arises by hydrolysis of the tetrachloride is left with no convenient
suffix.
If equal volumes of the sensitive solution of vanadium compounds
and spinal fluid are mixed, there is a precipitation of globulin when
syphilis is present. A volume of water equal to the total volume on
the slide is added to eliminate hypor-alfeuminosls. This globulin is
precipitated at a pH of 5*9* This pH value is much higher than that
found by Boyland (267}.with mixtures of vanadio acid and pure globulins,
or globulins protected by albumins.
By an inapeotion of the works of Moore (87) and Kahn (102) it is
seen that there must be some specific globulins produced by the
disease syphilis. These globulins apparently take the place of the
normal globulins. This is the only explanation for the disappearance
of excess globulin and continued positive fassermann and Kahn
reactions after an inadequate course of treatment for neurosyphilis.
Boyland found that the only means of explaining the reaction of globu
lin and vanadio acid in the presence of albumin was to postulate a
possible specificity. We have shown that this speoifiolty goes even


81
TABLE III
RESULTS OF TO* ABB J^C*e0T-H80* REACTIOHS
WITH SPIRAL PLtTCDS
SpiiM'Bm rir '" lMD 'Wx^ ',rhg<^07-M6*
SMgraent 7
US
#
<*>
<*
na
m
*
117
**
a
m
na
rt*
44
m
119
¡
120
**
121
m
r
188
-
o
123
T
m
184
T
4
183
T

126
T
4
127
T
188
T
4
129
T
4
130
T
-
131
4-
4
4
132
4
4
133
4
4
134
4
4
135
4
4
4
13
4
4
137
4
4
4
138
4
4
Shiment 8
Sfen
reaction
13
4*
m
not ran
140
m

hereafter
141
44
m
*
142
m
4*
*
143
m
m
#
144
44
m

145
m
m
*
146
*
M4
#


33
of dilute Had solution. The tubes are centrifuged a second tine and
the solution poured off. The sediment remaining Is shaken with phy
siologic saline* Flocculation is positire, a hazy suspension is nega
tive and a combination of the two extremes is called doubtful. The
compsrisen of this test with the Waaesraann is not good but it is
more sensitive, perhaps too such so.
fiahr (146) reports the Chcdiak-Pabr, Dahr or Chdiak reaction.
The nasoe of the reaction depends on the country in which the work is
dons. One drop of defibrinated dried blood ia used. To the blood is
added 0*015 cc* of a solution containing 0*3# JfajCQ^ and 3*5# Ha Cl, A
1*10 dilution of the Me inioke K la rungs Eeaktion antigen with this sane
solution of M.2C03 sod B.C1 is pUc.d on a second slide. The usunt
of antigen dilution used is 0.03 so. The two slides are heated to 5&C,
and their contenta mixed, the elide with the mixture is kept at 37C.
in a aolot chamber for half an hour. The reaction is read at the end
of this time. The appearance of black floes and clots is a positive
reaction, brown granules is a negative reaction. If o modified antigen,
M.K.R. II (modified),is used, the addition of the may be
emitted in the preparation of the diluting solution (147). Schuck (148)
used one drop of blood, 0*3 cc* of 3*5# Ha01 and 0*03 ce* of the
antigen dilution. The appearance of the reaction it the asms as before*
The reaction is strongly recommended as an additional aid In diag&o*l*
by Porto and de Britto (149), Althot^h it has received no mention in
the Onitod State, this test has gained a great amount of literature


9
meningitis. They only advised the use of each a teat in the epical
fluid. Morions (32) found that there as no parallel between tide test
and the albumin content, but he does not mention the amount of globulin
the more Important factor. Thia test is not mentioned again in the
literature, but It is used as the basis of several other works notably
the Hecht-Gradwohl test and ite modifications.
Lange published the results of his work in 1912 (33). By deductive
reasoning he worked out one of the most important tests applied to
spinal fluid even today. It was an entirely new reaction and came as a
result of his observing that all eerodiagnostic tests to that time were
based on colloid properties of liquids. Since one of the best methods
for the investigation of colloidal solutions was the gold number of
Zsigmondy, he applied this technique* The method Lange used van to make
dilutions of the fluid being investigated and add them to tubes containing
a solution of colloidal gold. If the gold was unaltered the reaction was
negative. When 10 dilutions were used erase would react and some fail.
Graphing those results gave curves, and it is in the interpretation of
these curves that the differential diagnosis of the varied manifesta
tions of neural syphilis lisa* Kafka (34) confirmed the work of Lange,
and Kryle, Brandt and Krns (35) nade this eramaent on it* The test
(Lange) runs parallel to the globulin content but is independent of it.
It appears when other fail* and just as well my fail when others appear"
Glascoe and Scrum (36) did some work on the sol itself and found that the
sensitivity decreased with increasing pH and increased with the else of


T&MM 3a icomxmm)
Results of pH deteminatlons on (X) sad (Â¥) from
the preceding seetlont
pH found on (X)MtiM 2.80
pH found on it) .......... 2.68
Eo@u3.to on analysis of holf~saturat& solution
after It hod boon in eonfcoet with rubber*
*
Frovious analysis
Hew analysis..*..
15.948 grcsas VC1*/1
15.250 grans VC1*A


#80X$138S 04E&& f^*#|?Si8
SotaoxTCJ PC TO* I peAioooa apxntj Wf%
0*
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0. r- .
39
04.
ts
40
0k
09
4*
04
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69
**
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as
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'V' ; -
99
44
99 '
FW
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4
4
99
4
4
4
§9
4
4
4
89

4
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T
4
4
4
0
4
4
4
6*
4*
4
4
80
4
4
4
0

4
£
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£
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£
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£
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99
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t Iwotrp
Ww5I
.*gaasi
mpatmwrweet
S& SOL
'l^garstaigg
-gRUiuaafe
((EnaiXKoo) i mi


28
Kline and Kahn tests are the same in that the antigen i3 concentrated to
obtain diamino-phosphatids* mixed with as little impurity as possible.
This waxy material is then mixed with water, cholesterol and physiologic
salt solution. The manner of mixing determines the state of the emulsion.
In a reverse procedure to Kahn's, Kline adds the cholesterol to the water
first, each particle being thrown out as a small plate. The antigen is
added and forms a covering over each plate, giving great surface area.
To this suspension is now added the saline, its purpose being the same as
in all such reactions, to make the reaction more sharp.
Compared, the Kline and Kahn antigens are opposites of stability, the
Kline not precipitating for hours, the Kahn giving precipitation as soon
as it is made. Two tests are proposed for spinal fluid syphilis detec
tion with Kline antigen. The first is the exclusion test, the second the
diagnostic. Again the reverse of the Kahn procedure is used, the
antigen emulsion being concentrated in place of the spinal fluid. For
both Kline tests 4 cc. of the antigen are heated 15 minutes} that for
the exclusion test at 50C., the antigen for the diagnostic test at 35C.
After the heating the tubes are centrifuged at 2,200 r.p.m. for 15
minutes and then the liquid decanted. To each tube is now added 1 cc.
of physiologic saline, pH 6. After centrifuging the cells out of the
spinal fluid, it is tested with Bennedict's solution for the presence
of sugar. If sugar is present the fluid is good, if not it has been sub
jected to bacterial action and will not give a precipitation test of
^The alcohol soluble, acetone insoluble fraction of beef muscle.


15
Sach and Georgl (59) proposed the test bearing their moos in
1916. They ande use of an alcoholic extract of heart tissue to which
was added cholesterol. This antigen was mixed with the serum (inacti
vated) and allowed to stand 18 to 24 horn's, after a short incubation at
3?C. Flocculation wee a positive reaction. This is one of the most
important forerunners of the modem Kahn reaction, and in fact the
faults in it are responsible for Kahn starting his research. In the
some manner# the work of Heinicks is just as responsible.
ieinieke (60,61) mete use of a physical-chemical reaction for the
diagnosis of syphilis. The reaction was found to depend on the fact
that a mixture of lipoids (the antigen) and syphilitic antibodies fora
a precipitate, and that this precipitate is insoluble is salt solution.
To 0,2 cc. of the inactivated senes is added 1 cc. of the lipoldal
antigen (made of human heart extract), the tube is then incubated for
20 hours at 37c After the incubation the precipitate is shaken and
1 cc, of 2* JfoCl solution is added. If the precipitate is insoluble,
the reaction is positive. Fr. Reichert (62) found this reaction, after
its third modification, was still not applicable to the spinal fluid,
the precipitated Material, if any, not being vieible. The various Modi
fications made in this reaction by Heinleke were not able to increase
its usefulness, ami in 1922 he announced a new reaction, the Jfoinleke
turbidity reaction (li.T.R. or Helnieke II), The antigen used in this
U,T,R, (63) was designed to make the reaction clearer, and to do away
with the addition of the salt solution at the end. To the lipoldal


27
method, the second the reaction of L'utercdlch. Neither cf these was
new, or very effective. Sauer reports (122) on the Douris-Beck reac
tion at this time* This reaction need a mixture of formalin, sodium
olate and phosphoric acid. This aleo vas nothing new, except in the
combination of reagents, and it was rot effective. Tapi (123)
explained the Sclarra reaction, which is a Wnsseraann test with alcohol
pressnt. It has no particular advantage over the parent test. helcxer
and Bahwen developed a jaicro-aastic test, hut Kleiner ($8) found It
only as effective as the Lange test.
Karchioni and Allensteln noted (124,125), in 1932, that the diastase
content of the spinal fluid is involved in syphilis. The normal range
of this material in the spinal fluid is 11-40 mg. percent, but in
syphilis it is found to he only 0*0-14 This drop is independent
of the sugar content, which reranins normal. The over-lapping of the
values does not prevent this finding from being of use in some casco
diagnostically. The effect after treatment is not given, and should be
even more important.
Kline (126) started publishing preliminary reports of a slide test
for syphilis in 1929. It was not until 1932 that the test was ready for
general use with spinal fluids* At this time a book was published by
Kline. Kahn had the idea that if all other factors were equal, then the
more unstable the antigen-emulsion, the more sensitive and rapid the
test would be. Kline presents the view that the physical state of the
antigen emulsion has more importance than the stability. Both the



o

2J
f.0
r
nil
O
a
147365


95
DISCUSSION OF RESULTS AND CONCLUSIONS
The reaction of equal volume* of 2*5/ potassium dichromate*
I \
It100 nltrlo acid and spinal fluid has been studied* This resemblas
the reaction of Taocono (109, 110}* The sensitivity of the di*
chromate solution is increased with ge* The nitric sold solution
varied in strength with age* Mew solutions must be made up for each
series of detominations* With 8*6gS of the &ahn negative, non
syphilitic spinal fluids there is a positive reaction by this method*
If the solution strengths are varied to do away with these false posi
tive reactions, the Malm positive, syphilitic spinal fluids do not all
give positive dichromate reactions*
With the diehr coata method a groat many positive syphilitic fluids
do not give consistent results* In sons tests there are observed
granulas and no flocoulation* If the test be repeated flocculation is
as likely to take piase as granule formation* Granules arc apt to
appear in negative non-syphilitic spinal fluids* In the same manner,
there are apt to be a groat many vary weak positive reaction*! these
are judged to be doubtful*
In line with the work of Prunell (113), it appears that this id
a test for globulin and albumin in certain ratios* Mo attempt was made
to verify this, the main part of this investigation being occupied by
work on vanadium compounds. There is no difference in the flocculation
which result# from different fluids, as Taoemte claimed* *# may


63
if these false, oold-induoed reactions ere lloved to oomo to row
temperature, there would be & re-diasolving of the floe calato. It
was also found that true positive reactions were not re-dissolved by
cooling or heating, but remained until the water had ewpletely evap
orated. The evaporation of a mixture of sensitive dilution and nega
tive spinal fluid at row temperature did sot rosult in floooulation at
any tine in its course,
to this time the finer points of the investigation had been in
tentionally put aside. The nature of the flocculate, the pH at which
it occurred, and similar questions now required answers, a total of
one hundred mid sixty-two spinal fluid sstaples had been cheeked by the
VCljj dilution. In these tests one ^assenaann negative fluid had given
a positive remeton, and two osseraaaa positive fluids had given
doubtful reactions. From these samples 100 had been run by the di-
chrcBUit e-nit rio acid method. This latter method had been tapered off
for several reasons, in the first place it gave rise to too many falso
reactions, and too many doubtful reactions, la the second plaee the
solutions wore not constant, KpCrpOf, which is not supposed to chango
with ago, gets more and more sensitive, so that a fresh solution must
be made up each week, Mltrio acid is also able to change on storing,
both through nitrite fomation and vaporisation.
The nature of the spinal fluid is such that the precipitate ob
tained is limited in its possible composition. If It were less
abundant, it might bo any of a number of things, ?hs amount Is such


TABLE VI (CCHTINIM
IpKOCTT
alm
rar~~nra;
303
30?
308
309
310
311
312
g&toasRfc
m m m
m ** m
313 T
314 T
318 T
310 T
31? T
SIS T
319 T
320 T
321 T
322
323


f
f
+ 4*


51
5+ a yellow to colorless, and 6+ rod-brown.
With these solutions ns a guide, an eaonoly appeared to be taking
place. The dilutions nade were originally blue, definitely tet rare lent.
They then acquire a bluo-green color which natehes that of tri valent
vanadium. After a woek that oolor has left, and the solution is a
definite yellow color, pentavalent. Since blue and yellow will make
all shades of blue-green, green-yellow and yellow, a trivalent solution
was divided and one-half oxidised slowly. At a valence between four
and five the oxidised solution could not be distinguished from the tri-
valent control. Dilutions were stride of these more concentrated scln-
tione, and no color changa found. In coca* oases the oolor of an ion
in solution is dependent on the oocctmtratioa, but thia is not reported
or found true of vanadium, ftith vanadium solution the color depends
upon the valenoe state, not the concentration.
To prove conclusively the valence state of the sensitive dilution,
it was coropured In two reactions with a known solution in which the
valence of the vanadium was a fixture of i#t and 5+* Both solutions
are capable of forming a red color immediately when a drop of 3% hydro
gen peroxide is added. If the amount of %<){ is small enough both solu
tions will go only to a yellow color on shaking. When this test was
repeated with a solution of VClq the result was a blue solution.
The second experiment to show the valence state was the addition
of dilute HaOH. It there is pentavalont vanadium present it will
coagulate to the extent of darkening the solution when it i# neutral,


17
a new reaction for spinal finid, twine Collargol and HC1. The reac
tion is based on the fact that the protected silver chloride of the
collargol is precipitated by the addition of spinal fluid rich in glo
bulin. Hermann (69) states that the basis of the gold sol and collar-
gol reactions are different, and that for this reason differential
diagnosis should be possible tanking use of both reactions. Since both
are dependent on the globulin content of the fluid, the truth of this
statement is not apparent.
In 1?19, 3tinfield (?0) found that the action of spinal fluid on
a suspension of FbC^ depended on whether it xme pathologic, or not. lie
found that nomal fluid had no effect on the suspension, but that fluid
fro a syphilitic source gave varying amounts of agglutination and floc
culation. fo further report on this work is found.
Ouillain, Laroche and Leohe11a (71) introduced the use of bensoin
as a colloid substitute for gold in tb Lange test. Their tost is
similar to the first mastic test of Immanuel. They make the statement
that the results are not in accord with the albumin or cell count of the
fluid. Since the globulin ie the determining factor in this type of
reaction, such a statement is not too important} however, the cell count
should be higher in pathologic conditions than in ths normal. Wright
and Ransack (72) give an exhaustive treatment of the reaction with
bensoin, strongly advising the use of this colloid. Quart! (73) in 1928,
reports the use of gum myrrh in spinal fluid testing, but does not give


98
further* Only certain of the globulins are capable of being pre
cipitated.
The only doubtful results obtained with Kahn positive,
syphilitic spinal fluids occurred when bacterial contamination was
present, or some other agent had caused an almost complete floccu
lation of all the protein present* These aro noted in shipment six*
All save one of the doubtful reactions obtained with vanadium tetra
chloride solution came in this one shipment* Of the twenty-four
ampies, sixteen were In very bad shape* Four doubtful reactions
occurred with treated fluids of class XI*
We may say that this reaction is more sensitive in the de
tection of certain globulins than the Kahn or Wasseraaim reactions.
This is brought out clearly by a study of the results under the
class IX fluids* It may be that a group of globulins is generated by
syphilitic infection and that we arc testing for any one of this group.
Thus the specific globulin responsible for the Kahn test may dis
appear first, in which ease we are getting results because of non
specific globulin* It Is also possible that there is seme change in
the lipoid content of the spinal fluid which makes globulins more
easily prcclpitatable* The main part of the evidence points to the
fact that there are specific globulins, however*
The logical conclusion Is that the use of vanadium tetrachloride
in the following of treatment is indicated. Once nsurosyphills has
been diagnosed by a standard, proven diagnostic teat, the vanadium


SODIUM YAHAMOtU AS OOUOIBS
BoyXaaA (167) made an investigatloa of tbs reaction of vanadio
add and proteine. Ho deists this Is tbs first time tbs reaction of
lyephllie sol md protein has been studied. Bsaaoia, metis and gold
sols are all lyophobio, Ferric hydroxide (colloidal) Is lyophille,
hut has a different motion, since it produce* a eopredpitation. The
reason that Boyiaad selected vanadium lies in the f&ot that it is the
only one of the comae metals which ferae poly-acid*. In an add sol
ution the chief add formed is KhVgOjj. the statement is Made that
this and other vanadios pely-aoidt are positively barged. It is also
claimed that proteins are negatively barged in neutral and basic solu
tions, and that in add solutions the proteins become positively
charged.
In the experimental work the concentration of ajVc^ me one gram
atm per 300 liters, Various oddities were reached by the addition of
acetic add* It was found that albumin In a elution cf pH less than
4 precipitated* euglobulin in solutions sore add than pH 5*2 and globur*
Un la solutions of pH less than $ were precipitated, the amount of
euglobulin which a given amount of albumin dll protect is increased by
lowering the pH* Ho precipitation In this protection reaction dll tat
place at e pH of more than 4*
The difference between a globulin and an albumin Is dated as
being the difference in the number of barges it takes to neutralise
each and bangs the pH one unit in the vicinity of the iso-sleotri*


90
TABLE VIII
StMMHX OF TABULATED RJSStJLTS KIWI ScCPaO,- REACTIONS
Antigenic negatives, non-syphilitic ............... 55 X
Antigenic positives, syphilitic.................... 38 X
Antigenic negativas, treated, syphilitic............ %7 Z
Total spinal fluids tested ifch K*CrR0*.......... .^IsSST^
S*Qr*Q* results with X. S , 1
Percentage disagreement.................... absolute 8*6£
ECrcQ? results with X* ........ ...... 38 +
Percentage disagreement * *................ absolute . o .00#
SaCSpaO^ results with 15 +, *?
Percentage dlaagreessont..... absolute 81*5#


68
Vanadium Corporation of America, no thought was given to securing
a second saraplo until near the end of the experimental work. At the
time samples in the eleventh shipment were being examined, the ques
tion was raised as to whether the solution was just a chanoe result,
made from impure material* If a matter of ehanoe entered the making
of the solution, then a very difficult analysis, on almost a micro
scale, would be required to duplicate the solution* Examination of
the saturated solution gave hope that this quantitative analysis would
not be necessary for anything but vanadium. On evaporation of the sol
ution the only residue was VgO^. The solution gave a slight flame
test for ^a, and nothing else*
An order for solid VC^t was sent the Vanadium Corporation and
their reply was quite disheartening. Since there was no sale for
solid VCl^, thoy had discontinued the preparation and had no idea that
there was any available in the country. Enquiries were then sent
several houses dealing in rare chemicals, before attempting to prepare
any of the material* A small supply was finally located at the
A* P. Maekay Company, New York* An ounce of their VCl^ (solid) was
purchased* This was found to be praetieally insoluble In water) and
the small amount that went into Solution gave the green-yellow color
of a mixture of pentavaleat and tetravalent vanadium*
A mall sample of the material was placed la a crucible and
ignited* At a very low temperature there was a slight amount of
material volatilised* The volatile portion fumed in the air* Vanadium


m& of spwal rests
he exact composition of tha spinal fluid Tarta* frota aso to
o&e*. Such authorities as Soot and Baylor (196) and bright (157} girt
raporta not wholly in agreement as to the composition of the spinal
finid
ffce pial finid arises in the toroid plexos of the brain end
leaves through the venan sinuses of the brain fhs source 1* the blood
stream. fbe componente aro not In any given ratio compared to the
blood however, this foot* added to the faot that the hydrostatic
pressure is not great enough to account for the pressure of the fluid
leads to the conclusion that the colls in the toroid plexus met do
tone work, there is not as ultrafiltration of blood alone. Bicarbon
ate Is the only substance having exactly the mm percentage in the
blood as the spinal fluid.
Protein* constitute some 16*38 mg. percent of the normal spinal
fluid, these protein* are about equally divided between globulins and
albumins, the protein* do net appear to be able to paos the meningitic
filter, or. If they are able to pase the barrier, the exchange 1* ex*
oeedlngly slow, at will become apparent later, lecovetoo (158) found
two proteins in the spinal fluid, one of which behaved a* a globulin,
the other being soluble In distilled water, 'Today this latter protein
would be called an albumin.
For may years following Xsrcoveseoa work authorities claimed that
there was no globulin in the spinal fluid of a normal person, fhe teats


93
mmLn of ahai^hs' of m vox* soxmoiw
The color!->etrte method making use of sulfuric cid
ana' thro percent hydrogen peroxide we need. The color
standard ere mede from a pure sample of irnmnlum
mot a-vaasdote*
riginal sensitive solution*.* (X) 1*44X0 grasas VC1*A*
(g) 1.4410 grme ¥C1*/1*
(5) 1.3040 grams VC1*A*
(4) 1.4410 grass* VC1*A*
Results of the analysis of solution after the com
pletion of tests and rosioval of precipitate fey the
centrifuge *
Stssnle A... {1} 0.0750 gFaa VC1*A*
(2) 0.6750 grans VC1*A*
Sample B U) 0.0070 grama VCl*/!.
(2 0.6750 grms VC1*A.
By straight dilution these solutions should hare a
vanadium content of 0.7B08 grans ¥01* per liter* She dif-
fcreme la due to the adsorption by the flocculate*
suits of the vanadium analysis of the new solution
prepared, and of half-saturated V01*i
Hew solution. (1) £1.38 greens ¥01*A*
() £1.38 gram# ¥01*/1.
Half-saturated VOX* (1) 13.945 grams VC1*/1.
(£) 13.045 grams VC1*A-
Results of the vanadium analysis of sensitive dilution
prepared from now end old half-saturated stock solutions*
From new solution (X)*.........*il) 1.4410 grams VC1*A*
(2) 1.4410 grams ¥G1*A*
Fro old solution Of)**. .(1) 1.4410 grams VC&*A*


23
of NaCl aided the reaction* Cholesterol is used as a sensitizing agent.
Inactive serum is used, since heating the serum increases its reactivity.
Used on spinal fluid, the reaction is carried out as follows. The
fluid is inactivated, and then mixed with 2/3 its volume of saturated
(NH^)230^ solution and allowed to set 1 hour. The tubes aro then cen
trifuged and the supernatant liquid poured off, leaving the globulin.
To this globulin is added l/lG tho volm? of the original fluid of physio
logic salt solution. Thia concentrated globulin solution is divided into
three parts, two of which are used with antigen* tho third, for a blank.
The anticon ie added to physiologic salt solution, the tubes shaken and
then the globulin solution ie added. Shaking is continued for a time and
then the tubes are read, l oaitive tests ore shown by the presence of
flocculation, negative by the fact that the tubes remain opalascoot.
Epstein end Raul (103), as wo11 as others, have investigated the pre
cipitate, or flocculate, formed in precipitation reactions. They find
there is little evidence of any but included protein being present, and
that tho min pert is lipoid* Brack, concerning tho mechanism of these
tests, (104) states that they depend on the agglutination of finely
divided lipoid particles, this being stimulated by some constituents of
the luetic serum. Kahn, in & store modern work, (105), presents the view
that a lipoid due to the Spirochete is responsible for the production of
the globulin antibody, nnd that this globulin will then precipitate e like
lipoid, no miter whet tho source.
In 1926, several reactions were brought out. Kadisch (106)


6l
a period of two monthb.
The concentration of vanadium* as vanadium tetrachloride, vas
determined in the sensitive dilution. After a consideration of the
many methods for vanadium analysis, the colorimetric method iras chosen*
In the first place a very dilute solution as at hand, and this would
require the use of raicro-equipsiaat for gravimetric or volumetric
analysis, his equipment was not available, in the cocona piece, a
pure sample of ammonium mota-vanadate was on hand for preparation of
known colorimetric standards, A direct comparator with split field
viewing was used for this determination. An Hell go type instrument
would have been much moro efficient, but none was available,
A known weight of the KH^Vlh; was made to volume in a volumetric
flask, Into one cell of the comparator was placed 1 ml, of the
sensitive dilution, 0*5 ml, of is6 HgSQjj and 1 ml, of hydrogen
peroxide. Into the second cell wore placed equal amounts of the
KgiiGk and %0g, followed by a definite volume of the known solution,
made to 1 ml, with distilled water, *vhen the approximate strength
of the solution had been found a new unknown was used and the standard
mixed in the same order as the unknown, When a color match was at
last found, the determination was seen to be accurate to 35?£, since
It was reproducible to 0,01 ml, of the standard, The concentration of
VCijt was 1,U*10 grams per liter, + Later analyses verified this
result*
The reason for aa.e early difficulties was accidentally discovered


A STUDY OF SPINAL FLUIDS;
AQUEOUS VANADIUM TETRACHLORIDE
AND ITS USE IN THE DIAGNOSIS
OF SYPHILIS
By
ROBERT SUMMERS LEOPOLD
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
September, 1942


24
developed o rapid ^asaeraatm test, but this never gained any favorable
reaction frotn pathologists. Hosenfeld (107) made a study of the Influ
ence of spinal fluid on electrolytic precipitation of positivo and
negative sola, at a definite pH. He found that if the pH of the eplml
fluid were adjusted to 5*0, then the normal fluid would protect night-
blue against flocculation. He alee found that if there were
hyporalbuminooia, the nightblue reaction was sensitised, end that in
eningitia the result was flocculation. 0 the cthsr hand, hyperglo-
bulinosis had an opposite effect, the nightblue being held in solution
completely, Using nightblue, Hosenfeld was able to distinguish between
meningitis and syphilis or other affections producing an excess of glo
bulin.
Sleeper and Welker (108) investigated the method published by Bolts.
They proved that the use of acetic anhydride or a salt of glyoxalic acid
with concentrated HgSO^ and spinal fluid was merely a test for trypto
phane $ that la ms an indirect test for increased protein, the amount
of reagents being such that the color did not appear in the norml and
did appear at a value just post normal,
Tnecone (109,110) advised the use of 5$ KjCrjO-y with one drop of
CCl-j-COCfl per cc. He states that with spinal fluid this solution gives
a white turbid ring ai the interface, and that this ring is more intense
and permanent with syphilitic than normal fluids. He states that the
differences in intensity and structure of the turbidity allow a differ
ential diagnosis. In his second paper Treceno uays that the dichromts


ioU
23. X* fccobathal, s. XamunltAt*, £, 107-X2S (1910)
24* Brock and ild&ka 8* lamnitat*, £ 476*82 (X$ll)
25* Stvwi* X jfcftgit* Frcncalaej 3cb#-t* wch#ehr.* *j&, 395*6.
26. Str# SfttiMfo, ised. foebcebr., 51. 624*5 (17*1)
27. Sabbatini, SI renslere md.t 1911, no* 3*5*
28. aoguchi, J. lap. lied., 557-68 (1711)
29. tma*m*l, Alexandre* cad FVSedMtttft* Kuneh. sjed, Soohochr,, £1
1271-4 (1714)
30* 1*. Fopoff, 2* Sraramltat#. M 218*54 (1?U)
31, f. Veil 4 v, Kafka* lied, mau, 2* 13X4*8 (1911)
32* ffcuwa Serte# Beutech Z* Henrenlaatlk, £2 169-93
33* Cferl Unge, 2* Choswtherojsie, £ 44*77 (1912)
34* 7* Sarria,* nerratnl, '^echamtohr. ^r@e*tinghft, ££, 52
35. Kryja* Brandt end aras Wei*. kUn. feiMwfer 33, 1-6 (1920)
36. Glaeeoe and Senses* J. Sab. Clin* Ked*t £g* 534 (1940)
37. Sjset-in and Paul, Kolleid 2*, |£, 310*4 (19)
38. J* Sadek, Berl, kli, Veefcccbr.* S, §93*5 (1915)
39* *# tlletti5# Sentr. Bleebe, Biej&ys. 4, 813
40. Vile and Moke# J. Oat. Di,t ££, 607-23
41. J. A. Kolaer, J.A.H.A.* j&, 1461*3 (1915)
42. d* Brenfetibrenaar and * Seeks*, Meehea. Bull* £, 377*380
43. Alfred Garden, fi.Y* M#d. d.* §£, 343*5 (1915)
44. H. 4* Krebe, Bent. ned. ^ochecbr., £1, 1771 (1925)
45. v* 3ttO£ra, Bunch* mcd. VeefaMhr** 6£* 1212*3 (1915)


26
bath gats too hot, there ill be clearing in all tubes, but no sedimen
tation,
duller proposed a conglobation, or conglomeration reaction in 1926,
Mayaer (11?) found this was not as good as the Wassersa&nn, Sachs-Georgl
or Kienicks tests. He applied it to 635 sera, Troutor (118) also
found that this Muller reaction gave for too many false positive reac
tions to bo of use unless checked by the Weeseraann method. In 1930
Muller brought out a reaction known as the K.B.R. 11 or the Muller Bel-
lungs Roaktion (119), results on 937 spinal fluids give agreement
in 683 (positive and negativo) and 39 positive reactions on ^'nsseraann
negativo fluids, 15 negative reactions with Wassonnn positive fluids.
The antigen is a beef heart extract with a very heavy cholosterinization.
I'ullnr and Mnndula (120) explained that if to constant amounts of heart
extract 'mro added increasing amounts of cholesterol, tvo zones of
optimum precipitation resultedt these basing separated by a sons of clear
colloid solution. The balling reaction takes place in the zone with the
large concentration of cholesterol, the ordinary flocculation reactions
in tas sows of low cholesterol concentration. Filan (121) reports that
*
the false positive reactions of the M.8.R. II are easily distinguished
from the true positives, because they are a course precipitate and not
a true ball. This balling is in reality an ogglutinisation of the anti
gen emulsion, the stronger the reaction the store completely it goes Into
one ball, leaving a clear solution.
In 1929 and 1930 five reactions appeared, the first being the Tsu


39
Wood reaction and a positive spinal fluid meiion?
Malloy* &ahn and %etall (l6a) have notad that the antigenic
lipoids* la precipitation tests* are precipitated by the globulin*
Shis was pointed out long 'before by %stein and Paul (103). bat Mal
loy* Kisfca and ttestail did find of special interest vas that the glo
bulin was not changed in any way* It the globulin is Isolated and used
in & Kahn test, the precipitate my be removed id the globulin sol*
tion still retain its power to giro a gold sol or maetic test.
?ha presence at excess lipoids in the syphilitic spinal fluid has
been noted by Kaaser (163). these lipoids have sons effect on the col
loid teste* and this was proven by irradiation* If the spinal fluid is
treated with ultra-violet light and then a gold teet ran, the curve will
have shifted to the left. Irradiation is known to effect lipoid (l6h).
A part of the imperative reason for testing the spinal fluid is
understood from the above. Moore plainly states th# rest of the case
(£?) Moore and the mm working with Mm at the Johns Hopkins Hospital
have found that the nervous system is not 4mediately involved in
syphilie* She invasion of this part of the body is a later step, but
the serologic tests show the pressnoe of this invasion long before ay
symptoms appear* the first sign at involvement is the appearance of
mononulcoor lymphocytes la excessive numbers. She second Change is the
appearance of a positive globulin teet* lastly there is a positive
serologic reaction*
As these steps go on the treatment must be stepped up, since the


35
certain dilutions, active, syphilitic aeran should prevent this by
binding the antigen* The technique recommended ia to add a 3-$f*
JaCl solution to the active senes contained in several tubes. Increas
ing amounts of heart lipoid are added to the tubes. After a few hours
incubation at 37C. the cholesterol is added in the for* of 0,1$ solu
tion. To obtain optimum conditions a mixture of dilute HC1 and acetone
is then added. There is flocculation only in the negative sera. This
test is merely proposed and no results given as yet.
By no means all of the tests in the literature have been covered.
When eadtted a test did not embody any new or original idea. As an
example, we might have considered the use of shellac, ge* guale and
several oilier substances capable of forming colloidal solutions. To do
this would be a waste of time, once the mastic reactions have been
covered. Store recently there have been quite numerous publications on
tests which are all indistinguishable from the Dahr or Chsdi&k reaction.


20
referred to as a ekln test* If a skin dose of neoaraphenaa&ne ms
administered, several sen made the claim that it had the same action as
tuberculin. This ms found to bo a falso assumption, and the reaction
is no defined as the aggravation of pathologic processes following anti
syphilitic therapy. It is found, for example, that old cases with a
negative Wnsaenaann reaction may give a positive reaction after a heavy
dose of noenraphenesine. This does net often take place, and it is not
a diagnostic method. It le theraputic shock and to be avoided, according
to Hoore.
Tashiro and Levinson reported the use of a mixture of sulfo-sali-
.jrUc Mid a4 !sca2 in tte tosmBl* of vjbiiU In th. brcplnnl
fluid. KaSahara (80) found this test positive with all forms of meningitic
involvement. He states it le never specific. It should be noted that
sulfo-salicylic acid la used in the quantitative estimation of albumin
(8?).
In 1922, Kahn started publishing the resulte of his research, am!
his methods (90) However, this toot was not fully ready until 1925*
only the antigen being used before that time. Wang (91) proposed a test
using alcoholic extract of human heart at the same time. His results were
in good agreement with the Wasoenaann test, but the test never gained
any following. It land no special advantage over any of the other pre
cipitation reactions of that time.
Almost as soon as Kahn published directions for preparation of his
antigen, Kerrold (92, 93) mads use of it; first in e reaction with serum,


3
equal writ were possessed by the heart muscle of almost all mammals.
Hone of these was speciflot whether the animal had syphilis or not was
unimportant. Since the lira* of the original teat several modifications
in antigen have been the sole alterations* Wassenaann and Plant (3)
applied the complement fixation reaction to the spinal fluid with good
results and this is the first test for syphilis using spinal fluid.
L'ichaelis (4) in 1$07, announced the first precipitation test making
use of the some antigen as the Wassermann reaction, the patients sen
end saline. By experiment fee found that antigen and antibody, in a salt
dilution of the proper strength, gave a visable precipitate if allowed to
incubate for several hours. In an article (5) proceeding the announce*
sent of his test, Ulehaelis proved experimentally that the Waeeenaann
reaction probably used a non-specific antigen) therefore, hie own test is
bound by the earns limit, in spite of his claims for its specificity.
In the course of the work on antigens. Forges & Staler (6), working
under the direction of Wseermann, found that pure lecithin could be used
in place of the antigen, and complement would still be fixed. Along the
earns line, was the discovery of Fleischaann (7) that cholesterol gave
even better results than lecithin. Varney (8} also made use of an anti
genic substance in a precipitation reaction. He found that the addition
of a few drops of If taurine to 1 cc. of the patient's sens gave a preci
pitate in the same manner as the Michaelis test end did not require as
long a time of incubation. However, taurine was not effective enough to
ever be of much use? It led to too many false negative reactions.


12
Hecht (46) worked out an antigen for use in both a precipitation
reaction and Wassons nn reaction. When diluted for the precipitation
reaction it wae turbid, but allowed the passage of light. The nenas
and ccEtplomnt were mixed before addition of the antigen and allowed to
react for a tints. After the addition of antigen the tubes were incubated
for eight hours. A positive reaction was shown by the foraation of a
heavy precipitate; in a negative the antigen emulsion resained unchanged.
Hecht-Gradwohl is the muse given a reaction first proposed by Grad-
wohl (47) in 1914, It as a heraolyain reaction and was modified by Bruce
(4E) four years lator. It is applicable in about 98$ of the patients
tested, the others not having sufficient hesaolytic power in their senas.
In the first place the hesaolytic index of the patients sen is found*
The units of the hemolytic index are volume of standard sheep cell sus
pension heiaolyxed psr unit volume of sema. Mow l/2 this amount of sheep
cells is added to a mixture of Hoguchi antigen and sen. The tube is
incubated at 37C. for an hour. If the reaction is negative, hemolysis
is usually templets at the end of this time. The tubes are kept over
night in the icebox and read the following morning,
Emanuel (49), finding the gold sol of Lange hard to prepare in a
reproducible Manner, proposed the use of a colloidal solution of gum
mstic. Five tubes were set up with the colloid and to the first four
were added graduated amounts of spinal fluid, the fifth receiving only
distilled water* If the reaction was positive the mastic in all five
tubes precipitated, if negativo only that in the blank precipitated.


102
globulina appear before the 8peai.fio ones. The solution should be
of certain use in suoh eases*


58
that this whole box of fluid* should have been discorded due to bac
terial contamination. However, trey were tested, and the results are
not so far out of line es to warrant striking then froa the record.
This sens series of samples was tested with dichromate end HSOjf.
The eight positive syphilitic and the eight negativo nca-ryphilitic
fluids gave perfect resulte. A deviation from previous experience was
found in the class II steeples. Three of the eight were negative, four
were doubtful and only one was a true positive. The three negatives
and one doubtful corresponded to the four negative results obtained
with Wife.
At this tiste an attempt was made to prepare a sensitive dilution
of VClfe from the original saturated solution. Ho reason was seen for
not using this, mixed with an equal volume of water, since the half-
saturated solution in use had been made from it In the sane Banner.
The sensitive dilution was being made now in smaller amounts, the usual
technique being to take 6 el. of distilled water, add to it 10 drops
of one-half saturated VClfe solution and then sensitise by the addition
of an old (oxidised} sensitive dilution. Trial testing still was used
to find the specifio amount of old dilution to be added.
To make use of the saturated solution of Wife, 5 drops each of
the solution and distilled water were added to 6 ail. distilled water,
ithile the fact that drops arc not accurate for measurement wee
recognised, it was supposed that this would be accurate enough for the
purpose. Although several attempts were made to sensitise solutions


21
later In one with spiral fluid* Herrold diluted the antigen It12 and
added toma fro a pipette so as to for two layara, A white ring in
the serum la a positive reaction. With spinal fluid, he found it neces
sary to concentrate the globulin fraction, using half saturation with
After removal of the liquid, the globulin was dissolved in
a snail velum of physiologic saline and then tested as above* While
the results were not too good, Kahn did not successfully run a spinal
fluid until after he ronde uoe of its concentration by this method*
Bruch (%) in 1922, returned to precipitation tests* He nade use
of a new antigen, heavy concentration of salt, and centrifugation* Hie
antigen is non-choleeteriaised extract of beef heart. Inactivated soma
(0,2 cc.) is added to 0,8 cc, of 10$ HsgSO^ solution and then 0.2 cc, of
antigen emulsion added* The tubes are centrifuged 20 sinutes. An insoluble
precipitate is positive; e negative serum also gives a precipitate, but
this goes back into solution on shaking. Teicfcmnn (95) notes that the
reaction must be run on positive and negative sera and gives tire method.
Becker (96) tested 12,000 blood specimens with this method, and found
almost perfect agreement between it and the Wassermim test.
Schwarts and Orunewald (97) wade use of colloidal silicon dioxide In
the examination of spinal fluid* A solution of S102 (0.25$) 1 used. Ten
drope of this are mixed with 1 cc. of spiral fluid, and it is noted that
luetic fluids flocculate in a few hours, or easily on heating* A second
method proposed is the dropsies addition of spiral fluid to 1 cc. of the
reagent. Fluids froa parcels, cerebral lues, and tabes flocculate in


fid* dissertation ms prepared under the direction of the Chain**
of the candidate*# Supervisory Committee end has been approved by all
member* of the Ccenltteo It tea submitted to the Graduate Council
and me approved ae partial fulfilment of the requirements for the
degree of Doctor of Philosophy*
Date
S5SH
SUPERVISORY COSKITTSB*
Chairman


66
fluid so precipitate was found* Hevartheleas, tho si ido toot* ware
showing falso positive reactions. If this was an excess albumin, it
should bo soluble in water. For sane tine it had been known that
water did not ohange the aspect of a true positive reaction, once it
had taken place. Thus a modification was made in the procedure. To
0.1 n&. of the spinal fluid was added 0.1 ral. of the sensitive dilu
tion in the ssne manner as before. If there was then floeoulatloa,
this was allowed to set for a minute and 0,2 ml. of distilled water
added. After stirring and allowing another minute to pass, the slide
was examined. Flocculation was persistent only in the positive fluids.
All fluids from number 13k were run microscopically by this method.
One fluid, marked non-syphilitic, ^assorraaim negative, gave a
true positive reaction with vanadium tetrachloride. The fact that the
precipitate was a mixture of globulin and VpOq was verified, While
the amount of globulin got by centrifuging was pite all, its
identity was unquestionably proves. The cense of this reaction is
discussed later.
Although this phase of tho investigation covers sons 92 spinal
fluids, only ?0 of these wars of sufficient volume to allow pH
determinations. The decision was made at the outset that no special
equipment be purchased for pH work. It is possible to take a pH read
ing from each slide with the proper equipment. After the probable er
ror of the mean had boon calculated it waa seen that tho method used
had been satisfactory in all details* The large deviation between


67
individual ampio i not hard to understand, Each fluid hue a dif
ferent alkaline reserve and original pS, While there ia little varia
tion at the time the fluids are drawn, standing causes then to
become alkaline. This takes place spontaneously, but depends on the
tightness of the stopper, heat and i# nature of the individual fluid*
Since all work of this type is statistical, large deviations are not
unexpected.
For the preparation of tide original saturated solution of VC1J4, a
sample which had boon on hand at this laboratory for years was used.
This was the so called solid salt, VC1 is in reality a red liquid,
highly deliquescent, Mo literature reference could be found to the
material sold under the name Vanadium Tetrachloride, C,P. (solid).
The original sample of salt was taken by a member of the inorganic
preparations class for purification. This sample was a dark blue-green
mass of non-crystal line material. It looked highly impure and this
student had the idea that it could be purified. At that time the use
of this salt had just been begun, and there was no reason to prevent
such a proposed undertaking. In the course of the purification all the
material was ruined. This unfortunate occurrence left the laboratory
with 25 ml, of the saturated solution and a like amount of half-
saturated solution. Over 250 fluids were examined before mere VClj,
was obtained, and oven then thorn was sufficient solution on band to
run about 100 simples.
Since the solid salt, VClj,, was listed in the catalogue of the


96
report that the us of diohrotvate and nitric acid id not of impor
tance In 'tee diagnosis of syphilis* Hoither Is it of any particular
value in following the course of treatment
hith respect to an aqueous solution of vanadium tetrachloride,
ire are able to moke several statements. A solution containing around
13 grans of VC lit Pr litsr has a definite sensitivity toward the glo
bulins of syphilitic spinal fluids if it is dilutsd to lJbUlO grams
per liter and partially oxidised. The solution containing 13 graos
per liter is slowly oxidised by atmospheric oxygen. Poly-vanadio
acids result from this oxidation. This solution has a definite tins
of mixing which leads to ssnsitivity and not the foraation of vanadio
acid of definite partile sise, sinos most of the oxidised vanadium
is formed after dilution to a concentration of 1*14*10 g. VC1}¡ per
liter.
The partially oxidised solution of vanadiusi tetrachloride content
corresponding to l*l*UlO grams per liter Bust be nade from the solution
with a content of 13 grams VCl^ per liter* The oxidation may be
carried out In either of two methods. By standing with atmospheric
oxygon over It for twelve hours, the content of pentavalent vanadium
is raised to the proper level. A more rapid method, and more satis
factory method, consists of allowing a solution with vanadium tetra
chloride content equal to 1*14*10 grams per liter to go completely to
the pent aval on t state* A definite volume Is then added from this solu
tion to a definite volume of freshly wade solution having the same
vanadium content, in this way a sensitive solution results without


109
138. Bauer and Woeaeek, Had. Klin., &, 107$ (1935)
139. Laughlen, Cenad. K. A. J., 3, W U935)
140. Moor*, J. Lab. Clin. Med., £4, 973 (1938)
v
141. Robinson end Stroud, 10|, 1170 (1937)
142. Thomason, Jord. Med., 8, 1972 (1940)j Abstr.i Chora. Abate., 21*
3710 (1941)
*
143. A. Vernos, Coajjrfc, rend., 203. $84 (193$)
144. Tasn and Takagi, T* Orient. Med*, 34 329 (1941)* Abetr.t Chea.
Abete. 21* 7440 (1941)
145* F. Ryt*, J, Lab. Clin, Med., gg, 82 (193$)
14$. P. Dahr, Beut. raed, Tochachr,, 3O, 94 (1934)
147. P* Dahr, Munch, raed, Woehsohr., &, 1723 (1934)
148. J. 3chuck, Arch. Gymkcl., JO, $16-25 (1940)
149. Porto and de Britto 0 Silva, Rev. Assoc* fhulista Med. (Sao Paulo),
1$* 87-98 (1940)
150. S. Ide and T. Ida, Fr, 814,052, June 14, 1937
151. van Kverdingen, Chora, hentr,, 1940 I. 2354
152. Rosegger, Klin, Woehochr., J, 501 (1938)
153* Kaehucharov, 2, Ian&aiiate*, 22. 410 (1938)
154. A. M&xxini, to. J. Clin. Path., J, 163 (1939)
155* Frohlieh, Arch, Deraratel. Syphilis, 180, 109 (1940)
15$. Beet and Tfeylor, Jh& &Z£*k9tei&S&l fijflU g£ Pmctlcj^ SUe
lian and Wilkins, Baltimore, Md (1940)
157. Sorason Wright, Applied Physiology. 7th Ed., Oxford Chiversity Press,
Hew York, 0940T
158. H, Iscoveaeo, Compt, rend. eoc. biol., 6g, 181-2 (190$)


22
that order. This method was found by Kleiner to be $2^ as effective as
that of Lange* and he recommends it for rapid orientation (98)*
Helia (99) found that pathologic spinal fluid, especially luetic,
formed foam when mixed with an equal volume of beneene or toluene. This
is presented as evidence of the colloidal change in the spinal fluid
during disease.
An investigation of the passage of Uranin (the sodium salt of flores*
coin) into the spinal fluid, following its injection, or ingestion, was
made by enroll (100). It ws found that the color of the spinal fluid
was related in some of the cases to the pathology, in others there was no
correlation.
In 1921 Kahn (101) published an article on the effect of heat on the
reactivity of antibodies. At this time he apparently found that heat had
a deleterious effect. At 5&C, he found a loss of $f. in strong positive
sera, and $0?. in weak positives. He also stated that in a great many cases
complete destruction resulted fro heating to 62C. In his book (102)
Kahn reports that heat lias an enhancing value, helping to promote the
reaction of sera. He explains all the reasoning that he and his co-work
ers lave used la the development of the Kahn test* In the flret place a
concentrated antigen is used to give better reactions with weakly positive
sera. Secondly, this antigen is made into an unstable emulsin, the more
unstable this is, the faster it reacts* in the third place shaking of the
antigen emulsion and the sertas is introduced, since this also speeds the
reaction. Salt solution is added because Kahn found the proper concentration


53
la euoh that oxidation la rapid In diluto solution. A stabilised solu
tion was diluted to the turn vanadium content and showed no oxidation
after three months.
After finding the mixture of valonee to be the sensitising prin
ciple, there was no need of making a dilution ten hours before use.
The old, overly oxidised solution was saved and added book to the
fresh solution to produce the mixture at the time it was needed.
The fourteen pooled spinal fluida in the first shipment were
tested with the dlchromate mid nitric add method. Class X fluids
were all positive in their reaction. Class XX fluids were all posi
tive or doubtful in their reaction. Class XII, whieh should have all
been negative, gave all negative results, with one exception. The
false positive at this point, coupled with the fact that Taoeone never
got any recognition for the dichromate reaction, all but led to the
stopping of experimentation on this method. It was decided that a
total of 100 tests would be made with this technique, and the results
cheeked to seo whether It should be continued.
On receipt of a second box of fourteen pooled spinal fluids, the
samples were cheeked with a sensitive dilution of VClJj. The dilution
was prepared in the following manner 1) two class X and two class XXI
fluids were selected at randoaj 2) to 12 ml. of distilled water was
added 20 drops of half-saturated VCl^ solutionj 3) an old dilution of
the same strength was added five drops at a time to the fresh dilution;
U) after each addition of five drops of old solution a test was run on


55
characteristic water white, but a turbid white.
The third shipment of pial fluids received contained eight
fluids in each class, all individual samples. The shipment was de
livered late in the afternoon, and was kept on lee overnight. In the
wonting an attempt was mode to prepare a sensitive dilution of VCl|j.
The sane method as used before was used, with the exception that only
six ml. of HpO and 10 drops of half-saturated VCljt were taken to make
tho fresh dilution. Both ciase XII samples taken for testing the solu
tion gave a great deal of trouble. A sensitive dilution was not suc
cessfully prepared. The fluids were left on the desk until late in
the afternoon.
At this time four other samples were selected for testing the
sensitive dilution. The afternoon trial at sensitization of a VClj,
dilution was successful. He trouble was encountered in any way, After
the soaoitivo dilution was obtained, all tho fluids were tested. All
class X fluids agreed. Class XI fluids gave five positive and three
negative reactions. Class XXX fluids gave seven negative and one posi
tive reaction. This positive was one of the fluids used in the morning
attempt to sensitiza VClj, and accounted for part of the trouble, at
least. It receive* treatment in the section on Discussion of Results,
By the dichromate method all elass X and XI fluids were positive,
as well as the false positive which carao out of class XXX with VCljt,
All ether class III fluids were negative,
ifith fresh fluids on hand it was decided to chock all the metal


16
extract, balsa of tolu me added, and then the turbid emulsion placed
in tubes with the inactive serna. If the reaction were positive, the
turbidity Increased, end mil floccules appeared. Although the H.T.R*
only required eos eight hours to go to completion, even Heinicke
admitted that it was not ns good for diagnostic work aa hie original
method.
Still searching for a better method, Keinicke (64) propoeed the
Welnicko Clearing Reaction (H.K.R. from ti German, or Keinleke III
reaction) in 1929. In this reaction he made use of a beef heart, lipoid
antigon and added three times as much tolu balsam as in the U.T.R.
Just prior to use, the extract of beef heart and the baleara are heated
to 45C, and 1C volumes of IfeCl solution at the samo temperature
are added, rapid mixing being essential, hen the mixture cools to 37C*
active serum la added, A positive serum results in a precipitation of
the colloid, leaving a clear solution. The colloid is not changed by
a negative, non-syphilitic sen. This last test of Heinicke is apparent
ly his boot, but it is not used in this country to any extent.
£llingr (65,66) tried a patented germicidal colloid of silver for
studying the spinal fluid. The preparation, Collargol, is made by a
secret patent process, but Alekseovski (67) prepared a duplicate material.
He ms engaged in breaking Goman patents for the Russian government.
He suggests that the final solution is not a colloid at all, but a mix
ture of complex silver salts of the amine acids in a dispersed state.
Rubeling (68) in 1938, ten years after Ellinger, published what he called


6o
saturated solution was again used to make a dilution, and again it was
imposslbla to sensitise it* If one discounts the fact that a definite
nixing timo is required* the only alternative is partila sise* This
could not enter in, since there is no polyvanadic acid domed until
after oxidation to the peatavalont state has taken place* Most, if
indeed not all, of the peatavalont vanadium is added in the sensitiza
tion, and this has be*m the same in each case*
Several questions as to technique and concentration were taken
up* The use of droppers for measurements was decidedly a thing to be
avoided, sc 25 pipettes, 1 tal., graduated in 0*1 and 0*01 mis. were
then employed* Approximately equal volumes of spinal fluid und sensi
tive dilution of VClj, hud been used in all tos ting, and it was found
that exactly 0*1 ml* of each gave a test which was easy to read ana
handle* Tills volume relation was used throughout the remainder of the
work* A separate, clean pipette was used for each sample* After us,
each pipette was cleaned with cleaning solution and rinsed six times
with distilled water* After rinsing, the pipettes were dried by draw
ing worn air thmifjh thorn with a water pump.
In order to save time, the ratio of oxidised (old) solution
needed to sensitise a given volume of fresh solution was determined*
Up to this tita sensitisation had required upwards of an hour each time
tests were to be run. Trial testing was used to determina the ratio of
old to frssh solution, and it was found to be 1*5 This ratio of 1 ml*
of old dilution to 5 ml* of the freshly made was used with success for


Uo
meningeal mt allow only a small part of bo total medication to
pass Into tho spinal fluid. It Is to bo noted that be disappearance
of be manifestation* of aeorosyphUis are In the same order as their
appearance, the cells and globulin disappear easily, rea under an in*
coraplete treatment* fhe ssrologic reaction persiste, tinder serologic
reaction in this ase is included the gold test, fhis would tend to
disprove any theories as to the meehanisa of the gold test, however,
it nay depend on a certain pert ef the global in exceeding lte normal
amount, not all the globulin,
Kahn, Kline And any other workers have found that precipitation
tests do not reverse as rapidly as complement fixation tests. Although,
in this connection, neither do the precipitation tests bacons fast, as
does tho baeserwann* JJy fast is meant that no amount of treatment will
cause a reversal, own though the disease is clinically cured. Host
authorities agree that a precipitation test, a complement fixation test
and a colloid test should be made with spinal fluids for diagnosis and
following treatment.
In summarising this section, we hare the following consideration}
1) rarely Is the spinel fluid test a primary diagnostic test, if the
physician is given a proper chance to follow the disease, fhe spinal
fluid tests how asmas tissue involvement long before lesions are
evident and should be periodically continued as long * the distas#
exists, 2) there is no true antibody formed, but a seal-specific glo
bulin la its placo, fhe tests depend on globulin-lipoid combinations I


62
during the course of routine tasting# Fluids wore being usad which
hod .just boon renovad from the ice-box, and th eeneltive dilution,
mad by a definite volume relationship gave thro falso positiva re
actions in succession. In the routine work, throe or four slides wore
lined up on the desk, spinal fluid from an equal number of samples
pipetted onto the loft hand side of the slide and dilution pipetted
onto the right hand side, ^he two liquid* were then drawn together
with a email glass stirring rod, stirred end the slides rotated by
hand for thirty eeeonde. As each slide was mixed, it was replaced on
th desk and allowed to remain there a few minutos before reading. In
e majority of the positive reactions th flocculation is easily seen
in an indirect light with the naked eye as soon as mixing is complete.
In the Instance referred to above three Kahn negative samples
had been so treated, and all gave a visible flocculation, the work was
being done under a desk lamp, tilted close to th top of the desk to
provide indirect light, tfhile starting tc make up a second dilution,
believing an error had been made, these elides wers left under this
light. Ihile making a now sensitive dilution, it was noted that the
false positives had apparently cleared, Microscopic examination con
firmed this fact.
In order to bo satisfied on this matter, slides were made with
spinel fluid at 10C, end 25C, The reaction of flocculation took
place In positive and some negative fluids alike et lOOC. Only Kahn
or Wasscrmann fluids gave this reaotion at 25C. It was found that


37
used for globulin depond on the ratio of globulin to albumin, and what
these non have found is that the globulin in a normal fluid it not de-
tooted by tho methods they used. Tho ratio of globulin to albumin in
a normal fluid is such that there is no precipitation under tho condi
tions of the tests. Therefore, when it is stated that a traee of glo
bulin is pathologic, tho moaning 1st the globulin-albumin ratio for
oomplote protection is passed and a part of tho globulin thrown down,
tho laok of protection being pathologic. The more modern authors reoog-
nise this fast.
The nature of tho globulin found in syphilis is somewhat different
from that found in the normal ease, Kahn glees a eery olear picture of
this fact (10$), He first explains that tho antigenic substance in
syphilis is most likely a lipoid-protein combination, Tho lipoid is
regarded as an incomplete antigen (called hapten). If it wore possible
to secure a pure protein from the spirochete, this would probably glee
tauoh noro satisfactory tests.
The antigen of syphilis is not capable of causing true antibody
fo mat ion, but glees rise to two globulina. The first of these is in
soluble and remains in tho cells, the second is soluble and is washed
into the blood stream. This soluble globulin is the antibody whi oh
reacts with tho lipoids in serologic tests. The origin of the globulin-
exciting lipoids is uncertain. They may ooae from either tho splro-
ohetea or the tissues of the host, In any case It is necessary that the
spirochetes be present for these lipoids to appear, Kahn claims that


77
TABLE I
THE RESULTS OF V01* AMD %8r*** REACTIONS
OH SPIHAL FLUIDS
1pffiTfCTT~
!f?l.l.P ....
ftassemsim
reaction
**liT
reaction ...
...JWHULtri...
1 (fooled)
+\
4
4
8 (pooled)
*
8
4*
4 ** 4*
4
4
4*
4 *
4
3
**
!
4
6
*
1 4#

7

**
*
a
T*
w
4
8
T
**
4
10
T
*
4
11
T
4
12
T
4 4 4
4
13
4
4
4
14

4
4
IS
4
4
4
IS
4 4
4
17
*
44
m
18
a*
**

ID
**
4*
£0
*
**
44
£1
f
4
4
T
m

£3
T
4
Jt
24
T
**
4*
£3
4
4
4
£S
4
4
4
£7
4
" 4
4
m
4 ;'
. 4
4
£9
4
4
4
30
4
4
4
*+ a syphilitic, *&ssersaaa positive#
- no-syphilitic, wg^swnaami negative*
aMultiple teste, mt a ol$n of strength of reaction.
*T a treated syphilitic, Waecemaan negative.
doubtful.


TABLE OP CONTENTS
Page
Aeknwledgaottts * 11
Introduction.......... v
Tests Proposed by Other Authors............................... 1
The Importance of Spinal Fluids Tests.... 36
Sodium Vanadate and Colloids....1*2
Experimental. 1*5
The Preparation of a Sensitive Vanadium Solution for
Syphilis Detection in Spinal Fluids............ 75
Results
Table I. The Results of VCl}, and KpCr^Oy-HHO*
P.eaotions on Spinal Fluids... 77
Tabls H. Results of VClf^ and K^CrpOy-HNO^
Reactions with Spinal Fluids Showing Ageing Effect
on 1/2 Saturated VClj,. 90
Table III* Results of VCl^ and KjjCrgO^-IIHOj| Reactions
with Spinal Fluids.....*....... 31
Table IV* Results of Vdj? Reactions with Spinal
Fluids. Showing pH and Composition of Precipitate
if l omad ... 83
Table V* Results of the VClj. Reaction with Spinal
Fluids.. 86
Table VI. Results of VCl], Reactions with Spinal
Fluido. Showing Reproducibility of Solutions. 87
Table VII. The VClj, Reaction with Spinal Fluids
from Unknown Diseases. 89
Table VIII* Summary of Tabulated Results with
KpCr^Or-uO^ Reactions 90
Table XX* Summary of Tabulated Results with
VClj( Reactions.*.. 91
lii


hotter than the ovar-al! resulte, there is a lack of sensitivity*
Seri aleo tried to doteminn albumin and globulin in the same
sample by hanging the pi! of the vanadio acid-spinal fluid mixture,
This is admitted to be a failure.
The pH of the no mal spinal fluid is 7*35 to ?*& Thi* changes
on standing to a value between 3*0 and 3,1, The alkalinity is gener
ated by the large carbonate reserve found in the spinal fluid (156,
157). In disease the same pH values are found with one exception,
aeningocoeous meningitis* In meningococcus meningitis the pH is
7&7*U on withdrawal and the value Is not changed on standing (169),
The iso-elestrtc point depends on the salt concentration and lies
between pH h and pH 5 for the meat part.


2
and is carried out in a 56C* water bath for 30 minutes. It destroys the
patients complement. The second step is the addition of a known amount of
complement. This complement is secured fro the sent of a guinea pig, and
in reality is just the serum of that animal, titrated in such a way as to
know how much antigen it will take per unit of volume. The third step is
the addition of the antigen. The tube is then placed in a 37C. water
bath for thirty minutes. At the samo time inactivated, corpuscle-immune
rabbit serum and a suspension of sheep cells is alto put into a tubs and
incubated. Equivalent quantities are used in this system, as determined
by titration. After both tubes have been in the incubation bath for thirty
minutes they are removed and the hemolytic system added to the antigen-
antibody tube. Incubation is again required for a length of timo equal to
the first period. At the end of this second incubation the tubs is removed
and the result read. If there is a complete lack of hemolysis then the
reaction is positive. On the other hand, a complete hemolysis indicates a
negative reaction, the complement having been unaffected. In between
these limits the amount of positivenees is estimated by the operator.
It Is obvious that several blanks must be run cn each series of tests
and that In order to be certain of the result, a known positive and nega
tive serum must also be included. In addition to these minor difficulties
must be stated the fact that the antigen which is specific for syphilis has
never been found. Wasseraann and his co-workers used a saline extract of
the liver of syphilitic fetus, thinking that this did contain the actual
antigen. It did not. later it was found that antigenic substances of


TASU XII (COHTIHOED)
Spinal fluid lialn V01* .aOraOrvoo
aiBBl9 a*ar rocotlon ruaotlon raaotlon
147
iyi
(Ooatnwoa)
4
*
140
ir
*
*
140
T
4

150
T

*
151
T
4

15B
T
m

155
T
m
#
154
f
m

158
4
4-
*
156
4
+

157
4*
4
#
158
4
4

159
4
4
*
160
4
4

161
4
4

im

4



76
of the mixturo. The two solutions oro mixed with a small gloss rod
end observed under the low power of the microscope.
To each slide on which is deposited a floeoulent precipitate is
added 0*2 tal. of distilled water. At the end of a minute each of
these slides is again observed.
Of the mixtures of fresh and old vanadium dilutions, one should
give a very sensitive reaction, differentiating the Kahn positive
epinal fluids by flocculation from the %hn negative spinal fluids
which do not flocculate.
The pH of the sensitive mixture should be 2.6-2.7* The ratio of
dilutions which is most sensitive may be used for at least a month
without being re-determined. The more concentrated solution (13-11*
g. VCl|t per liter) should be stable for three to six months if kept
tightly stoppered and in a oool place. Rubber is not recommended for
stoppering vanadium solutions it has some catalytic properties for
their oxidation apparently.


A STUDY OF SPINAL FLUIDS;
AQUEOUS VANADIUM TETRACHLORIDE
AND ITS USE IN THE DIAGNOSIS
OF SYPHILIS
By
ROBERT SUMMERS LEOPOLD
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
September, 1942


o

2J
f.0
r
nil
O
a
147365

ACKSffLKIXMZKTB
The author wishes to exproa* hie gratitud* to
Dr. C. 0. Pollard, without whoso guidance thi* invest!-
got ion would have boon impossible, It was at the sug
gestion of Dr* Pollard that this work was begun and
carried on to completion*
Th author would also like to give credit to
Dr8. Therrel1 and Heudlger of the Florida State Hospital,
Dr. Lcmglno of the Georgia State Hospital for the Insane,
Dr* J. 0. Patterson and the Kahn Staff of the Florida
State Board of Health, Drs. L, Y. Dyronforth and H, Mar
shall Taylor of Jacksonville, and Dr* K. D* Thompson of
the State Tuberculosis Sanatorium, all for their
splendid cooperation,
The author's grateful thanks also go to the staff
of the Department of Chemistry of the University, Their
suggestions on equipment and methods have proven
invaluable.
11

TABLE OP CONTENTS
Page
Aeknwledgaottts * 11
Introduction.......... v
Tests Proposed by Other Authors............................... 1
The Importance of Spinal Fluids Tests.... 36
Sodium Vanadate and Colloids....1*2
Experimental. 1*5
The Preparation of a Sensitive Vanadium Solution for
Syphilis Detection in Spinal Fluids............ 75
Results
Table I. The Results of VCl}, and KpCr^Oy-HHO*
P.eaotions on Spinal Fluids... 77
Tabls H. Results of VClf^ and K^CrpOy-HNO^
Reactions with Spinal Fluids Showing Ageing Effect
on 1/2 Saturated VClj,. 90
Table III* Results of VCl^ and KjjCrgO^-IIHOj| Reactions
with Spinal Fluids.....*....... 31
Table IV* Results of Vdj? Reactions with Spinal
Fluids. Showing pH and Composition of Precipitate
if l omad ... 83
Table V* Results of the VClj. Reaction with Spinal
Fluids.. 86
Table VI. Results of VCl], Reactions with Spinal
Fluido. Showing Reproducibility of Solutions. 87
Table VII. The VClj, Reaction with Spinal Fluids
from Unknown Diseases. 89
Table VIII* Summary of Tabulated Results with
KpCr^Or-uO^ Reactions 90
Table XX* Summary of Tabulated Results with
VClj( Reactions.*.. 91
lii

table of Content (Continued)
Page
table X. Summary of tabulated Result on the pH
of the VClfj-Spinal Fluid Mixtures *..**.. 98
fable XI* Result of Analyses of the Various VCljt
Solutions..***. 99
Di semes! on of Results and Conclusions*.*.....................* 95
Summary of Conclusions**** 101
Bibliography**.105
iv

INTRODUCTION
When It was proposed that the author undertake the development
of an original test for syphilis in the spinal fluid the difficulties
of supply were not fully realized. This obstacle has been overese to
the best of our ability.
An investigation of this type ia almost invariably carried out in
a large hospital, or very near one. The clinical and laboratory evi
dence of each ease is examined minutely. In this manner the true
value of a reaction is determined. The new reaction my be best under
certain conditions which lead to failures with all previously known
reactions. At any rate the value of a teat should be determined by
both the laboratory and medicll staffs.
In direct contrast to the norm! state of affairs, wo were forced
to use a source of spinal fluid samples ccmo two hundred miles distant.
The samples were shipped by express and their time in transit was a
minimum of twenty-four hours. The data furnished with each sample
included only the result of the Kahn or lassenaann test and the clini
cal presence or absence of syphilis.
The fact that Florida does not have a medical college necessitated
visiting other libraries for a study of the literature.
Prior to 1906 (1), all but one of the leading syphilologists in
this country believed the appearance of secondary lesions to be the
only means of diagnosing the disease of syphilis. In I906, Waasers&nn,

Neisser and Bruck (2), made one of the two most important advances
in the ultimate defeat of syphilis. The other was the later discovery
of salvarsan by Ehrlich.
Although the theory that was proposed by Wassermann et al. was
not in accord with the modern work, the test itself has undergone only
minor changes since its introduction. Despite the time required to
make a Wassermann test, it is still one of the leading reactions
for the diagnosis of syphilis.
vi

TESTS PROPOSED BY DIFFSREKT AUTHORS
FOR THE DIAGNOSIS OF SYPHILIS
In the coapleiaont fixation teat, or Whsaenrann test (2), the
mechanism ie postulated to be me follows 1) An antibody is built
up in some concentration in the patient's system. 2} Tills antibody
sill combine with and destroy some specific part of the affecting
organism* which is called antigen. 3) In order for the antibody-anti
gen reaction to take place it is necessary that a reaction promoter, or
linking agent, bo present this agent is called complement. 4) If the
antigen be added to a sample of serum which has no complement* and then
just enough of this complement is added for that antigen to react then
proving the presence of the unaffected complement would prove the
absence of antibody. 5) To prove the presence or absence of this
bridging material it is necessary that we have a cm other reaction
which aleo requires complement. Such a reaction ie the hemolysis of
foreign blood corpuscles by the sertas of any corpuacle-tesunc animal.
6) To get ouch a system, a rabbit ie injected with the red blood cor
puscles of the sheep, so that his blood will contain an antibody specif ie
for the hemolysis of sheep corpuscles. ?) Complement is the molabile;
that is, heating it at 56C. will destroy it| on the other hand, anti
body is not so destroyed and ie called thonaoatabil.
With these facts in mind, it ie easy to visualise the Wesesraann
Test for syphilis. Hie first step is to heat the patient's blood serum,
the cells having been removed. This heating is known as inactivation

2
and is carried out in a 56C* water bath for 30 minutes. It destroys the
patients complement. The second step is the addition of a known amount of
complement. This complement is secured fro the sent of a guinea pig, and
in reality is just the serum of that animal, titrated in such a way as to
know how much antigen it will take per unit of volume. The third step is
the addition of the antigen. The tube is then placed in a 37C. water
bath for thirty minutes. At the samo time inactivated, corpuscle-immune
rabbit serum and a suspension of sheep cells is alto put into a tubs and
incubated. Equivalent quantities are used in this system, as determined
by titration. After both tubes have been in the incubation bath for thirty
minutes they are removed and the hemolytic system added to the antigen-
antibody tube. Incubation is again required for a length of timo equal to
the first period. At the end of this second incubation the tubs is removed
and the result read. If there is a complete lack of hemolysis then the
reaction is positive. On the other hand, a complete hemolysis indicates a
negative reaction, the complement having been unaffected. In between
these limits the amount of positivenees is estimated by the operator.
It Is obvious that several blanks must be run cn each series of tests
and that In order to be certain of the result, a known positive and nega
tive serum must also be included. In addition to these minor difficulties
must be stated the fact that the antigen which is specific for syphilis has
never been found. Wasseraann and his co-workers used a saline extract of
the liver of syphilitic fetus, thinking that this did contain the actual
antigen. It did not. later it was found that antigenic substances of

3
equal writ were possessed by the heart muscle of almost all mammals.
Hone of these was speciflot whether the animal had syphilis or not was
unimportant. Since the lira* of the original teat several modifications
in antigen have been the sole alterations* Wassenaann and Plant (3)
applied the complement fixation reaction to the spinal fluid with good
results and this is the first test for syphilis using spinal fluid.
L'ichaelis (4) in 1$07, announced the first precipitation test making
use of the some antigen as the Wassermann reaction, the patients sen
end saline. By experiment fee found that antigen and antibody, in a salt
dilution of the proper strength, gave a visable precipitate if allowed to
incubate for several hours. In an article (5) proceeding the announce*
sent of his test, Ulehaelis proved experimentally that the Waeeenaann
reaction probably used a non-specific antigen) therefore, hie own test is
bound by the earns limit, in spite of his claims for its specificity.
In the course of the work on antigens. Forges & Staler (6), working
under the direction of Wseermann, found that pure lecithin could be used
in place of the antigen, and complement would still be fixed. Along the
earns line, was the discovery of Fleischaann (7) that cholesterol gave
even better results than lecithin. Varney (8} also made use of an anti
genic substance in a precipitation reaction. He found that the addition
of a few drops of If taurine to 1 cc. of the patient's sens gave a preci
pitate in the same manner as the Michaelis test end did not require as
long a time of incubation. However, taurine was not effective enough to
ever be of much use? It led to too many false negative reactions.

4
Format, la 1907 proposed o tost b&sod on his laboratory*s ex
perience that a syphilitic scrum would cause precipitation If added to
a normal scrum* Plant, Houck and Fosal (9) investigated this, and found
that any two sera, regardless of affection by syphilis, might give rise
to a precipitate. The mechanism of this reaction is not olsar, but it
may have something to do with blood types, sins# it may taka place be*
tween two normal aera*
The Aborderholden reaction was applied to the diagnosis of syphilis*
Varney and Morse (10) claimed it was specific* They noted that the
reaction did not approximate the Wassexmana reaction in usefulness. This
is a reaction depending on ensyme, or foment action* The serum of the
affected person, mixed with a substrate of protein from Spirochete
pallida, Should give rise to free amino acids by hydrolytic action*
Bronfenbromer (11) found that a pure lipoid could be used for substrate
just as well as a protein* the protein being acted on was In the sern
itself* It was also found that almost any scrum would give rise to coas
free amino acids*
Klauaner (12, 13) gave his name to the reaction which takes place on
dilution of sera with distilled water* The syphilitic sera yield a pro*
olpltate, the non-syphllitio fail to do so* It was admitted, however,
that the reaction was also positive with typhoid fever, lupus, and
pneumonia* Daranyi used the same reaction, but made the substitution of
alcohol for distilled water* Both of these tests are based on the protein
content of the serum* Krameke, In 1923 was still making use of the

5
Baranyi test (14) in following the course of syphilitic treatment.
A tost of very obscur mechanists was proposed by Sehuraan (15)*
Sonsa is diluted with 3-5 parts of physiologic salt solution and a
drop of 30£ hydrogen peroxide added* How a very dilute solution of
FeCl^ and phenol is added and the color read. Brown to black is a
positive reaction* while a green color is normal. This test is not
mentioned again ia the literature, and must have been guilty of too
many false reactions.
Independently* and using different nothods, Monne (16) and Noguchi
(17) did all they could to get the globulin contest of the spinal fluid
into the diagnostic picture. They point cut that the spinal fluid is
not encumbered by the large concentration of other materials found ia
the blood. Nona made use of the fact that globulin is not soluble in
a solution which is half-saturated with (NH4)2 SO^. Noguchi claimed that
the addition of a 10^ solution of butyric acid showed the presence of
excess globulin much more clearly. Belts (IS) otates that a positive
Bonne reaction (also called Roso-Jones reaction and (IS^JjSO^ reaction)
is a confirmation of syphilitic diagnosis, but that a negative reaction
le not conclusive* Also that tuberculous and suppurative affections of
the meninges give positive reactions. Jonas (1$) in a study of general
paralysis (paresis) stated that increased globulin almost invariably
occurs} that the iVaasermann antibody is associated with this globulin}
and that the excess globulin is the cause of the Bonne and Noguchi teste.
He regarded these last two tests as the best method of separating general

6
nervous and syphilitic affliction.
Brelger and Hen (20) sought to tacdlfy the assonannn reaction by
th, . ot KClOj . Mm.ly.tn. In a .end articl. (21) tMy mk, th.
statement that the active hemolytic agent is mainly in the ccopienat
and the KClOj aetivntoo it as ell as the use of specific rabbit serum.
While their reaction checked with the Bhoaermrm in 65 caeos, it did
not receive any favor, and is not mentioned again in the literature.
Jacobsthal (22,23) evolved an ultramicroscopio test, based on his
observations that 1) extract of syphilitic livor shows minute, equally
sized particles; 2) syphilitic senes contains less numerous, fine,
motile partidos; 3) the addition of these two liquids give rise to
the formation of large, irregularly shaped flakes, which are composed
of fino particles 4) centrifugation gives a top layer with no comple
ment binding ability, a bottom layer with very strong complement bind
ing ability; 5) the formation of these flakes, and the coiaplenent bind
ing ability are a specific test for syphilis.
Jncobsthal adratted that the percentage of positive tests was not
as high by his method as by the Wassetmna, but stated that it was 100/'
sure on negative results. Be also stated that heart muscle and ether
artificial antigens were of no use in this reaction. It ma indicated
that the Maaanmrm la also 9 precipitation reaction.
Bruck and Hidaka (24) mda an investigation of the test proposed by
Jacobotbel, and in part confirmed it. They also found it was not constant
enough to be of use. However, modifications made the precipitate visible

7
to the naked oye* Working at 24+C*, and allowing the reaction 2k
hour* for completion, they found that centrifuging gave a precipitate
of maeroeoopio also* This reaction occurred with from 72 to 99?* of the
positive ifuaenum asm, and from 5/£ to 13% of the negative sera* The
preolpitate was found to differ from that of Kleusner (globulin) and
that of Forges and Mador (lecithin). While the globulin is not expected
in this reaotlon, they were most certainly getting a lipoid, adulterated
with protein. The test Is not used today, because of the long reaction
period and the non-confonaity of results.
About this samo time a reaction called the Bsllostl, or Butenko,
reaction appeared. The reaction used as reagent a 10% solution of HgHOj
with a slight excess of free Hg end a little HKOq. It was proposed
for any body fluid and Boveri (25) found that it gave negative remite
with some 15 sera from afflicted patients. Stern (26) aleo checked
this reaction and found it hod no specificity with the urine.
abb&tini, in 1911. reported on the method of Rivalta (27)* The
method le to put equal volumes of spinal fluid and very dilute HagCO^
solution into a tube and then pour down the tide several drops of 2%
acotio aoid. In 1 to 5 minutes the appearance of a white ring shows a
positive reaction. There are positive results in paralysis, tabes, eto.,
and the results are claimed parallel to those with the Sonne, Noguchi,
and Wassomann reactions. This reaotlon may be as good as that of Hoaao
and Noguohi, but it would probably not parallel the Hfassenoann. It is

8
ro reported again in tho literature.
Hoguohi, a tireless Investigator* next looked into the possibility
of an extract made frost a pure culture of 2. pallida (28). He used this
in a skin test, both with rabbits and sen. A well narked inflammation
developed when the extract vas used after the Injection of the ansale
with either a live or killed culture. i?o reaction was obtained in
rabbits suffering active orchitis of syphilitic origin* or in rabbits in
which this condition had been cured four months previously. In man it
was found that ike reaction was positive in tertiary and congenital* but
not in lc or 2C syphilis. If there as any reaction at all in the pri
mary and secondary caaes* it was very weak. ifenu-Suscel* Alexandreecu
and Friedmann (29) found that this reaction was a true confirmation, if
positive* but when negative had no special significance.
Pcpcff (30) made the observation that the senas of man is hemolytic
toward the red corpuscles of the guinea pig, and that inside certain
limits tliis property is constant. He studied a series of diseases, and
found that the hemolytic value of serum was altered only in syphilis. In
this case the syphilis, by producing freo lipoids in the blood, apparently
tied up a great deal of the complement, and this hindered the homolytic
action. Ho found also that a great deal of the hemolytic value of a serum
vas dependent on its complement content. fell-Kafka is the name given to
a diagnostic test depending on this loss of hemolytic power, ^eil and
Kafka (31} found that in the apinal fluid there was almost a total loss
of complement during syphilis, and a normal level was maintained in

9
meningitis. They only advised the use of each a teat in the epical
fluid. Morions (32) found that there as no parallel between tide test
and the albumin content, but he does not mention the amount of globulin
the more Important factor. Thia test is not mentioned again in the
literature, but It is used as the basis of several other works notably
the Hecht-Gradwohl test and ite modifications.
Lange published the results of his work in 1912 (33). By deductive
reasoning he worked out one of the most important tests applied to
spinal fluid even today. It was an entirely new reaction and came as a
result of his observing that all eerodiagnostic tests to that time were
based on colloid properties of liquids. Since one of the best methods
for the investigation of colloidal solutions was the gold number of
Zsigmondy, he applied this technique* The method Lange used van to make
dilutions of the fluid being investigated and add them to tubes containing
a solution of colloidal gold. If the gold was unaltered the reaction was
negative. When 10 dilutions were used erase would react and some fail.
Graphing those results gave curves, and it is in the interpretation of
these curves that the differential diagnosis of the varied manifesta
tions of neural syphilis lisa* Kafka (34) confirmed the work of Lange,
and Kryle, Brandt and Krns (35) nade this eramaent on it* The test
(Lange) runs parallel to the globulin content but is independent of it.
It appears when other fail* and just as well my fail when others appear"
Glascoe and Scrum (36) did some work on the sol itself and found that the
sensitivity decreased with increasing pH and increased with the else of

10
the particle*
Epeteln and Paul randa sotso comments (37) on the theory of eyphille
aorodiagnoeia, and in effect aid that all tete are baaed on electric
charge change in surface tension adsorption and coagulation. They
further add that the alteration of chemical structure of raacion sub
stances* or the formation of new compounds hardly entere into t
problem. Whether thle can be accepted at its face value or not is a
natter of debate.
While no record of the original work on the Hermann-Perots reaction
is found is our literature, it le a reaction using the sodium salt of
glycocholie acid with added cholesterol ae antigen. Zadek (38} found
this to be a non-specific reaction where there were the following affec
tions present* pulmonary tuberculosis, eclampsia, carcinoma, typhoid,
sepsis and uremia, when testing o enea 5 and when testing the spinal fluid
urania and aclamis interf erred. A large number of controls gave 74.4*
positive Wfissenannn reactions and 8positive Henmnn-Peruts reactions.
In connection with this test v. ^Hermann (39) states that it is not
positive with tabes or paresis, but only with true syphilis. V&ny such
substances as the glycocholatcs have been used as antigens in precipita
tion reactions, but none of them have been successful.
At this timo a comparison of the various teats and their indications
was published by Wile and Stokes (40). Their deduction is that the
c aso nest indication of neural involvement is increased globulin and
albumin, the positive Wassenoinn reaction and lymphocytosis, in the

11
order given.
Kolmer (41) gives us n record of the reactions of Landau. The
first method was to overlay scrura or fluid tilth paraffin oil, contain
ing iodine, the second ana to put the iodine ir. CCl^ solution and run
this under the liquid in question. It was claimed that in either case
a syphilitic affection gave the liquid the power to decolorise the
iodine. It was found by Koltaor that this was a very poor method,
giving far too many false positive reactions. Bronfenbrenner aw! Rock-
man (42) found this gave 30,1 false positives and only fi$)t positive
reactions with syphilitic controls,
Gordon (43) proposed a test using %C12 in a dilute solution (1*100).
Tho sera or fluids vare placed In tubes and a few drops of the reagent
poured into them. With a positive liquid a cloudiness appeared, with
the normal a clear solution resulted. Woichbrodt later modified this
test and used it for the detection of globulin increase. Krebs (44)
found that the aaaiaum globulin precipitation in this tsat occurred at
an albumin percentage of C.25. This is not a common test in this country,
but should bo as good as tho Nonas or Noguchi method.
v, Dungern (45) proposed a tact involving the use of a solution of
indigo, sulfuric acid and Foiling solution B in the proper proportions.
When added to serum a completo gel resulted if syphilis were present.
The observation me aleo made that tuberculosis of t5j bone might cause
a false positive, as this condition leads to Increased protein, just as
syphilis does.

12
Hecht (46) worked out an antigen for use in both a precipitation
reaction and Wassons nn reaction. When diluted for the precipitation
reaction it wae turbid, but allowed the passage of light. The nenas
and ccEtplomnt were mixed before addition of the antigen and allowed to
react for a tints. After the addition of antigen the tubes were incubated
for eight hours. A positive reaction was shown by the foraation of a
heavy precipitate; in a negative the antigen emulsion resained unchanged.
Hecht-Gradwohl is the muse given a reaction first proposed by Grad-
wohl (47) in 1914, It as a heraolyain reaction and was modified by Bruce
(4E) four years lator. It is applicable in about 98$ of the patients
tested, the others not having sufficient hesaolytic power in their senas.
In the first place the hesaolytic index of the patients sen is found*
The units of the hemolytic index are volume of standard sheep cell sus
pension heiaolyxed psr unit volume of sema. Mow l/2 this amount of sheep
cells is added to a mixture of Hoguchi antigen and sen. The tube is
incubated at 37C. for an hour. If the reaction is negative, hemolysis
is usually templets at the end of this time. The tubes are kept over
night in the icebox and read the following morning,
Emanuel (49), finding the gold sol of Lange hard to prepare in a
reproducible Manner, proposed the use of a colloidal solution of gum
mstic. Five tubes were set up with the colloid and to the first four
were added graduated amounts of spinal fluid, the fifth receiving only
distilled water* If the reaction was positive the mastic in all five
tubes precipitated, if negativo only that in the blank precipitated.

13
In 1917 Cutting (50) noted that the salt solution (spinel fluid) in
a negative reaction did not allow the gum mastic to precipitate. In
order for the blank to remin a colloidal solution, he found that the
efficacy of the test was not altered by the addition of 1 co, of 0.5$
KgCCh solution to each 99 cc, of the mastic made up In alcohol. The
KgCO^ did, however, keep the blank from precipitating.
In 1930, Hoaenfeld (51) proposed a change in the mastic test in
order that curves similar to these of the gold sol test would result.
He prepared t colloidal solution fro buffered &Cl solution, pR 7*5
and an alcoholic bass solution of mastic. In this manner lie claimed to
have a reproducible solution and to get curves which wars of more use
than those in the Lange test, Brandt (52) was still not satisfied with
the sensitivity of the mastic test. He found that the speed of mixing
had mors te do with sensitivity and reproduction of the test than was
usually assumed. His test is run in the following manner. A series of
tubes containing the etnas amount of mastis is set up, ami as rapidly as
possible graduated amounts of water are added. After setting for a
while each tube is nade to the same volume with more water. Once the
particle else is determined by the original addition of water, more water
does not change it. In this way a reproducible colloid of the same sen
sitivity is always obtainable. In a period of 14 years some 70,000 tests
were run by this method. Since the normal and syphilitic curve both
depend on the globuliryfobumin ratio, there can never be a colloid reaction
which differentiates syphilitic from non-syphilitic, if the globulin goes

l4
to the asm level. There can not poaaibly he a safety sons, as seas
authors oala, but always a gradation fresa non-ayphilitie to syphilitic.
Pfeiffer, Kober and Field (53) sede an investigation of th# pro
teins in the spinal fluid, using nepholosaatry for their determinations.
They found that if the normal value for euglobulin as token as 0.004J,
then fluids giving a positive Ifesaermrm test -sent over this value in
14 out of 16 casca, and negative fluids were under it in 33 out of 35
cases. They also admitted that in two of these disagreeing eases their
measurements were doubtful. It is concluded that the euglobulin con
tent and the Wasseraann test are closely related.
As a test for pathogenicity, Hofftean and Schwarts proposed the
ue of the permanganate number (54). This is the number of cc. of 0.1*1
KHn04 reduced by 1 cc. of the spinal fluid. Genoese (55) found that
this reaction was also capable of being carried out with methylene blue,
ke everlayed 1 cc, of the fluid with 1 cc. of reagent and the yellow
color at the Interface, followed by the whole liquide becoming yellow
after shaking, was supposed to denote a pathologic fluid. Bovori,
after who th permanganate reaction is named, found that this was in
reality a test for albumin (56), and that 1 cc. of spinal fluid should
not decolorise 1 cc# of ltlO,COO KKnO^, Gulllain and Libert (57) also
report that hyperalbusainoois always gives a positive permanganate test.
Lowery (56) found that neither tho permanganate nor the mastic teet was
of sufficient value for the routine examination of spinal fluid. His
work was dono prior to 1$1?# and while tfc permanganate test is not used,
as ho advisee, the mastic test is being used acre and more.

15
Sach and Georgl (59) proposed the test bearing their moos in
1916. They ande use of an alcoholic extract of heart tissue to which
was added cholesterol. This antigen was mixed with the serum (inacti
vated) and allowed to stand 18 to 24 horn's, after a short incubation at
3?C. Flocculation wee a positive reaction. This is one of the most
important forerunners of the modem Kahn reaction, and in fact the
faults in it are responsible for Kahn starting his research. In the
some manner# the work of Heinicks is just as responsible.
ieinieke (60,61) mete use of a physical-chemical reaction for the
diagnosis of syphilis. The reaction was found to depend on the fact
that a mixture of lipoids (the antigen) and syphilitic antibodies fora
a precipitate, and that this precipitate is insoluble is salt solution.
To 0,2 cc. of the inactivated senes is added 1 cc. of the lipoldal
antigen (made of human heart extract), the tube is then incubated for
20 hours at 37c After the incubation the precipitate is shaken and
1 cc, of 2* JfoCl solution is added. If the precipitate is insoluble,
the reaction is positive. Fr. Reichert (62) found this reaction, after
its third modification, was still not applicable to the spinal fluid,
the precipitated Material, if any, not being vieible. The various Modi
fications made in this reaction by Heinleke were not able to increase
its usefulness, ami in 1922 he announced a new reaction, the Jfoinleke
turbidity reaction (li.T.R. or Helnieke II), The antigen used in this
U,T,R, (63) was designed to make the reaction clearer, and to do away
with the addition of the salt solution at the end. To the lipoldal

16
extract, balsa of tolu me added, and then the turbid emulsion placed
in tubes with the inactive serna. If the reaction were positive, the
turbidity Increased, end mil floccules appeared. Although the H.T.R*
only required eos eight hours to go to completion, even Heinicke
admitted that it was not ns good for diagnostic work aa hie original
method.
Still searching for a better method, Keinicke (64) propoeed the
Welnicko Clearing Reaction (H.K.R. from ti German, or Keinleke III
reaction) in 1929. In this reaction he made use of a beef heart, lipoid
antigon and added three times as much tolu balsam as in the U.T.R.
Just prior to use, the extract of beef heart and the baleara are heated
to 45C, and 1C volumes of IfeCl solution at the samo temperature
are added, rapid mixing being essential, hen the mixture cools to 37C*
active serum la added, A positive serum results in a precipitation of
the colloid, leaving a clear solution. The colloid is not changed by
a negative, non-syphilitic sen. This last test of Heinicke is apparent
ly his boot, but it is not used in this country to any extent.
£llingr (65,66) tried a patented germicidal colloid of silver for
studying the spinal fluid. The preparation, Collargol, is made by a
secret patent process, but Alekseovski (67) prepared a duplicate material.
He ms engaged in breaking Goman patents for the Russian government.
He suggests that the final solution is not a colloid at all, but a mix
ture of complex silver salts of the amine acids in a dispersed state.
Rubeling (68) in 1938, ten years after Ellinger, published what he called

17
a new reaction for spinal finid, twine Collargol and HC1. The reac
tion is based on the fact that the protected silver chloride of the
collargol is precipitated by the addition of spinal fluid rich in glo
bulin. Hermann (69) states that the basis of the gold sol and collar-
gol reactions are different, and that for this reason differential
diagnosis should be possible tanking use of both reactions. Since both
are dependent on the globulin content of the fluid, the truth of this
statement is not apparent.
In 1?19, 3tinfield (?0) found that the action of spinal fluid on
a suspension of FbC^ depended on whether it xme pathologic, or not. lie
found that nomal fluid had no effect on the suspension, but that fluid
fro a syphilitic source gave varying amounts of agglutination and floc
culation. fo further report on this work is found.
Ouillain, Laroche and Leohe11a (71) introduced the use of bensoin
as a colloid substitute for gold in tb Lange test. Their tost is
similar to the first mastic test of Immanuel. They make the statement
that the results are not in accord with the albumin or cell count of the
fluid. Since the globulin ie the determining factor in this type of
reaction, such a statement is not too important} however, the cell count
should be higher in pathologic conditions than in ths normal. Wright
and Ransack (72) give an exhaustive treatment of the reaction with
bensoin, strongly advising the use of this colloid. Quart! (73) in 1928,
reports the use of gum myrrh in spinal fluid testing, but does not give

18
any conclusions as to the worth of the teat. It is clear that any
colloid my be uad In the oxaniontion of cpiml fluid and tiiat the
resulta ill depend on the care with which the technique la followed.
Vemos (74*75*76*77) began work in 1917 on the diagnosis of syphilis.
At first he raid use of a colloidal iron solution containing Fe(CH)y
FetCjHjOg)^. The results were net good enough, so he made use of an
antigen front horse heart, which he called perethynol. Since this gave
a reaction which he could not see, he added a pig sarun-shesp coll
hemolytic ayate, for indirect reading. This test wee similar to the
Wassermnn, hurt not as good, so that in 1925 he published his last modi
fication, a precipitation test making use of the perethynol, inactive
aerura and an instmsent for reading the turbidity. This instrument ie a
photometer with a special seal designed to show pathogenicity by the
numerical reading. Cornwall (70,79) discusses these tests in detail
and praises the last modification highly for use in the following of
treatment*
hold (BO) started publishing a series of tests in 1921, but none of
them was very successful The antigen he used was similar to that of
Sachs and Osorgl and hsinicke, and diluted It 10 with salt solution prior
to use. The serum was then added to this opalescent solution and incu
bation carried out for two hours at 37C. After another two hour at
room temperature the result was road. If the solution was still in its
original state, the result was negative! if turbidity developed, then
the reaction was positive. Qtreaspel (81) found this reaction lees

19
sensitive than either the flachs-Georgi or the Ifeinieke III) he found it
gave false negative results*
Bold (82) found that formlin enhanced the reactivity of sema
especially syphilitic and in 1922 made a simplification of his original
test, using formaldehyde, antigen and serum. In order to read this test
it was still necessary for the incubation to go on two hours and the tubes
wore read after 2 and 22 hours in the icebox. In line with this was the
formaldehyde-gel test proposed around this timo by Cate and Papaccsta.
Johnston (83) made an investigation of this test and found it mas never
to bo used with any surety. The technique is to add a drop of formlin
to 1 cc, of blood serum. The tubes are allowed to stand 48 hours and then
examined for coagulation. In 100 cases it as positive in 23, the Wasser-
raann in 27. There was agreeaent in 11 of these, all the others conflict
ing. Considering thio, perhaps the addition of formalin to the Bold
technique was a mistake, loading to erroneous results,
Dreyer and ard, in 1921 (84), published a test which is in use in
England today. A eholeeterinised antigen of beef heart extract ia prepared
and two dilutions ade of it. iline dilutions of the oerus are then rand
up and five added to the stronger antigen dilution, four to the weaker.
After a short incubation the turbidity in each tube is read by moans of a
special agglutinoscope. The results are expressed in standardized Sita
units. The necessity of using a special instrument keeps this reaction,
just as It does the Vernos reaction, in a limited field.
The Berxheimr reaction (85, 86, 87), or provocative teat, was first

20
referred to as a ekln test* If a skin dose of neoaraphenaa&ne ms
administered, several sen made the claim that it had the same action as
tuberculin. This ms found to bo a falso assumption, and the reaction
is no defined as the aggravation of pathologic processes following anti
syphilitic therapy. It is found, for example, that old cases with a
negative Wnsaenaann reaction may give a positive reaction after a heavy
dose of noenraphenesine. This does net often take place, and it is not
a diagnostic method. It le theraputic shock and to be avoided, according
to Hoore.
Tashiro and Levinson reported the use of a mixture of sulfo-sali-
.jrUc Mid a4 !sca2 in tte tosmBl* of vjbiiU In th. brcplnnl
fluid. KaSahara (80) found this test positive with all forms of meningitic
involvement. He states it le never specific. It should be noted that
sulfo-salicylic acid la used in the quantitative estimation of albumin
(8?).
In 1922, Kahn started publishing the resulte of his research, am!
his methods (90) However, this toot was not fully ready until 1925*
only the antigen being used before that time. Wang (91) proposed a test
using alcoholic extract of human heart at the same time. His results were
in good agreement with the Wasoenaann test, but the test never gained
any following. It land no special advantage over any of the other pre
cipitation reactions of that time.
Almost as soon as Kahn published directions for preparation of his
antigen, Kerrold (92, 93) mads use of it; first in e reaction with serum,

21
later In one with spiral fluid* Herrold diluted the antigen It12 and
added toma fro a pipette so as to for two layara, A white ring in
the serum la a positive reaction. With spinal fluid, he found it neces
sary to concentrate the globulin fraction, using half saturation with
After removal of the liquid, the globulin was dissolved in
a snail velum of physiologic saline and then tested as above* While
the results were not too good, Kahn did not successfully run a spinal
fluid until after he ronde uoe of its concentration by this method*
Bruch (%) in 1922, returned to precipitation tests* He nade use
of a new antigen, heavy concentration of salt, and centrifugation* Hie
antigen is non-choleeteriaised extract of beef heart. Inactivated soma
(0,2 cc.) is added to 0,8 cc, of 10$ HsgSO^ solution and then 0.2 cc, of
antigen emulsion added* The tubes are centrifuged 20 sinutes. An insoluble
precipitate is positive; e negative serum also gives a precipitate, but
this goes back into solution on shaking. Teicfcmnn (95) notes that the
reaction must be run on positive and negative sera and gives tire method.
Becker (96) tested 12,000 blood specimens with this method, and found
almost perfect agreement between it and the Wassermim test.
Schwarts and Orunewald (97) wade use of colloidal silicon dioxide In
the examination of spinal fluid* A solution of S102 (0.25$) 1 used. Ten
drope of this are mixed with 1 cc. of spiral fluid, and it is noted that
luetic fluids flocculate in a few hours, or easily on heating* A second
method proposed is the dropsies addition of spiral fluid to 1 cc. of the
reagent. Fluids froa parcels, cerebral lues, and tabes flocculate in

22
that order. This method was found by Kleiner to be $2^ as effective as
that of Lange* and he recommends it for rapid orientation (98)*
Helia (99) found that pathologic spinal fluid, especially luetic,
formed foam when mixed with an equal volume of beneene or toluene. This
is presented as evidence of the colloidal change in the spinal fluid
during disease.
An investigation of the passage of Uranin (the sodium salt of flores*
coin) into the spinal fluid, following its injection, or ingestion, was
made by enroll (100). It ws found that the color of the spinal fluid
was related in some of the cases to the pathology, in others there was no
correlation.
In 1921 Kahn (101) published an article on the effect of heat on the
reactivity of antibodies. At this time he apparently found that heat had
a deleterious effect. At 5&C, he found a loss of $f. in strong positive
sera, and $0?. in weak positives. He also stated that in a great many cases
complete destruction resulted fro heating to 62C. In his book (102)
Kahn reports that heat lias an enhancing value, helping to promote the
reaction of sera. He explains all the reasoning that he and his co-work
ers lave used la the development of the Kahn test* In the flret place a
concentrated antigen is used to give better reactions with weakly positive
sera. Secondly, this antigen is made into an unstable emulsin, the more
unstable this is, the faster it reacts* in the third place shaking of the
antigen emulsion and the sertas is introduced, since this also speeds the
reaction. Salt solution is added because Kahn found the proper concentration

23
of NaCl aided the reaction* Cholesterol is used as a sensitizing agent.
Inactive serum is used, since heating the serum increases its reactivity.
Used on spinal fluid, the reaction is carried out as follows. The
fluid is inactivated, and then mixed with 2/3 its volume of saturated
(NH^)230^ solution and allowed to set 1 hour. The tubes aro then cen
trifuged and the supernatant liquid poured off, leaving the globulin.
To this globulin is added l/lG tho volm? of the original fluid of physio
logic salt solution. Thia concentrated globulin solution is divided into
three parts, two of which are used with antigen* tho third, for a blank.
The anticon ie added to physiologic salt solution, the tubes shaken and
then the globulin solution ie added. Shaking is continued for a time and
then the tubes are read, l oaitive tests ore shown by the presence of
flocculation, negative by the fact that the tubes remain opalascoot.
Epstein end Raul (103), as wo11 as others, have investigated the pre
cipitate, or flocculate, formed in precipitation reactions. They find
there is little evidence of any but included protein being present, and
that tho min pert is lipoid* Brack, concerning tho mechanism of these
tests, (104) states that they depend on the agglutination of finely
divided lipoid particles, this being stimulated by some constituents of
the luetic serum. Kahn, in & store modern work, (105), presents the view
that a lipoid due to the Spirochete is responsible for the production of
the globulin antibody, nnd that this globulin will then precipitate e like
lipoid, no miter whet tho source.
In 1926, several reactions were brought out. Kadisch (106)

24
developed o rapid ^asaeraatm test, but this never gained any favorable
reaction frotn pathologists. Hosenfeld (107) made a study of the Influ
ence of spinal fluid on electrolytic precipitation of positivo and
negative sola, at a definite pH. He found that if the pH of the eplml
fluid were adjusted to 5*0, then the normal fluid would protect night-
blue against flocculation. He alee found that if there were
hyporalbuminooia, the nightblue reaction was sensitised, end that in
eningitia the result was flocculation. 0 the cthsr hand, hyperglo-
bulinosis had an opposite effect, the nightblue being held in solution
completely, Using nightblue, Hosenfeld was able to distinguish between
meningitis and syphilis or other affections producing an excess of glo
bulin.
Sleeper and Welker (108) investigated the method published by Bolts.
They proved that the use of acetic anhydride or a salt of glyoxalic acid
with concentrated HgSO^ and spinal fluid was merely a test for trypto
phane $ that la ms an indirect test for increased protein, the amount
of reagents being such that the color did not appear in the norml and
did appear at a value just post normal,
Tnecone (109,110) advised the use of 5$ KjCrjO-y with one drop of
CCl-j-COCfl per cc. He states that with spinal fluid this solution gives
a white turbid ring ai the interface, and that this ring is more intense
and permanent with syphilitic than normal fluids. He states that the
differences in intensity and structure of the turbidity allow a differ
ential diagnosis. In his second paper Treceno uays that the dichromts

25
reaction is the most sensitive knctm; that it positive with 92^
of children having syphilis of th spinal chord, and that 45/' of these
children had no apparent lesione of the nervous system. Prunall had
provimmly found that CClj-CO* o a pr.clplt.tton <** for lWdn.
(111).
later Prunell (112) investigated the reaction of ?akata-£ra (113)*
This reaction is carried out with 1 cc. of spinal fluid, 1 drop of &2
CO^ (10/Q and 0.3 cc. of a very dilute solution of JigCl2 (G,2$f,) and
diamond fuchsln (0.0l£). It was claimed that the color nial flocculation
gave a toot for syphilis. Prunell found it indicated a pathologic con
dition hut not espocailly syphilis, that it probably depended on
albwiin. Staub end Jealer found this Takata-Ara reaction depended on
globulin, and was of use in diagnosis of hepatic cirrhosis. It la no
better than the gold ieet or ether tests depending on globulin (114).
In 1926, Sachs and litebsky (115) proposed the citochol reaction.
They concentrated beef heart antigen three times and then choleeterinised
it, Th reaction is very rapid. They also investigated the possibility
of a lentochol reaction, using lecithin in place of cholesterol, but
this was not as successful*
Hinton (116) made use of an antigen emulsion which consisted of
alcoholic beef heart extract, cholesterol, glycerol and HaCl solution*
la testing spinal fluid five unheated, graduated sables are used. If
the reaction is positive there Is a clearing of the antigenic emulsion
and sedimentation. The reaction is carried out at 3?C., and if the

26
bath gats too hot, there ill be clearing in all tubes, but no sedimen
tation,
duller proposed a conglobation, or conglomeration reaction in 1926,
Mayaer (11?) found this was not as good as the Wassersa&nn, Sachs-Georgl
or Kienicks tests. He applied it to 635 sera, Troutor (118) also
found that this Muller reaction gave for too many false positive reac
tions to bo of use unless checked by the Weeseraann method. In 1930
Muller brought out a reaction known as the K.B.R. 11 or the Muller Bel-
lungs Roaktion (119), results on 937 spinal fluids give agreement
in 683 (positive and negativo) and 39 positive reactions on ^'nsseraann
negativo fluids, 15 negative reactions with Wassonnn positive fluids.
The antigen is a beef heart extract with a very heavy cholosterinization.
I'ullnr and Mnndula (120) explained that if to constant amounts of heart
extract 'mro added increasing amounts of cholesterol, tvo zones of
optimum precipitation resultedt these basing separated by a sons of clear
colloid solution. The balling reaction takes place in the zone with the
large concentration of cholesterol, the ordinary flocculation reactions
in tas sows of low cholesterol concentration. Filan (121) reports that
*
the false positive reactions of the M.8.R. II are easily distinguished
from the true positives, because they are a course precipitate and not
a true ball. This balling is in reality an ogglutinisation of the anti
gen emulsion, the stronger the reaction the store completely it goes Into
one ball, leaving a clear solution.
In 1929 and 1930 five reactions appeared, the first being the Tsu

27
method, the second the reaction of L'utercdlch. Neither cf these was
new, or very effective. Sauer reports (122) on the Douris-Beck reac
tion at this time* This reaction need a mixture of formalin, sodium
olate and phosphoric acid. This aleo vas nothing new, except in the
combination of reagents, and it was rot effective. Tapi (123)
explained the Sclarra reaction, which is a Wnsseraann test with alcohol
pressnt. It has no particular advantage over the parent test. helcxer
and Bahwen developed a jaicro-aastic test, hut Kleiner ($8) found It
only as effective as the Lange test.
Karchioni and Allensteln noted (124,125), in 1932, that the diastase
content of the spinal fluid is involved in syphilis. The normal range
of this material in the spinal fluid is 11-40 mg. percent, but in
syphilis it is found to he only 0*0-14 This drop is independent
of the sugar content, which reranins normal. The over-lapping of the
values does not prevent this finding from being of use in some casco
diagnostically. The effect after treatment is not given, and should be
even more important.
Kline (126) started publishing preliminary reports of a slide test
for syphilis in 1929. It was not until 1932 that the test was ready for
general use with spinal fluids* At this time a book was published by
Kline. Kahn had the idea that if all other factors were equal, then the
more unstable the antigen-emulsion, the more sensitive and rapid the
test would be. Kline presents the view that the physical state of the
antigen emulsion has more importance than the stability. Both the

28
Kline and Kahn tests are the same in that the antigen i3 concentrated to
obtain diamino-phosphatids* mixed with as little impurity as possible.
This waxy material is then mixed with water, cholesterol and physiologic
salt solution. The manner of mixing determines the state of the emulsion.
In a reverse procedure to Kahn's, Kline adds the cholesterol to the water
first, each particle being thrown out as a small plate. The antigen is
added and forms a covering over each plate, giving great surface area.
To this suspension is now added the saline, its purpose being the same as
in all such reactions, to make the reaction more sharp.
Compared, the Kline and Kahn antigens are opposites of stability, the
Kline not precipitating for hours, the Kahn giving precipitation as soon
as it is made. Two tests are proposed for spinal fluid syphilis detec
tion with Kline antigen. The first is the exclusion test, the second the
diagnostic. Again the reverse of the Kahn procedure is used, the
antigen emulsion being concentrated in place of the spinal fluid. For
both Kline tests 4 cc. of the antigen are heated 15 minutes} that for
the exclusion test at 50C., the antigen for the diagnostic test at 35C.
After the heating the tubes are centrifuged at 2,200 r.p.m. for 15
minutes and then the liquid decanted. To each tube is now added 1 cc.
of physiologic saline, pH 6. After centrifuging the cells out of the
spinal fluid, it is tested with Bennedict's solution for the presence
of sugar. If sugar is present the fluid is good, if not it has been sub
jected to bacterial action and will not give a precipitation test of
^The alcohol soluble, acetone insoluble fraction of beef muscle.

29
any value. If the fluid is usable. 0*03 oc. of 1% acetic acid Is
pl&oed on a slide and 0.25 so. of fluid and 0.008 oc. of antigen added.
For either exclusion or diagnosis the quantities are the seme. The
slides are rotated for completo mixing and read under the microscope
iranodi&tely. If the exclusion test is negative, it is claimed that
there is no ehanee of neural involvement. The dumping of material is
a positive test.
In the book Kline also reports preliminary work on a balling reac
tion. The antigen Is strongly cholostorinised, and when emulsified has
the appearance of long needles of cholesterol partially cove rod by anti
gen. This has the same appearance as the emulsion for the l.B.h. II.
Since no further report of this tost has bean made, one may judge it
turned out no more successfully than the &.B.R. II,
Kiss (127) proposed a balling reaction using s oephalin (phosphatid
from brain) antigen with active sonsa. This test did not get a very good
reception. The statement Is made by Kiss that lecithin is good only
when used with inactive serum, oephalin only with aotive serum. Kahn
has proven the former assertion, but the latter has yet to be proven.
Sohube and Hams (123) proposed a colloidal carbon test. To the
spinal fluid is edited distilled water and then a dilute solution of oxa
lic add. After standing a time, diluted India ink is added. Precipi
tation of the carbon is a positive result. Looney (129) had previously
tried to use this reaction with colloidal carbon made by the Bredig are
method, but had been unsuccessful. Sohube (130) explains the mechanism

30
of the reaction* First enough distilled voter is added to render
inert any protective colloid action by the proteins of the fluid.
In the second place oxalic acid, which hears a positive charge, is
added, and this charge is neutralised hy the free negative charges of
the proteins in a normal fluid. Then the third step is the addition
of the colloidal carbon, which remains in suspension. If there Is
neuresyphilia, the oxalic acid is not neutralised, the free chargee
of the proteins being less maw roue, at! this unneutrallied oxalic acid
causes flocculation of the carbon which has a negative charge, Boadrnn,
Elliot and Smith report (131) on this reaction* "The results using
the method of Schubo and Harms agree with the Wassensann and other
colloid tests," Although the reasoning on the mechanism is rather hard
to understand, the teat undoubtedly works, asunto, Burack and Kreisler
(132) claim it is specific for syphilis,
Ingle, in 1932, proposed a new test (133). The antigen is made of
beef heart extracted with ethanol and then treated with § mixturo of
equal parts of corn gera sterol and cholesterol. This is emulsified
In 4? f'feiCl and ripened 46 hours before using. Inactive serum is used
with an equal volume Cf emulsion, the tubes shaken and incubated four
heure at 37C* After the incubation the tubes are centrifuged and 1,2$
itoCl solution added. Strong agglutination is a positive test, homo
geneity is a negative teat, all others are doubtful. All doubtful tests
aro centrifuged again) if a white flake results in the bottom of the
tube, the reaction is positive) if not, negative, Simpson (134)

31
applied the Eagle teat to 171 epinal fluids, the resulta being about
parallel to the laeseraatm reaction. This test runa about 20?* store
sensitive than either the Kahn or tfaesenaann, and gives fever doubtful
resulte when used with **ra'
Grushin began a series of papera in 1931 entitled Studies in
Cerebrospinal Fluid (135*136). In the first he nado use of the lose of
color of starch-iodine mixture, when it was added to spinal fluidj in
the second he used glycogen-iodine mixture. It vaa claimed that the
decolorieatlo was so graded that a differential diagnosis became possible.
On the basis of the results obtained by Kolaer, studying the landau test,
this work seems to have boon ill fated before it as started (41). It
nay be that the adsorption makes this a more successful reaction, but no
further reporte have been published on it.
Kopaezevakl (137) claims that although lactic acid will gel serums
which contain increased globulin, this is not true with syphilis. The gel
reaction was soldo found with syphilis. This was not published as a
diagnostic procedure, but merely to show that there appeared to be some
difference in syphilitic globulin and other globulins.
The Bauer (136) reaction, an KgClg flocculation of serum or spinal
fluid, is claimed to be as delicate a test for Increased globulin as the
Takate-Am reaction. These tests were both proposed for syphilis, but
proved to give positive reactions with any affection increasing the glo
bulin content of the epiml fluid.
The Laughlon test (139) was published in 1935* Moore (140) found

32
it to bo 18$ In error with the Kuhn toot and 20$ with the saseeraann.
Using inactivated sema It was only 10$ in error compared to the Kahn
test. The reagent is a mixture of bensoin, cholesterol, scarlet red
and physiological salt solution, this is added to an extract of beef
heart. One drop of the reagent emulsion is placed on a slide and a
drop of serum (or spinal fluid) is added. The appearance of red floes,
soon in indirect light, is a positive reaction. Robinson and Stroud
(lip.) found it 93$ effective as against their ffassermann and 97$ against
their Kahn. Thcmaasen (11*2) used it on 3,000 sera end got good results.
The lunger it is used, tho better this test is.
Vernes (li;j), in 1936, stated the finding of a spool fio material
in the spinal fluid was a diagnostic test. The material is oalled
pellidin and is obtained by precipitation* The spinal fluid is precipi
tated with CgCljj and this precipitate is extracted with ether or ace
tone. The extract yields a substance which gives a positive reaction
if added to a negative spinal fluid. Yasitsa and TakagL found the ma
terial pallldin only In the later stages of syphilis (Il4t), and that it
is not demonstrable until some time after the secondary stage is over.
fiyt* reported what he oalled a rapid floooulation reaction in 1936
(ll*5) This can hardly be oalled rapid. To the active spinal fluid is
added ammonium sulfate and NaCl solutions. An antigen emulsion is then
run over this. The density is now such that the proteins In the fluid
are forced into the antigen emulsin on centrifugation. After centri
fuging, the solution is made lighter than the flooculate by the addition

33
of dilute Had solution. The tubes are centrifuged a second tine and
the solution poured off. The sediment remaining Is shaken with phy
siologic saline* Flocculation is positire, a hazy suspension is nega
tive and a combination of the two extremes is called doubtful. The
compsrisen of this test with the Waaesraann is not good but it is
more sensitive, perhaps too such so.
fiahr (146) reports the Chcdiak-Pabr, Dahr or Chdiak reaction.
The nasoe of the reaction depends on the country in which the work is
dons. One drop of defibrinated dried blood ia used. To the blood is
added 0*015 cc* of a solution containing 0*3# JfajCQ^ and 3*5# Ha Cl, A
1*10 dilution of the Me inioke K la rungs Eeaktion antigen with this sane
solution of M.2C03 sod B.C1 is pUc.d on a second slide. The usunt
of antigen dilution used is 0.03 so. The two slides are heated to 5&C,
and their contenta mixed, the elide with the mixture is kept at 37C.
in a aolot chamber for half an hour. The reaction is read at the end
of this time. The appearance of black floes and clots is a positive
reaction, brown granules is a negative reaction. If o modified antigen,
M.K.R. II (modified),is used, the addition of the may be
emitted in the preparation of the diluting solution (147). Schuck (148)
used one drop of blood, 0*3 cc* of 3*5# Ha01 and 0*03 ce* of the
antigen dilution. The appearance of the reaction it the asms as before*
The reaction is strongly recommended as an additional aid In diag&o*l*
by Porto and de Britto (149), Althot^h it has received no mention in
the Onitod State, this test has gained a great amount of literature

34
treatment in the rest of the world*
Ide end Ido were granted a French patent for a diagnostic reagent
in 1937' The reagent ia an extract of beef heart atusle, to which is
added cholesterol and crystal violet, or methylene blue (150). Van
Sverdingen (131) found this reaction lose sensitive than the Molnioke,
Kahn and eltoohoi reactions, but more sensitive then the Wessermann or
Saehe-Georgi reaction, lie states the specificity is the Mae as thet
of the ifaasertaann reaction* It gives a great number of doubtful reac
tions, but simplicity la its advantage*
Rosegger, in 1938, (138) proposed the use of a constant amount of
spinal fluid nixed with varying amounts of calclua chloride* These mix
tures are then heated and the flocculation curves used in diagnosis*
This test has received no further mention*
In another of the new and untried teats, Hlrsohfeld and Klinger
propose that the antibody be used to bind thrembokinaee* If there is
antibody present, then the thronbokin&ae will be unable to coagulate
plasm* Kaehueharov found this to be an impractical test (153)*
Hastial makes use of an antigen got by the extraction of a mixture
of egg yolk and beef heart muscle* It is claimed that the egg yolk
allows the minimal amount of antigen to be used without loss in speoifi-
clty (13^)* In this roaction the egg yolk is probably acting in the
manner as an addition of lecithin* This test is also in the trial stage*
Prohlioh (155) while critically studying the H*B.R, II, hit upon
an idea of a new type test* Since cholesterol and antigen flocculate in

35
certain dilutions, active, syphilitic aeran should prevent this by
binding the antigen* The technique recommended ia to add a 3-$f*
JaCl solution to the active senes contained in several tubes. Increas
ing amounts of heart lipoid are added to the tubes. After a few hours
incubation at 37C. the cholesterol is added in the for* of 0,1$ solu
tion. To obtain optimum conditions a mixture of dilute HC1 and acetone
is then added. There is flocculation only in the negative sera. This
test is merely proposed and no results given as yet.
By no means all of the tests in the literature have been covered.
When eadtted a test did not embody any new or original idea. As an
example, we might have considered the use of shellac, ge* guale and
several oilier substances capable of forming colloidal solutions. To do
this would be a waste of time, once the mastic reactions have been
covered. Store recently there have been quite numerous publications on
tests which are all indistinguishable from the Dahr or Chsdi&k reaction.

m& of spwal rests
he exact composition of tha spinal fluid Tarta* frota aso to
o&e*. Such authorities as Soot and Baylor (196) and bright (157} girt
raporta not wholly in agreement as to the composition of the spinal
finid
ffce pial finid arises in the toroid plexos of the brain end
leaves through the venan sinuses of the brain fhs source 1* the blood
stream. fbe componente aro not In any given ratio compared to the
blood however, this foot* added to the faot that the hydrostatic
pressure is not great enough to account for the pressure of the fluid
leads to the conclusion that the colls in the toroid plexus met do
tone work, there is not as ultrafiltration of blood alone. Bicarbon
ate Is the only substance having exactly the mm percentage in the
blood as the spinal fluid.
Protein* constitute some 16*38 mg. percent of the normal spinal
fluid, these protein* are about equally divided between globulins and
albumins, the protein* do net appear to be able to paos the meningitic
filter, or. If they are able to pase the barrier, the exchange 1* ex*
oeedlngly slow, at will become apparent later, lecovetoo (158) found
two proteins in the spinal fluid, one of which behaved a* a globulin,
the other being soluble In distilled water, 'Today this latter protein
would be called an albumin.
For may years following Xsrcoveseoa work authorities claimed that
there was no globulin in the spinal fluid of a normal person, fhe teats

37
used for globulin depond on the ratio of globulin to albumin, and what
these non have found is that the globulin in a normal fluid it not de-
tooted by tho methods they used. Tho ratio of globulin to albumin in
a normal fluid is such that there is no precipitation under tho condi
tions of the tests. Therefore, when it is stated that a traee of glo
bulin is pathologic, tho moaning 1st the globulin-albumin ratio for
oomplote protection is passed and a part of tho globulin thrown down,
tho laok of protection being pathologic. The more modern authors reoog-
nise this fast.
The nature of tho globulin found in syphilis is somewhat different
from that found in the normal ease, Kahn glees a eery olear picture of
this fact (10$), He first explains that tho antigenic substance in
syphilis is most likely a lipoid-protein combination, Tho lipoid is
regarded as an incomplete antigen (called hapten). If it wore possible
to secure a pure protein from the spirochete, this would probably glee
tauoh noro satisfactory tests.
The antigen of syphilis is not capable of causing true antibody
fo mat ion, but glees rise to two globulina. The first of these is in
soluble and remains in tho cells, the second is soluble and is washed
into the blood stream. This soluble globulin is the antibody whi oh
reacts with tho lipoids in serologic tests. The origin of the globulin-
exciting lipoids is uncertain. They may ooae from either tho splro-
ohetea or the tissues of the host, In any case It is necessary that the
spirochetes be present for these lipoids to appear, Kahn claims that

38
all lasts for syphilis aro based on the soluble globulin. The foot
that this soluble globulin disappear as sow as the exalting influence
is geno plains why serologic loots sometime* fail I the latent stages
of syphilis.
la the same way In which certain diseases generala non-specific
antibodies, it is probable that syphilis dose not produce one specific
globulin* but others In addition*
The fact that true antibodies are act generated in persons affected
by syphilis is shown by the occurrence of positive blood reactions end
simultaneous negative spinal fluid reactions, flam (139) demonstrated
that true antibodies easily pass the meningitic barrier and are found
immediately in the spinal fluid A second point to consider Is the
work of Hoof old (l6o), who proved that the spinal fluid system is unable
to manufacture its o antibodies.
In eases of aeurosyphilis the globulin-antibody amt be generated
in the spinal fluid system, since it is net always found in the blood
On the other hand* true antibodies muid always be present in both the
spinal fluid and blood streams We may say that the spinal fluid and
the blood systems are both capable of producing globulln-satibodies for
syphilis and that these systems work independently.
Almost and Tarbouroich (l&L) state that they have proven there is
always more antibody in the blood stream than in the pial fluid This
report is evidently made tas an incomplete work hew else may we view
their work in the light of the many eases In which there is a negative

39
Wood reaction and a positive spinal fluid meiion?
Malloy* &ahn and %etall (l6a) have notad that the antigenic
lipoids* la precipitation tests* are precipitated by the globulin*
Shis was pointed out long 'before by %stein and Paul (103). bat Mal
loy* Kisfca and ttestail did find of special interest vas that the glo
bulin was not changed in any way* It the globulin is Isolated and used
in & Kahn test, the precipitate my be removed id the globulin sol*
tion still retain its power to giro a gold sol or maetic test.
?ha presence at excess lipoids in the syphilitic spinal fluid has
been noted by Kaaser (163). these lipoids have sons effect on the col
loid teste* and this was proven by irradiation* If the spinal fluid is
treated with ultra-violet light and then a gold teet ran, the curve will
have shifted to the left. Irradiation is known to effect lipoid (l6h).
A part of the imperative reason for testing the spinal fluid is
understood from the above. Moore plainly states th# rest of the case
(£?) Moore and the mm working with Mm at the Johns Hopkins Hospital
have found that the nervous system is not 4mediately involved in
syphilie* She invasion of this part of the body is a later step, but
the serologic tests show the pressnoe of this invasion long before ay
symptoms appear* the first sign at involvement is the appearance of
mononulcoor lymphocytes la excessive numbers. She second Change is the
appearance of a positive globulin teet* lastly there is a positive
serologic reaction*
As these steps go on the treatment must be stepped up, since the

Uo
meningeal mt allow only a small part of bo total medication to
pass Into tho spinal fluid. It Is to bo noted that be disappearance
of be manifestation* of aeorosyphUis are In the same order as their
appearance, the cells and globulin disappear easily, rea under an in*
coraplete treatment* fhe ssrologic reaction persiste, tinder serologic
reaction in this ase is included the gold test, fhis would tend to
disprove any theories as to the meehanisa of the gold test, however,
it nay depend on a certain pert ef the global in exceeding lte normal
amount, not all the globulin,
Kahn, Kline And any other workers have found that precipitation
tests do not reverse as rapidly as complement fixation tests. Although,
in this connection, neither do the precipitation tests bacons fast, as
does tho baeserwann* JJy fast is meant that no amount of treatment will
cause a reversal, own though the disease is clinically cured. Host
authorities agree that a precipitation test, a complement fixation test
and a colloid test should be made with spinal fluids for diagnosis and
following treatment.
In summarising this section, we hare the following consideration}
1) rarely Is the spinel fluid test a primary diagnostic test, if the
physician is given a proper chance to follow the disease, fhe spinal
fluid tests how asmas tissue involvement long before lesions are
evident and should be periodically continued as long * the distas#
exists, 2) there is no true antibody formed, but a seal-specific glo
bulin la its placo, fhe tests depend on globulin-lipoid combinations I

la
the presence of a apiernen t, lipoid flocculation by the globulin, and
colloid flocculation by the globulin) 3) the existing teats for globu
lin are not in any way specific for the especial globulin generated by
syphilis, but are merely dependent on the total globulin-albumin ratio)
4) a combination of tests is most effective in the spinal fluid. One
test of each type should be used, -ha precipitation test is more
valuable than any other type for following the progress of treatment)
3) it is essential that testing of the spinal fluid be started as soon
as a positive blood test appears. If the spinal fluid is positive by
any test the treatment must be for neurosyphilis.
In excess globulin in the spinal fluid may be caused by many other
pathologic conditions in addition to syphilis, tuberculosis and menin
gitis (meningococcus) both lead to an excess protein, the former giving
riee to globulin, the latter to albumin (I07), dementia praecox may
give rise to hypertension, hyperglobuLlnosia and hyperalbsminosis.
Any or all of these conditions may be found, or the spinal fluid may be
normal (165, 166)#
We have also seen that some globulin tests are misleading in the
presence of eclampsia, hepatic cirrhosis, uremia, typhoid fever, lupus
and pneumonia (13# 3* 113)
In globulin precipitation the pH at the time of precipitation ie
of importance# This will be discussed in the section on Sodium,,Vanadate

SODIUM YAHAMOtU AS OOUOIBS
BoyXaaA (167) made an investigatloa of tbs reaction of vanadio
add and proteine. Ho deists this Is tbs first time tbs reaction of
lyephllie sol md protein has been studied. Bsaaoia, metis and gold
sols are all lyophobio, Ferric hydroxide (colloidal) Is lyophille,
hut has a different motion, since it produce* a eopredpitation. The
reason that Boyiaad selected vanadium lies in the f&ot that it is the
only one of the comae metals which ferae poly-acid*. In an add sol
ution the chief add formed is KhVgOjj. the statement is Made that
this and other vanadios pely-aoidt are positively barged. It is also
claimed that proteins are negatively barged in neutral and basic solu
tions, and that in add solutions the proteins become positively
charged.
In the experimental work the concentration of ajVc^ me one gram
atm per 300 liters, Various oddities were reached by the addition of
acetic add* It was found that albumin In a elution cf pH less than
4 precipitated* euglobulin in solutions sore add than pH 5*2 and globur*
Un la solutions of pH less than $ were precipitated, the amount of
euglobulin which a given amount of albumin dll protect is increased by
lowering the pH* Ho precipitation In this protection reaction dll tat
place at e pH of more than 4*
The difference between a globulin and an albumin Is dated as
being the difference in the number of barges it takes to neutralise
each and bangs the pH one unit in the vicinity of the iso-sleotri*

point* it toko six times the numbs)r of hydrogen ion* to satee tills
change with an alborada no with a global In. For this reason th albumin
is meh harder to precipitate by neutralisation. fhero la no known
roaaoa why these effects nr* not additive in the protection reactions*
fhsy nr# not found to bo of such a nature* boylaad proposed that it
sight Ho in a compound formation V tho aglobo!in and salt* this eom-
pouad hawing mm specific reaction with tho poly-van&die add*
Precipitation Is cannot! by the neutralisation of tho charges of
tho protein with an excess of vanadio add. It then Is not an orneas
of the acid, bat an roots of tho protoin, then tho poly-acids should
precipitate vhm they are neutralised, the fact that this does not
happen it probably doe to the outlier partidlo siso, as because the
vanadio add particles only weigh about one fiftieth as such*
Seri (l68) attempted to apply the work of 3eyland directly to the
spinal fluid. He nado use of a solution of sodios vanadate end acetic
acid* His test does act parallel the Waseoraana reaction very well,
only fifty fluids were rm la caparison, twenty-one being basseraann
positive. Of the twenty-one positive fluids, the teet ef eri may be
judged positive with IT Of the twenty-nine %#seraann negative fluids
there it a positive reaction with 10,
fhc method of Start consista of adding spinel fluid Aropwise to
exactly 5 al* of the vanadio add solution* If lose than 10 drape pro
duce flocculation, the test is probably positive* $h pH of the vanadio
acid solution used mat be 3,94, Although the results with paresis arc

hotter than the ovar-al! resulte, there is a lack of sensitivity*
Seri aleo tried to doteminn albumin and globulin in the same
sample by hanging the pi! of the vanadio acid-spinal fluid mixture,
This is admitted to be a failure.
The pH of the no mal spinal fluid is 7*35 to ?*& Thi* changes
on standing to a value between 3*0 and 3,1, The alkalinity is gener
ated by the large carbonate reserve found in the spinal fluid (156,
157). In disease the same pH values are found with one exception,
aeningocoeous meningitis* In meningococcus meningitis the pH is
7&7*U on withdrawal and the value Is not changed on standing (169),
The iso-elestrtc point depends on the salt concentration and lies
between pH h and pH 5 for the meat part.

KXPEHIMKNm.
4 aingle-ooapound, spinal fluid reaction of syphilis was the ob
jective elected* In order to be successful this reaction should show
a clear differentiation between the spinal fluids of syphilitic pa
tients and those of non-syphilitic*. In order to save tie the labora
tory and literature work were begun simultaneously*
the best starting point was suggested to be an extension of
Pfeiffer* work (170)* Reports had been heard of his diagnosis by
crystal patterns* Fortunately no copy of Pfeiffer* s book was avallabia
for sons three months* The present work eould net have developed from
attempts to use the rather ayatie methods he recomends.
Having no idea what might result from attempting to crystallise
metal salts in the presence of spinal fluid, several metal salt solu
tions wore made up* These solutions were made from those alts which
happened to be in the laboratory, no conscious selection was exercised.
The solutions prepared worst KgCrgOy (5, 0*5 & 1j£), Co(HO^)g (10, 5 A
1%), FdHHl^CpOfi) (10, 5, & 1%), CuCLg (10, 5 lj£). VC% (saturated &
half-saturated), AspOg (saturated in 05K SCI), and Bicljj (saturated,
clear solution in 0*5S HCl)
Those solutions were made up wit!) rough weights of materials* It
was recognised that any solution found to be useful would have to be
analysed for metal content and purity*
hile the organisation of materials in the laboratory was progress
ing, an agreement was made with the Florida State Hospital for spiral

fluid ampios. If wa would furni ah shipping material and expense, they
wotild furnish us with from 20-25 freah samples of pinol fluid weekly,
a* long te they were able, Two boxes wore built for the shipping of
spinel fluid sample*. Baoh box was built to carry Sh oft gises test
tubos with eark stoppers. As long as ths tubes were stoppered, the
lid clamped them in such a way that breakage was impossible uniese the
whole box wee broken. All shipping wse by express, the trip requi ring
am 2h-?6 hours.
Peking use of sosas aged spinel fluids roaaining fro s finished
research of ihr* Pollard* s, work wee started* fhs results with KgOpO?
and Co(M0g)g were of no use in the differentiation of syphilitic and
non-syphilitic spinal fluids. Therefore a series of secondary reagents
was also prepared. Thee were made with two objects in mind. The first
was to ehanpo the solubility, tho second to change the pH, For the
former ethanol and acetone ware used, for the latter purpose concen
trated end dilute (1*6) HSO^, HgS0|tt hCl and SE^cH were tried. All
reagent* were put into ene oimoe gises bottles with medicine droppor
tops. The screw tops were plstic.
After making the secondary reagents, work was strain started using
the dichromate Solutions. There wars three aolutioaa cf KpCrpOy, and
ton secondary reagents* Os t ag each combination with both a syphilitic
and non-syphilitio spina) fluid, there remtltad 60 test* to bo es*iidned
after mixing said than again after drying. With throe exceptions this
work gawe no results worthy of mention.
It was found that a drop of syphilitio spinal fluid to which had

kl
bean added a drop of any strength EgCrgO? gave a flocculation when a
drop of 1*6 HHOj? was also added* This reaction did not take place If
the spinal fluid was itomal (non-syphilitie). There was no differen
tiation with any of the other metal salt solutions, and these particu
lar spinal fluids, with the exception of the half saturated vanadium
tetrachloride solution* While the flocculation in this ease had a
different appearance, the reaction appeared to be the saae* This
reaction of VCl^ did not look as promising as that of the KgCrgCty* The
non-syphilitic samples flocculated inside of five dimites.
Que to the age of the spinal fluids, these reactions were looked
on with distrust* Any number of changes could be imaginad to have
token place in the six months the fluids had been in storage* As this
testing was nearing completion, two fresh samples were received* These
were pooled, that Is, each was a mixture of a large number of saaples,
all giving the same asseraann reaction. One of these saaples was
Wassemann positive, the other negative* With both of these samples
the KgCrpO^-HKOj and ?Q1| reactions occurred. Whether the sample was
positive or negative, a flocculation resulted In every trial*
It appeared that the most likely change in a fluid would be loss
in strength ae it aged* For this reason the reagents must have been
made too strong* It was recalled that changing the concentration of
the KgCrgOp five-fold had had no effeet on the reaction, eo ths 1*6
nitric acid was diluted* At a dilution of about 1*100 HHO5 and making
use of 2*5 or 1% KgOr^, a olear differentiation was again obtained a

1+8
good part of the tine*
This reaction had one serious fault* In too many trials the mix
ing was not just right, and Instead of floeoulatlon the result was a
hard granular precipitate* hilo the positive sample always gave
flooeulatloB or granules, the negative sample at tinea gave granules
also* It was seen that repeated tests would be necessary for a clear
differentiation when using the KgCrgOyHKO^ method*
At soon as this reaction was discovered, sn intensive search of
the literature under potassium dlchromato was conducted* Two references
(109* HO) to the work of Taoeone were found* Although the technique
was different, the principle was the same* In placa of the trichloro
acetic sold, we were staking use of nltrlo aoid, but the notion of theae
two acids la vary touch the aaste* Both of theee acida have been used in
the estimation of albumin* Brunei1 mentions triohloro-acotio (111) and
nitric was used by Dourls (171)* neither method is to be recommended
for the estimation of albumin, but the albumin excess is the cause of
the reaction* In the light of these findings, it was decided that
work on the ?C1|, should be the min object, end that the dichromate
method be used to determine whether Its results were as reliable as
those reported by Taoeone*
Several dilutions were made from the half-saturated solution of
vanadium tetrachloride* hone of theee wae able to give a satisfactory
differentiation of syphilitic from non-syphilltio spinal fluid. The
solutions were allowed to stand on the desk overnight, and tried again
the next day* One of these, ho* 11, gave a olear differentiation of

b9
syphilis. The solution, especially the more dilute one, had changed
color In this length of time* At the timo they were made each had a
blue color. After ton hours tho more dilute one were blue-green* the
more the dilution, the sore green and less blue the solutions appeared,
At this time fourteen fresh samples of spinal fluid were received.
Through a misunderstanding those were all pooled. This misunderstand
ing was later cleared up, and individual samples received. Since a
sensitive solution was at hand, these fourteen samples were checked.
All save one gave a reaction which was in accord with its Wassormarm
reaction. The sensitive dilution of VC1}4 was mads by the addition of
20 drops of half-saturated VCljij solution to 12 ml. of distilled water.
The sensitisation was allowed to develop by letting the solution stand
for 10-12 hours.
This shipment of fourteen samples was divided into three groups,
or olasses. The tern class is used by the hospital to denote the aero
loga reaction and the presence or absence of syphilis. Thus class I
fluids are has semana or Kahn positive and syphilitic. Class II fluid
are Wassoraann or Kahn negative and derived from treated syphilitic
patients. Class IZ1 fluids are those tros, non-syphilitio patients and
are Kahn and hassermana negative. The one fluid which did not agrss
in its VCljj reaction (positive) and Wassermaim reaction (negative)
belonged to oaos II. A great deal of interest was taken in this find
ing, sines it indicated that the VCl^ test might be a precipitation
reaction which did not reverse ae soon as the /assoraann reaction.

50
The foot that the vanadium tetrachloride dilution had to bo nade
a day before use was a disturbing natter* This bahart or was studied
and found due to an oxidation of a part of the vanadium. That there
actually was not an oxidation of all this vanadium was made olear by
attempts to uae the solution two and three days after its preparation*
So results obtained on either the second or third day had any crrela-
r
tion with either the %ssenaann report* or the same dilution on the
first day following preparation* On the second and third days the
reaction with a olass X1Z fluid was as likely to be positive as that
with a olass X fluid*
As stated before, freshly made VC1J dilution is blue* Whether it
is allowed to stand in tit# open, or in a tightly elosed vessel, the
color at the end of twelve hours is a blue-green. After $6*1$ hours
the color is green-yellow, and after a weeks time the solution is
almost a pure yellow* When the yellow color is reached, no other change
occurs. After a month and a half one of these solutions was found to
still be the setae yellow*
The colors of the vanadium ions in the various vlenos states
are well known. These were experimentally produoed to determine
exactly what changes wore taking placo. The lowest state of valency
vanadium assumes Is 2+ and the highest is 6+ A definite and dis
tinctly colored solution exists corresponding to each of the valonee
states between two and six* Thus it was found that the colors and
corresponding valneles are* 2+ lavender, 3+ r blue-green, 1++ * blue.

51
5+ a yellow to colorless, and 6+ rod-brown.
With these solutions ns a guide, an eaonoly appeared to be taking
place. The dilutions nade were originally blue, definitely tet rare lent.
They then acquire a bluo-green color which natehes that of tri valent
vanadium. After a woek that oolor has left, and the solution is a
definite yellow color, pentavalent. Since blue and yellow will make
all shades of blue-green, green-yellow and yellow, a trivalent solution
was divided and one-half oxidised slowly. At a valence between four
and five the oxidised solution could not be distinguished from the tri-
valent control. Dilutions were stride of these more concentrated scln-
tione, and no color changa found. In coca* oases the oolor of an ion
in solution is dependent on the oocctmtratioa, but thia is not reported
or found true of vanadium, ftith vanadium solution the color depends
upon the valenoe state, not the concentration.
To prove conclusively the valence state of the sensitive dilution,
it was coropured In two reactions with a known solution in which the
valence of the vanadium was a fixture of i#t and 5+* Both solutions
are capable of forming a red color immediately when a drop of 3% hydro
gen peroxide is added. If the amount of %<){ is small enough both solu
tions will go only to a yellow color on shaking. When this test was
repeated with a solution of VClq the result was a blue solution.
The second experiment to show the valence state was the addition
of dilute HaOH. It there is pentavalont vanadium present it will
coagulate to the extent of darkening the solution when it i# neutral,

52
end turn yellow when the solution is basic* With the known solution
this raaction readily took place, but the resulting solution was
groen, duo to the totravelont vanadium ions still present* the un
known behaved in exactly the same manner* Trival ant vanadium does not
change in basic solution* the coagulation and darkening of the sola*
tion at the neutral point is due to the formation of polymers with
the eaparioal formula VjgOj;.
The formation of VgO5, or its polymers, is prevented in a solu
tion made from VCl^* Such a solution is aeid due to hydrolysis and
this leads to the formation of polyvanadio aeid, probably i|tV^0i7*
solubility of VCl|t is not found in the literature, only the fact that
it decomposes in water* This decomposition is in accord with the
following equation
Wl|,+ HgO s V0++ + 2H+ + liCl" .
The oxidation of the vanadyl ion (V0++) is Inhibited by the
presence of hydrogen ion* To stabilise solutions of VCljt, excel 1 HC1
is added. It was found in the course of our experiments that the atore
concentrated solutions did not oxidise with nearly the speed of the
dilute solutions* This is probably due to a lower solubility of
oxygen and a lowered activity of vanadyl ions* The law of mase
action shows why hydrogen ion is effective in prevention of oxidation
in tiie following equation:
4V0++ + Og + li(H)~ + 0HpO s kHVOg + |*B+
The aeidlty of a solution made from a mixture of VOCl^ and VC1|(

53
la euoh that oxidation la rapid In diluto solution. A stabilised solu
tion was diluted to the turn vanadium content and showed no oxidation
after three months.
After finding the mixture of valonee to be the sensitising prin
ciple, there was no need of making a dilution ten hours before use.
The old, overly oxidised solution was saved and added book to the
fresh solution to produce the mixture at the time it was needed.
The fourteen pooled spinal fluida in the first shipment were
tested with the dlchromate mid nitric add method. Class X fluids
were all positive in their reaction. Class XX fluids were all posi
tive or doubtful in their reaction. Class XII, whieh should have all
been negative, gave all negative results, with one exception. The
false positive at this point, coupled with the fact that Taoeone never
got any recognition for the dichromate reaction, all but led to the
stopping of experimentation on this method. It was decided that a
total of 100 tests would be made with this technique, and the results
cheeked to seo whether It should be continued.
On receipt of a second box of fourteen pooled spinal fluids, the
samples were cheeked with a sensitive dilution of VClJj. The dilution
was prepared in the following manner 1) two class X and two class XXI
fluids were selected at randoaj 2) to 12 ml. of distilled water was
added 20 drops of half-saturated VCl^ solutionj 3) an old dilution of
the same strength was added five drops at a time to the fresh dilution;
U) after each addition of five drops of old solution a test was run on

all four fluida selected.
The four selected fluida gave the proper raaction after a total
of Uo drops of old dilution had been added to the fresh dilution.
Maximum sensitivity was shows after the addition of 50 drops, and at
60 drops the ciaeseraaxm negative fluids gave positive reactions* When
the solution was at a fairly sensitive point (after the addition of
h5 drops of old dilution) all the samples In the box were tested* All
class X and III fluids agreed with their Vaeeeraan reports* Of the
four class 11 fluids in the shipment, two were positive and two
negative to VCl^.
these same fluids were then tested with the di chrome. te-HH0?$
method* All class X fluids oheoked. The class IX fluids gave two
negative results, one positive and one doubtful fro the four fluids*
Class III gave one doubtful result and the reminder negative* the
presence of all these doubtful reactions may be the degree of positiv
ity to which taoeonc refers, but careful examination under the
microscope failed to reveal any difference in structure of the
turbidity. At any rate, these reactions appear to be graded from
positive to negative by the mount of protein present*
Up to this time all fluids tested had been pooled samples. There
is a reaction on the addition of certain spinal fluids to others.
Plaut, Iteuoh and Rossi found this reaction unpredictable, and not
related to syphilis (9)* hile this fact was not known at the tas,
the fluids were known to be altered in some manner* they wer not the

55
characteristic water white, but a turbid white.
The third shipment of pial fluids received contained eight
fluids in each class, all individual samples. The shipment was de
livered late in the afternoon, and was kept on lee overnight. In the
wonting an attempt was mode to prepare a sensitive dilution of VCl|j.
The sane method as used before was used, with the exception that only
six ml. of HpO and 10 drops of half-saturated VCljt were taken to make
tho fresh dilution. Both ciase XII samples taken for testing the solu
tion gave a great deal of trouble. A sensitive dilution was not suc
cessfully prepared. The fluids were left on the desk until late in
the afternoon.
At this time four other samples were selected for testing the
sensitive dilution. The afternoon trial at sensitization of a VClj,
dilution was successful. He trouble was encountered in any way, After
the soaoitivo dilution was obtained, all tho fluids were tested. All
class X fluids agreed. Class XI fluids gave five positive and three
negative reactions. Class XXX fluids gave seven negative and one posi
tive reaction. This positive was one of the fluids used in the morning
attempt to sensitiza VClj, and accounted for part of the trouble, at
least. It receive* treatment in the section on Discussion of Results,
By the dichromate method all elass X and XI fluids were positive,
as well as the false positive which carao out of class XXX with VCljt,
All ether class III fluids were negative,
ifith fresh fluids on hand it was decided to chock all the metal

56
solutions previously prepared plus a fon moro which it wo* thought
might ho effective. CobIt mis nitrato 110%) foiled to give a reaction
with either class l or HI fluid. Saturated arsenic trioxide also was
without effect.
Cupric chloride gave a reaction with both class I and III down to
C.1? dilution, and then failed to give a reaction with either syphili
tio or non-syphilitio fluids. Cuprous chloride (saturated; gives a
reaction with either class I or III fluid, and when made half-saturated
reacts with neither class I nor III. Between these concentrations it
is possible that CupClp will differentiate syphilitic from non
syphilitic fluid, but the instability is eueh thst further work was not
indiosted at the time,
Bismuth trichloride in dilute BCl might be used to good advantage,
but ary reaction of the metal is masked by the FloCi precipitated, fhe
amount of HCJ needed te clear this precipitate is sufficient to floccu
late the protein of a non-syphi 11 tic fluid. Ferric ammonium oxalate is
not a stable compound in solution, and it presents tho sane difficulty
as #ici^. When added to the spinal fluid the reaotion is masked by
the Fe(oH)^ precipitated. Vemos (?U) used colloidal Fe(OH)a¡ in his
first test and this proved unsuccessful.
Molybdic acid gives a precipitate with negative spinal fluids,
even at trace concentrations. It is dear that a great majority of
these metal ions are able to precipitate albumin, as well as globulin.
Silver nitrate is capable of giving s precipitate with either class I

57
or III fluid*. A* the strength of the solution is out down, the pro*
cipitation stops and a milky agglutination takes Its place. Apparently
protection of the silver is nsedsdy Collargol, containing protected
silver, may be of scam use (69).
Tho next fluids received were tram St. Mikes Hospital, Jackson
ville, /hilo these were all sots whet cloudy, they checked with the
vanadium chloride. lino of these was marked only syphilitic, gold sol,
positive. This also gave a positiva reaction with VCl[t. The weekly
box of fluids from tho State Hospital gave good clear cheeks with class
X and III fluids. As usual, class XI was divided, the eight samples
giving h+, 3 and 1 results. Fro the thirty-five samples re
ceived in this week, only fourteen were checked with dl chromate and
nitric acid. Those were in line with the ttaeseraans reports, with the
exception of those in class IX, all of which were positive.
The fifth box of spinal fluid samples from the State Hospital con
tained ample* which were, for the most pert, quite cloudy. In spite
of this, tho VCljj, solution, prepared end sensitised in the usual manner
was effective. Class XXX gave eight negative reactions, class XX, four
positive and four negative, class I, six positive and two which were
recorded as doubtful. These doubtful fluids contained bacterial con
tamination, beyond a doubt. The first of them gave two weak positive
reactions and one very weak positive reaction* The second questionable
sample gave one negative and two positive results, the positive reactions
being weak. Since studying other tests exhaustively, It is apparent

58
that this whole box of fluid* should have been discorded due to bac
terial contamination. However, trey were tested, and the results are
not so far out of line es to warrant striking then froa the record.
This sens series of samples was tested with dichromate end HSOjf.
The eight positive syphilitic and the eight negativo nca-ryphilitic
fluids gave perfect resulte. A deviation from previous experience was
found in the class II steeples. Three of the eight were negative, four
were doubtful and only one was a true positive. The three negatives
and one doubtful corresponded to the four negative results obtained
with Wife.
At this tiste an attempt was made to prepare a sensitive dilution
of VClfe from the original saturated solution. Ho reason was seen for
not using this, mixed with an equal volume of water, since the half-
saturated solution in use had been made from it In the sane Banner.
The sensitive dilution was being made now in smaller amounts, the usual
technique being to take 6 el. of distilled water, add to it 10 drops
of one-half saturated VClfe solution and then sensitise by the addition
of an old (oxidised} sensitive dilution. Trial testing still was used
to find the specifio amount of old dilution to be added.
To make use of the saturated solution of Wife, 5 drops each of
the solution and distilled water were added to 6 ail. distilled water,
ithile the fact that drops arc not accurate for measurement wee
recognised, it was supposed that this would be accurate enough for the
purpose. Although several attempts were made to sensitise solutions

59
made i this uanmrt tha best of the lot gave ene falta positiva
with tito sagatlva samples and two negativa results an Wessons am
positiva, syphilitic ampias. Thinking that this was not accurate
enough work, 20 ml, of exactly half satura tad solution was mads. This
was placed in tha ttse type bottle as the other halfsaturated sola*
tion and marked "1/2 SaVd VCl^ Ho. 2." The earn# dropping top was
then usad for making a dilution from this Ho, 2 solution as ms usad
on the original h lfsaturatad solution, Tha results in this casa
ware no bettor or worse 'than those got by use of the saturated solu
tion in the first place, Tbo fact that alcohol takas some tine to
mix with water and form a solution which may be duplicated has been
known to the liquor trade for years* However, no record of this it
found concerning wetal salts, That soma partial oxidation is able to
taka place in tho more dilute solution is possible, but it would have
no effeot on the results, As long as an oxidised solution is being
added for sonsitication, the only result would be the necessity of
using moro of this oxidised solution, whan preparing a sensitive dilu
tion from the saturated solution.
It was found that with tha next shipment of spinal fluids, either
tha He* 1 or Me* 2 half-saturated solution of ?Cl}j gave the s me re
sults, Close I and XII ware in accord with their Waesefwfe&n reporte*
olasa II wae equally divided, four positive and four negative reactions.
It must be that a definite period of aging is necessary after the
dilution of saturated vanadium tetrachloride. To cheek this, the

6o
saturated solution was again used to make a dilution, and again it was
imposslbla to sensitise it* If one discounts the fact that a definite
nixing timo is required* the only alternative is partila sise* This
could not enter in, since there is no polyvanadic acid domed until
after oxidation to the peatavalont state has taken place* Most, if
indeed not all, of the peatavalont vanadium is added in the sensitiza
tion, and this has be*m the same in each case*
Several questions as to technique and concentration were taken
up* The use of droppers for measurements was decidedly a thing to be
avoided, sc 25 pipettes, 1 tal., graduated in 0*1 and 0*01 mis. were
then employed* Approximately equal volumes of spinal fluid und sensi
tive dilution of VClj, hud been used in all tos ting, and it was found
that exactly 0*1 ml* of each gave a test which was easy to read ana
handle* Tills volume relation was used throughout the remainder of the
work* A separate, clean pipette was used for each sample* After us,
each pipette was cleaned with cleaning solution and rinsed six times
with distilled water* After rinsing, the pipettes were dried by draw
ing worn air thmifjh thorn with a water pump.
In order to save time, the ratio of oxidised (old) solution
needed to sensitise a given volume of fresh solution was determined*
Up to this tita sensitisation had required upwards of an hour each time
tests were to be run. Trial testing was used to determina the ratio of
old to frssh solution, and it was found to be 1*5 This ratio of 1 ml*
of old dilution to 5 ml* of the freshly made was used with success for

6l
a period of two monthb.
The concentration of vanadium* as vanadium tetrachloride, vas
determined in the sensitive dilution. After a consideration of the
many methods for vanadium analysis, the colorimetric method iras chosen*
In the first place a very dilute solution as at hand, and this would
require the use of raicro-equipsiaat for gravimetric or volumetric
analysis, his equipment was not available, in the cocona piece, a
pure sample of ammonium mota-vanadate was on hand for preparation of
known colorimetric standards, A direct comparator with split field
viewing was used for this determination. An Hell go type instrument
would have been much moro efficient, but none was available,
A known weight of the KH^Vlh; was made to volume in a volumetric
flask, Into one cell of the comparator was placed 1 ml, of the
sensitive dilution, 0*5 ml, of is6 HgSQjj and 1 ml, of hydrogen
peroxide. Into the second cell wore placed equal amounts of the
KgiiGk and %0g, followed by a definite volume of the known solution,
made to 1 ml, with distilled water, *vhen the approximate strength
of the solution had been found a new unknown was used and the standard
mixed in the same order as the unknown, When a color match was at
last found, the determination was seen to be accurate to 35?£, since
It was reproducible to 0,01 ml, of the standard, The concentration of
VCijt was 1,U*10 grams per liter, + Later analyses verified this
result*
The reason for aa.e early difficulties was accidentally discovered

62
during the course of routine tasting# Fluids wore being usad which
hod .just boon renovad from the ice-box, and th eeneltive dilution,
mad by a definite volume relationship gave thro falso positiva re
actions in succession. In the routine work, throe or four slides wore
lined up on the desk, spinal fluid from an equal number of samples
pipetted onto the loft hand side of the slide and dilution pipetted
onto the right hand side, ^he two liquid* were then drawn together
with a email glass stirring rod, stirred end the slides rotated by
hand for thirty eeeonde. As each slide was mixed, it was replaced on
th desk and allowed to remain there a few minutos before reading. In
e majority of the positive reactions th flocculation is easily seen
in an indirect light with the naked eye as soon as mixing is complete.
In the Instance referred to above three Kahn negative samples
had been so treated, and all gave a visible flocculation, the work was
being done under a desk lamp, tilted close to th top of the desk to
provide indirect light, tfhile starting tc make up a second dilution,
believing an error had been made, these elides wers left under this
light. Ihile making a now sensitive dilution, it was noted that the
false positives had apparently cleared, Microscopic examination con
firmed this fact.
In order to bo satisfied on this matter, slides were made with
spinel fluid at 10C, end 25C, The reaction of flocculation took
place In positive and some negative fluids alike et lOOC. Only Kahn
or Wasscrmann fluids gave this reaotion at 25C. It was found that

63
if these false, oold-induoed reactions ere lloved to oomo to row
temperature, there would be & re-diasolving of the floe calato. It
was also found that true positive reactions were not re-dissolved by
cooling or heating, but remained until the water had ewpletely evap
orated. The evaporation of a mixture of sensitive dilution and nega
tive spinal fluid at row temperature did sot rosult in floooulation at
any tine in its course,
to this time the finer points of the investigation had been in
tentionally put aside. The nature of the flocculate, the pH at which
it occurred, and similar questions now required answers, a total of
one hundred mid sixty-two spinal fluid sstaples had been cheeked by the
VCljj dilution. In these tests one ^assenaann negative fluid had given
a positive remeton, and two osseraaaa positive fluids had given
doubtful reactions. From these samples 100 had been run by the di-
chrcBUit e-nit rio acid method. This latter method had been tapered off
for several reasons, in the first place it gave rise to too many falso
reactions, and too many doubtful reactions, la the second plaee the
solutions wore not constant, KpCrpOf, which is not supposed to chango
with ago, gets more and more sensitive, so that a fresh solution must
be made up each week, Mltrio acid is also able to change on storing,
both through nitrite fomation and vaporisation.
The nature of the spinal fluid is such that the precipitate ob
tained is limited in its possible composition. If It were less
abundant, it might bo any of a number of things, ?hs amount Is such

that it is macroscopic in dimensions, and is obtained from 0.1 ml. of
the spinal fluid* Clearly it must be oapossd of either the proteins
or vanadio asid, or sorae combination of those materials* The proteins
have boon dismissed, and their significance in syphilis pointed out*
In order to determine whether the preoipitate was organ!o or inor
ganic, a series of maerosoopio teats were run*
Kith samples having a volume of 2*3 ml* or more, It was possible
to determine the nature of the preoipitate and find the pH of the solu
tion from which it oame* A Beckmann pH meter was employed for those
determinations* for its operation were needed 3*5 ml* of solution in
addition to 2 ml* for preliminary rinsing of the electrodes and cell*
Equal volumes of spinal fluid and sensitive dilution were mixed in a
centrifugo tube, and If flooculetion resulted, it was centrifuged out
at 3,000 r.p.m. The pH was then determined on the supernatant liquid
and the preoipitate examined separately* Before using samples in this
manner, microscopic tests were run to determine their reaction,
A total of 70 pH determinations were made, and 2 of these fluids
gave positive reactions both micro and raaoroseoploally. No signifi
cant difference in the pH of a positive reaction end a negative
reaction exists* Either reaction gives an average pH of 3*9*
After eentrifuging out the precipitate, its solubility was deter
mined, In no case was the solubility tost positive in distilled water*
and In every ease there was solubility in dilute sold, base or salt*

65
This Is eharaeterlstlQ of globulin* throe of the precipitates were
restored to crucibles and fused, The characteristic odor of burning
protein was very strong, and a very slight residue of yellow powder
remained. The residue was soluble in acid, and proved to be VgOg*
the precipitate was thus found to consist of globulin and vanadium
ponteadde.
To determine whether the VpO*; or hydrated vanadio acid was tied
into the precipitate in a compound, or just carried down by the
floooulate, four of the precipitates were washed with distilled water
repeatedly. After the third washing no test for vanadium was obtained
in the wash water, and the precipitate had changed from a grey color
to a pure white. Fusion of one of these samples of globulin gave no
residue* To check the amount of vanadium being adsorbed by the globu
lin, vanadium determinations were made on the supernatant liquid ir>
two oases* The liquid used was that from the pH meter cell, after pH
had been read* The liquid should have contained 0.7205 g/l*# end
was found to contain 0*6750 g/l* of VClj^. This amounts to a loss
of about 6/ Since the analysis is only accurate to et best, it
may bo stated that the amount of vanadium adsorbed is not the amount
in the pentavalent state. This is at least 16.675?, the amount added
for sensitisation.
While working microscopically with some of the negative fluids in
lis series, a peculiar occurrence was noted* In a few eases a floccu
lation took place, but on centrifuging the macrosoopie test of ths seme

66
fluid so precipitate was found* Hevartheleas, tho si ido toot* ware
showing falso positive reactions. If this was an excess albumin, it
should bo soluble in water. For sane tine it had been known that
water did not ohange the aspect of a true positive reaction, once it
had taken place. Thus a modification was made in the procedure. To
0.1 n&. of the spinal fluid was added 0.1 ral. of the sensitive dilu
tion in the ssne manner as before. If there was then floeoulatloa,
this was allowed to set for a minute and 0,2 ml. of distilled water
added. After stirring and allowing another minute to pass, the slide
was examined. Flocculation was persistent only in the positive fluids.
All fluids from number 13k were run microscopically by this method.
One fluid, marked non-syphilitic, ^assorraaim negative, gave a
true positive reaction with vanadium tetrachloride. The fact that the
precipitate was a mixture of globulin and VpOq was verified, While
the amount of globulin got by centrifuging was pite all, its
identity was unquestionably proves. The cense of this reaction is
discussed later.
Although this phase of tho investigation covers sons 92 spinal
fluids, only ?0 of these wars of sufficient volume to allow pH
determinations. The decision was made at the outset that no special
equipment be purchased for pH work. It is possible to take a pH read
ing from each slide with the proper equipment. After the probable er
ror of the mean had boon calculated it waa seen that tho method used
had been satisfactory in all details* The large deviation between

67
individual ampio i not hard to understand, Each fluid hue a dif
ferent alkaline reserve and original pS, While there ia little varia
tion at the time the fluids are drawn, standing causes then to
become alkaline. This takes place spontaneously, but depends on the
tightness of the stopper, heat and i# nature of the individual fluid*
Since all work of this type is statistical, large deviations are not
unexpected.
For the preparation of tide original saturated solution of VC1J4, a
sample which had boon on hand at this laboratory for years was used.
This was the so called solid salt, VC1 is in reality a red liquid,
highly deliquescent, Mo literature reference could be found to the
material sold under the name Vanadium Tetrachloride, C,P. (solid).
The original sample of salt was taken by a member of the inorganic
preparations class for purification. This sample was a dark blue-green
mass of non-crystal line material. It looked highly impure and this
student had the idea that it could be purified. At that time the use
of this salt had just been begun, and there was no reason to prevent
such a proposed undertaking. In the course of the purification all the
material was ruined. This unfortunate occurrence left the laboratory
with 25 ml, of the saturated solution and a like amount of half-
saturated solution. Over 250 fluids were examined before mere VClj,
was obtained, and oven then thorn was sufficient solution on band to
run about 100 simples.
Since the solid salt, VClj,, was listed in the catalogue of the

68
Vanadium Corporation of America, no thought was given to securing
a second saraplo until near the end of the experimental work. At the
time samples in the eleventh shipment were being examined, the ques
tion was raised as to whether the solution was just a chanoe result,
made from impure material* If a matter of ehanoe entered the making
of the solution, then a very difficult analysis, on almost a micro
scale, would be required to duplicate the solution* Examination of
the saturated solution gave hope that this quantitative analysis would
not be necessary for anything but vanadium. On evaporation of the sol
ution the only residue was VgO^. The solution gave a slight flame
test for ^a, and nothing else*
An order for solid VC^t was sent the Vanadium Corporation and
their reply was quite disheartening. Since there was no sale for
solid VCl^, thoy had discontinued the preparation and had no idea that
there was any available in the country. Enquiries were then sent
several houses dealing in rare chemicals, before attempting to prepare
any of the material* A small supply was finally located at the
A* P. Maekay Company, New York* An ounce of their VCl^ (solid) was
purchased* This was found to be praetieally insoluble In water) and
the small amount that went into Solution gave the green-yellow color
of a mixture of pentavaleat and tetravalent vanadium*
A mall sample of the material was placed la a crucible and
ignited* At a very low temperature there was a slight amount of
material volatilised* The volatile portion fumed in the air* Vanadium

69
totraohlorido le veletile at low temperature end very easily hy
drolysed, the conclusion was reached that the material purchased did
have a very low content of VCl^.
On continued heating a second portion of the sample volatilised
This took place just as a dull red heat eras reached. Deposited on a
cold watch glass, thla had the properties of vanadium oxychloride
(vanadyl chloride, VOClsj), When this material was dissolved in halogen
free nitric aeid the presence of vanadium and chloride ion were easily
demonstrated.
After the sublimation of VOCI3 k*n completed, there was no
change in the residual material, oven at a bright red color* Cooling
the crucible to roost temperature, there retrained an orange-yellow,
miorocryatal lino material* This was proven to be vanadium pent oxide
(VpOtj), the solid, C*F* VCl^ was therefore a mixture containing VCty,,
VCCln and VpO^, In addition to the three compounds proven present, a
nail percentage of VoClg is probably present.
Of the various methods it is possible to use for the preparation
of VCljj, the one used to make the sample purchased was ths passage of
a stream of chlorine gas over heated V#Ot*, This would yield all the
produets found, and that suspected* It was recalled that the original
sample of saturated VClj, was made from a material which was only
partially soluble* Bo doubt exists that It is till 9 mixture of several
salts which bears the trade name Vanadium tetrachloride, C.F
In this mixture of vanadium compounds, the only soluble salts art

70
the VC If, end VOClp, The VOGlg ie a very rare oompound and *o
unstable that only traces are likely to be present* as far as could
be predicted at the tine, however, any new solutions should be nade
from such a mixture, and not from pure VClj,, in addition to greater
ease of preparation, this also allowed for the difference In acidity
which would result from solution of a mixture of VOClp and VC If, and
solution of pure VClf,. It Is known that the very acid solutions of
Wlfj made at this laboratory several years ago do not undergo any
oxidation whoa diluted and left exposed to the air* this Is not a de
sirable effect for the present work. As far as is recalled by the
director of this earlier work, liquid YClft alone was used for the prep
aration of these samples* The early work was not ever completed, &/
written up, so no absolute knowledge of the solution referred to could
be got.
A simple apparatus was used for the conversion of the old material
on hand back to a mixture actually having some VC If, content. In order,
the apparatus consisted of a chlorine generator, CaGlg drying tube,
tube containing the a ample, a safety tube and an exit tube passing over
a ysf* solution of HaOK. For the generation of Clg, concentrated SCI
and hypochlorite were used* The sample tube was so arranged that it
oould be heated*
About 2 grams of the mixture was placed in the apparatus and
chlorine passed over it for a few minutes. With a steady stream of
Clp going through the apparatus, heat was applied to the sample tube*

t
71
For this purpose & moving Punten fimo* 1/2 inda in height* me used*
After the hooting bed been continued for ninety minutes the apparatus
ms taken down and the sample allowed to cool* During the course of
the heating a mall quantity of red liquid distilled from the reaction
tube to Hie safety tube* this was found to be VClj^, After cooling*
the sample ms removed end broken up*
It ms decided that no attempt would be made to produce a dupli
cate saturated solution, therefore the sample was treated in centri
fuge tubes with an excess water* A deep blue color developed in Hie
solution* The solid residue ms centrifuged out and the solution fil
tered, using very fine filter paper* This solution was labelled con
centrated VCl^ and put on the shelf* The next step would be the analy
sis of this and then its dilution to a vanadium content the same as
1/2 saturated Wl^. Vanadium analyses were made of the newly prepared
solution and of the half-saturated VCljt solution* Tho method ms the
sane as that previously outlined. After calculation had boon com
pleted the new solution was diluted so that its vanadium content was
equal to that of tho original half-saturated solution* This new half-
saturated solution was allowed to age for a week and then used in
parallel tests with old VClj^ solution*
Ho difficulty arose in running the last 70 semipis with sensitive
dilutions prepared from both of the half-saturated solutions on hand#
Both sensitive dilutions were analysed for vanadium content and found
to have l*iiUlO gmas of tClj^/liter* The average pH of the sensitive

72
dilution prepared from the old solution differed only 0.03 pH units
from that of idle new sensitive dilution. Considering errors nade in
dilution, the sensitivity of the Beokaann pH meter and ell faetors
involved, the two solutions were judged to be equivalent.
In order that ae little vanadium solution be wasted as possible,
the sensitive dilutions were prepared by the addition of 0*35 ml* of
half-saturated VCl[t to 3 ml* of HpO. The maximum sensitivity of this
solution was got by the addition of 0*62 ml. of yellow, oxidised solu
tion. While the oonoentratlon is the same ss always, the ratio of
new to old was changed by this work from 5*1 to 5>0*9U This changa
may be due to the use of a more oxidised solution of half-saturated
VClj, than originally used. The slightly lowered pH in the older solu
tion would indicate this feet. Bines a pura yellow solution was used
throughout the work, once the ratio was established, there is not much
oh anco that it would be more oxidised one time than another*
This change of ratio for sensitisation took place over a period
of three months. It amounts to 6$* If the sensitisation ratio wars
determined each month there would be no difficulty arising from the
gradual chango. Ae much as an eight percent change does not offset
the sensitivity to a groat extent, but on some few fluids, lass than
105$, the ratio arnst be accurate to25$ or the resulting reaction will
be weak enough to cause doubt*
During this investigation the question of the offset of sensitive
VClj^ dilution on spinal fluids from patients suffering from certain
diseases other than syphilis has been considered*

73
Enquiries were sent the State Board of Health mod tho State Tuber
culosis Sanatorium regarding tho furnishing of sample# from diseases
other than syphilis. It was found that at tho time there wore no suoh
samples available* laboratory work was stopped at tho end of May, to
bo resumed In July,
On the resumption of work moro enquiries wore made as to a possi
ble source of diseased, non-syphilitic spinal fluids* Tho State Board
of Health laboratory gave the only enoouraging reply. They stated
that any suoh fluido would be raved for our testing* The fluids wore
not hipped for fear of eontsmlsatioi,
Anticipating the test work to be done at Jacksonville, an
examination was node of the VCl}t solutions in the laboratory* A black
precipitate was found to have formed in each one* This say have been
due to the heat, or eoatnot with the rubber In the topper* The solu
tions hod been left In an upright position and had been overturned acci
dentally at some time in the muster* The precipitate was filtered out
of one of the solutions of half-saturated TGI}, and the solution
analysed for vanadium content.
Whereas the solution had originally contained 13*9U g* VCl}, per
liter, the aoneentratian was now found to b# 15*25 R* Pr liter. In
order to make a solution the concentration of the sensitive dilution
(lJ&lO g# per liter), 0*& ml# of this solution were made up to 5 ml.
This solution was allowed to oxidise for sensitisation use in the work
to be done later.

7k
Allowing the State Board of Health a week in which to save seat
plea, a trip was made to their laboratory to tost thorn. On arrival
it taras found that seventeen spinal fluids had been saved. These
were non-syphilitic steeples which had been sent in for Ashn. Eagle,
quantitative protein and globulin determinations.
A sensitive dilution of vanadium was prepared, using one ml.
of the solution prepared a week previously to sensitise five ml. of
the freshly made dilution* Is order to check this solution for sensi
tivity, three Kahn positive and three Kahn negative samples were
tested. All gave perfect confirmatory reactions.
The seventeen special samples were them run and each gave a
negative vanadium test, four of these fluids had exosss globulin con
tent reported to the physician attending the ease.
At this point laboratory work was stopped. It wss felt that the
test was ready for hospital work of a routine nature. Only by such
work can the true worth of ary test be established.

75
THE PKEPAiUTION OP A SSWSITIVK VAHAJDIUM SOLUTION
POR SYPHILIS DETECTION IN SPINAL FLUIDS
Vanadium tetrachloride, C*P*, solid is first troatod with dry
chlorine gas and mild heat for one to two hours* The solid material
resulting is leashed with water to yisld a blue solution* If 2 grams
are used as starting material* 50 ml* of water is sufficient for
leaching* The solution is analysed for vanadium content and diluted
to give a concentration of vanadium corresponding to 13-H* grass of
VCl^ per liter* This solution is allowed to age for one week*
After aging the solution* it is oolorimetrleally analysed for
vanadium as VCljj. Prepare 5-6 ml* of a dilution containing 1*14*10
+ 3/S grams of VCl|, per liter and allow this to age until a yellow solu
tion results* To hasten this reaotion of oxidation one drop of HpOg
may be used*
Prepare 10 ml* of fresh dilution containing 1*14*10 grams of VCl^
per liter* Secure several weakly Kahn positive spinal fluids and Kahn
negative spinal fluids. Divide the fresh dilution into 5 equal parts*
and to eaoh part add a known volume of the yellow* aged solution
which has the seme vanadium content. The amounts added should give
volume ratios of fresh dilution to old dilution of 5/3*9* 5/3*99
9/U 5A*5 ad 5/M*
Each of these mixtures is tested with the spinal fluids in the
following manner* Place 0*1 ml* of the spinal fluid on a microscope
slide and beside it* but not in contact with it* place an equal volume

76
of the mixturo. The two solutions oro mixed with a small gloss rod
end observed under the low power of the microscope.
To each slide on which is deposited a floeoulent precipitate is
added 0*2 tal. of distilled water. At the end of a minute each of
these slides is again observed.
Of the mixtures of fresh and old vanadium dilutions, one should
give a very sensitive reaction, differentiating the Kahn positive
epinal fluids by flocculation from the %hn negative spinal fluids
which do not flocculate.
The pH of the sensitive mixture should be 2.6-2.7* The ratio of
dilutions which is most sensitive may be used for at least a month
without being re-determined. The more concentrated solution (13-11*
g. VCl|t per liter) should be stable for three to six months if kept
tightly stoppered and in a oool place. Rubber is not recommended for
stoppering vanadium solutions it has some catalytic properties for
their oxidation apparently.

77
TABLE I
THE RESULTS OF V01* AMD %8r*** REACTIONS
OH SPIHAL FLUIDS
1pffiTfCTT~
!f?l.l.P ....
ftassemsim
reaction
**liT
reaction ...
...JWHULtri...
1 (fooled)
+\
4
4
8 (pooled)
*
8
4*
4 ** 4*
4
4
4*
4 *
4
3
**
!
4
6
*
1 4#

7

**
*
a
T*
w
4
8
T
**
4
10
T
*
4
11
T
4
12
T
4 4 4
4
13
4
4
4
14

4
4
IS
4
4
4
IS
4 4
4
17
*
44
m
18
a*
**

ID
**
4*
£0
*
**
44
£1
f
4
4
T
m

£3
T
4
Jt
24
T
**
4*
£3
4
4
4
£S
4
4
4
£7
4
" 4
4
m
4 ;'
. 4
4
£9
4
4
4
30
4
4
4
*+ a syphilitic, *&ssersaaa positive#
- no-syphilitic, wg^swnaami negative*
aMultiple teste, mt a ol$n of strength of reaction.
*T a treated syphilitic, Waecemaan negative.
doubtful.

#80X$138S 04E&& f^*#|?Si8
SotaoxTCJ PC TO* I peAioooa apxntj Wf%
0*
-' 4
' '' v't > :. ..*. \f- .<
0. r- .
39
04.
ts
40
0k
09
4*
04
4
69
**
' -
as
*
4
*4-
4%.
'V' ; -
99
44
99 '
FW
4
4
4
99
4
4
4
§9
4
4
4
89

4
4
T
4
4
4
0
4
4
4
6*
4*
4
4
80
4
4
4
0

4
£
09
4
4
1
9*
4
*
£
I*
4
4
£
90
Â¥
4
£
80
4
4
£
t0
4
04
00
4
4
£
ee
#
4*
4
99
4
**
04
9
0
*
-
99
4
*4
S2
4
**
04
09
0
m
'0
e
4 4
4 4
4 <*
m
44
m
4
12
m
t Iwotrp
Ww5I
.*gaasi
mpatmwrweet
S& SOL
'l^garstaigg
-gRUiuaafe
((EnaiXKoo) i mi

TABUS I (OHTISBD)
79
plual' jSbT"
mifeuatesaL
ItaEh'
TSXT
Xoaotlon reaction.
werr***
K0r07
potion.
63
64
T
as
a
66
T
f
66
T
a
67
T

4
08
T
4
60
T
4a
4
70
T

71


7£
4
+
4
76


74
4

76
4
4
4
76
4
4-
77
4
4
78

4
79
Sklrsaeni S
striSr^spitai
raECtion
** a*
4
80
a
m
a*
81
a
m
a*
88
gold sol a
4
4
83
+
4
4
84
4
4
85

4
86

> '
*. 4
87
4
4
86
+
4
4
88

4

80
TABLE II
RESULTS OF VG1 AS ^Of*Or*BSi0* REACTI0H3 XT8 SFXJtAL
fluids shoots aosisg effect o 1/2 saturated vex*
Spinal' f^uidt Weasemann
Vifr* reaction ktOPOr'^,
JKi
JMt.
jMftiL,jamaiaeL
90
**
m
m
we
91
m
*
98
ee
-
4#
*
93

m
4
94
*
4
m m <|#
P
96
*
m

a#
96
a*
m
ep
m
97

r
1*
m
98
' T
not
i .'?*- >
99
T
run
* i -
H*
100
T

4
4
101
T

p
HP
108
T
4
4
103
T .
*#
#
104
T
#
4
£
105
T

4
£
106
Kahn
reaction

4
4
4
107
4
4
4
4
108
4
4
4
4
109
4
m
4 4 44
4
no
4
m
** *
4
in
4
4
4
4
118
4
4
4
4
ns
4
4
4
4
114
Single fluid
from St# Luke#
laaeermsm
4
4
4
4
1Humbers 109 and 1X0 would bo reportad as doubtful#
the reset Iona being so uncertain and weak#

81
TABLE III
RESULTS OF TO* ABB J^C*e0T-H80* REACTIOHS
WITH SPIRAL PLtTCDS
SpiiM'Bm rir '" lMD 'Wx^ ',rhg<^07-M6*
SMgraent 7
US
#
<*>
<*
na
m
*
117
**
a
m
na
rt*
44
m
119
¡
120
**
121
m
r
188
-
o
123
T
m
184
T
4
183
T

126
T
4
127
T
188
T
4
129
T
4
130
T
-
131
4-
4
4
132
4
4
133
4
4
134
4
4
135
4
4
4
13
4
4
137
4
4
4
138
4
4
Shiment 8
Sfen
reaction
13
4*
m
not ran
140
m

hereafter
141
44
m
*
142
m
4*
*
143
m
m
#
144
44
m

145
m
m
*
146
*
M4
#

TASU XII (COHTIHOED)
Spinal fluid lialn V01* .aOraOrvoo
aiBBl9 a*ar rocotlon ruaotlon raaotlon
147
iyi
(Ooatnwoa)
4
*
140
ir
*
*
140
T
4

150
T

*
151
T
4

15B
T
m

155
T
m
#
154
f
m

158
4
4-
*
156
4
+

157
4*
4
#
158
4
4

159
4
4
*
160
4
4

161
4
4

im

4


83
TABLE IV
RESULTS OF VOX* REACTIONS WITH SPIRAL FLUIDS* SHOWISO
pH ASD COMPOSITION OF PRECIPITATE IF FORMED
Spinal finid
NKtita. nwabar...
Ha
mr
resetIon
asr**-
pa
Preciptate
163
afea..?
** m
6*7
164
m :
*
5*0

165
vs
m
6*0
as
166
> S..
vs
6*0
m
167
m
V
5,7

168

**
3.9
vs
169
4 m

5,9
*
170
', J
vs
5*7

171
-
*
#**

172
-
m
6*0
-
173
T
X
**#
4*
174
T
m
* *
175
T
m
5,7
as
176
T
m
* **
41*
177
T
m
5.7
m
178
T
as
3,9
m
179
T
V*
3.9
VS
180
+
' 4-
# ,
globulin +
181
4
*
#
globulin 4
18£
4
4-
, *
4
183
+
4
***
globulin 4
184
** *a
6,1
m
183
6*0
m
186
V*
*
6*0
as
187
' a*
a#
3*7
as
188
-

6,0
as
189
*
vs
6*0
as
190
*
4#
5*9
m
191
vs
m
5*95
v*
192
Â¥*
** ,
vs
193
vs
4V
3,8
4
194

V*
8.0
4,
19
V
**
6*15
VS
^Fluids la very bel shape on roeoipt.

table iv (Gomimrn)
Ipinci Huid
seraalt numbs*1
. ..reaction
reaction
pi
Precipitate
196
Mpaa?- M L%>tttlnu041
** #
m
197
T
e*
* *
m
im
f
*
5*85
*+
im
Â¥
m
6*0
m
POO
f
£
3*3
£01
f
m
5*9
nm
4
4
6*3
globulin 4
£03
4
4
6.1
globulin 4
5504
4
4
6*0
^.obulin 4
£05
4 4
Shimant 11
* *
not run
£06
*
m
6.0
ee
£07
m
6*1
-
£08
**
s*
5*6
m
£09
**
5.95
m
£10
#
m
5*85
*P
£11
M
m

£12
4*
m
*#
*
£13
*>
*
5*8
*
£14
T
4
5.7
globulin 4
£15
T
m
5*9
m
£16
Â¥
m
6*8

517
Â¥
m
5*9
m
£18
T
as
5*7

£19
T
m
5*8

£20
T
4
6*8
globulin 4 V
£21
JP
4
6*0
globulin 4 V
£28
4
4
5*3
globulin 4 V
: £3
4
4
6*3
globulin 4 V
£24
4
4
5*9
globulin 4 V
£25
4
4
6.8
globulin 4 V
&£
4
4
,6.6
globulin 4 V
a 7
4
4
5*9
globulin 4 V
£88
4
4
*
£29
4
4-
5*7
' globulin 4
llo floeeulat could be get out for touting by aeons
of centrifuging*

85
table iv (oomimm)
~rar~

eso
SkffttjRfwrfe It?
5.7
4*

Mr
£81
#
+
5.7
globulin 4
2S2

m
5.8
48
ess
8
m
5*8
M> '
£84
*
4
M
M
ess

ft
3,0
*
236
**
8
5*05
#
257
8
Mi
5*8
*
£38
T
4
3.95
M
£39
T

5*6
not osntrifugod
£40
T

* *
£41
T
Mr
* * *
*
£42
T
3*8
globulin 4
243
T
fr
5.3
globulin
£44
T
1
.
*
£45
f

* *
£40
+
* -*
*
247
*

5.7
globulin 7
£48
3.1
4*
£49
*
+
6,0
globulin
250

+
@.l
globulin
£51


6,1
globulin
252
4*

6.9
globulin
£53
*
+
.***.
.*****

9 NS *03 fO
*3 *3 *#**§ *§
+ + + + i
86$

II
8£8*P8S8SggS kpj
I t t t t t 1 I t I t
03
O

table n
KSSQVSS OF VCl* KSACJXOHS OT SFXSAL ifcSISS,
i'IW0 HEPROOTCXBILITt OF SOLtmCHTS
SpXal fluM
W
JL
£70
m
£77
m
<*
£79
m
@00
m
£01
m
£0£
£03
4
£04
V
£83
%'
£06
f
£87
T
£08
T
£89
f
m
T
£91
T
£0£
4
.£83
4
004
. 4
£93 *
4
893. ,
£97
. V
4
£98
*
£99
4
iMzmmt is
300
m
301
m>
302
m>
303
m
304
m
303
m
4
4
4
4
4
4
4
4
, :y
*Samples of this group m sub unkrouns*
44444444

TABLE VI (CCHTINIM
IpKOCTT
alm
rar~~nra;
303
30?
308
309
310
311
312
g&toasRfc
m m m
m ** m
313 T
314 T
318 T
310 T
31? T
SIS T
319 T
320 T
321 T
322
323


f
f
+ 4*

89
TABLE TO
TUB TO* HBAOTIOH WITH SPIRAL FLUIDS FROM
mrnrnm diseases
BCTT
number
Kahn
TO*
tw
524*

*
*
32S*
4

ses*
4

8
**
327*
*
40
3E8*
6*
m
...*
389*
OS
m
*#*6
330
4
m
m
331
'**
m

532
m
m
m
333
m
SO
334
m
OS
m
333
*
*>
4
336
m
4

337
m

m
338
m>
*
m
m
m
340
m
4
341
m
0

342
4*
40
4
343
m
m
so
344
m
m '

343
m
so
<*
346
m
P
#>
*3yphUitie m& aon^sypkUitie series used to
test sensitivity of TO* dilution*
*Hot reported at tine of testing*

90
TABLE VIII
StMMHX OF TABULATED RJSStJLTS KIWI ScCPaO,- REACTIONS
Antigenic negatives, non-syphilitic ............... 55 X
Antigenic positives, syphilitic.................... 38 X
Antigenic negativas, treated, syphilitic............ %7 Z
Total spinal fluids tested ifch K*CrR0*.......... .^IsSST^
S*Qr*Q* results with X. S , 1
Percentage disagreement.................... absolute 8*6£
ECrcQ? results with X* ........ ...... 38 +
Percentage disagreement * *................ absolute . o .00#
SaCSpaO^ results with 15 +, *?
Percentage dlaagreessont..... absolute 81*5#

91
TABLE XX
SOWAR* OF TAmAT'E) RE30LTS ITH VC1* HHACflOKS
Total Pfassemcnn end Kahn positivo,
syphilitic spinal fluid samples 100 A
Tot al Aassermarm and Eaten negative,
non-syphilitic spinal fluid samples **..,.* 145 B
Total Wasseiutfm end Kahn negative,
treaood syphilitic spinal fluid samples. ....... 101 C
Total number of samples .oieT
vCl* results Tilth A.$ +, ^
Percentage disagree:¡ont Absolute
Hoi stive *s
VCl* results with B...................... 143 -, £ 4
Percentage disagreement ....... Absolute 1.4$
Relative 1 4£>
VCI4 results with £ ea 35 , 5+
Percentage disagreement*......... ... Absolute 38.9#
Relative 39*9$
1Bacterio present.

92
TJSLS X
mmmt of taslas results oh the ps of
m voi4-spxjal flci mm¡m&
Total pS doteroinAtiona...... ........ 70
Arithmetic 6*9 pH
units
Average deviation of & single measurement....... 0,15 pH
imita
Probable error of a single measurement ..... 0.1S pH
units
Probable error of the mean....... 0*016 pE
units
.reolpttttta
' v>- 'y " i V ,
Total precipitates oarained 64
Precipitates behaving as globulin (solubility} 64
Precipitates examined for vanadium*............. 9
Precipitates found to contain vanadium...** 9
Additional precipitates examined for
vanadium by fusion*....*..*.*.,...*........., 6
Precipitates found to contain vanadium
by fusion*.. 6
^Colorimetric qualitative method used.

93
mmLn of ahai^hs' of m vox* soxmoiw
The color!->etrte method making use of sulfuric cid
ana' thro percent hydrogen peroxide we need. The color
standard ere mede from a pure sample of irnmnlum
mot a-vaasdote*
riginal sensitive solution*.* (X) 1*44X0 grasas VC1*A*
(g) 1.4410 grme ¥C1*/1*
(5) 1.3040 grams VC1*A*
(4) 1.4410 grass* VC1*A*
Results of the analysis of solution after the com
pletion of tests and rosioval of precipitate fey the
centrifuge *
Stssnle A... {1} 0.0750 gFaa VC1*A*
(2) 0.6750 grans VC1*A*
Sample B U) 0.0070 grama VCl*/!.
(2 0.6750 grms VC1*A.
By straight dilution these solutions should hare a
vanadium content of 0.7B08 grans ¥01* per liter* She dif-
fcreme la due to the adsorption by the flocculate*
suits of the vanadium analysis of the new solution
prepared, and of half-saturated V01*i
Hew solution. (1) £1.38 greens ¥01*A*
() £1.38 gram# ¥01*/1.
Half-saturated VOX* (1) 13.945 grams VC1*/1.
(£) 13.045 grams VC1*A-
Results of the vanadium analysis of sensitive dilution
prepared from now end old half-saturated stock solutions*
From new solution (X)*.........*il) 1.4410 grams VC1*A*
(2) 1.4410 grams ¥G1*A*
Fro old solution Of)**. .(1) 1.4410 grams VC&*A*

T&MM 3a icomxmm)
Results of pH deteminatlons on (X) sad (Â¥) from
the preceding seetlont
pH found on (X)MtiM 2.80
pH found on it) .......... 2.68
Eo@u3.to on analysis of holf~saturat& solution
after It hod boon in eonfcoet with rubber*
*
Frovious analysis
Hew analysis..*..
15.948 grcsas VC1*/1
15.250 grans VC1*A

95
DISCUSSION OF RESULTS AND CONCLUSIONS
The reaction of equal volume* of 2*5/ potassium dichromate*
I \
It100 nltrlo acid and spinal fluid has been studied* This resemblas
the reaction of Taocono (109, 110}* The sensitivity of the di*
chromate solution is increased with ge* The nitric sold solution
varied in strength with age* Mew solutions must be made up for each
series of detominations* With 8*6gS of the &ahn negative, non
syphilitic spinal fluids there is a positive reaction by this method*
If the solution strengths are varied to do away with these false posi
tive reactions, the Malm positive, syphilitic spinal fluids do not all
give positive dichromate reactions*
With the diehr coata method a groat many positive syphilitic fluids
do not give consistent results* In sons tests there are observed
granulas and no flocoulation* If the test be repeated flocculation is
as likely to take piase as granule formation* Granules arc apt to
appear in negative non-syphilitic spinal fluids* In the same manner,
there are apt to be a groat many vary weak positive reaction*! these
are judged to be doubtful*
In line with the work of Prunell (113), it appears that this id
a test for globulin and albumin in certain ratios* Mo attempt was made
to verify this, the main part of this investigation being occupied by
work on vanadium compounds. There is no difference in the flocculation
which result# from different fluids, as Taoemte claimed* *# may

96
report that the us of diohrotvate and nitric acid id not of impor
tance In 'tee diagnosis of syphilis* Hoither Is it of any particular
value in following the course of treatment
hith respect to an aqueous solution of vanadium tetrachloride,
ire are able to moke several statements. A solution containing around
13 grans of VC lit Pr litsr has a definite sensitivity toward the glo
bulins of syphilitic spinal fluids if it is dilutsd to lJbUlO grams
per liter and partially oxidised. The solution containing 13 graos
per liter is slowly oxidised by atmospheric oxygen. Poly-vanadio
acids result from this oxidation. This solution has a definite tins
of mixing which leads to ssnsitivity and not the foraation of vanadio
acid of definite partile sise, sinos most of the oxidised vanadium
is formed after dilution to a concentration of 1*14*10 g. VC1}¡ per
liter.
The partially oxidised solution of vanadiusi tetrachloride content
corresponding to l*l*UlO grams per liter Bust be nade from the solution
with a content of 13 grams VCl^ per liter* The oxidation may be
carried out In either of two methods. By standing with atmospheric
oxygon over It for twelve hours, the content of pentavalent vanadium
is raised to the proper level. A more rapid method, and more satis
factory method, consists of allowing a solution with vanadium tetra
chloride content equal to 1*14*10 grams per liter to go completely to
the pent aval on t state* A definite volume Is then added from this solu
tion to a definite volume of freshly wade solution having the same
vanadium content, in this way a sensitive solution results without

97
the wait for partial oxidation to talco place.
This sensitive dilution contains polyvaaedio aoids of the type
%V617* hydrogen ions, chloride ions, and a vanadyl ion (V0++). This
latter ion is not named in the literature. All VO ions, regardless
of charge, are called vanadyl, ~ous being added to ahow monovalence
and le being added to ahow trivalonee. The divalent form which
arises by hydrolysis of the tetrachloride is left with no convenient
suffix.
If equal volumes of the sensitive solution of vanadium compounds
and spinal fluid are mixed, there is a precipitation of globulin when
syphilis is present. A volume of water equal to the total volume on
the slide is added to eliminate hypor-alfeuminosls. This globulin is
precipitated at a pH of 5*9* This pH value is much higher than that
found by Boyland (267}.with mixtures of vanadio acid and pure globulins,
or globulins protected by albumins.
By an inapeotion of the works of Moore (87) and Kahn (102) it is
seen that there must be some specific globulins produced by the
disease syphilis. These globulins apparently take the place of the
normal globulins. This is the only explanation for the disappearance
of excess globulin and continued positive fassermann and Kahn
reactions after an inadequate course of treatment for neurosyphilis.
Boyland found that the only means of explaining the reaction of globu
lin and vanadio acid in the presence of albumin was to postulate a
possible specificity. We have shown that this speoifiolty goes even

98
further* Only certain of the globulins are capable of being pre
cipitated.
The only doubtful results obtained with Kahn positive,
syphilitic spinal fluids occurred when bacterial contamination was
present, or some other agent had caused an almost complete floccu
lation of all the protein present* These aro noted in shipment six*
All save one of the doubtful reactions obtained with vanadium tetra
chloride solution came in this one shipment* Of the twenty-four
ampies, sixteen were In very bad shape* Four doubtful reactions
occurred with treated fluids of class XI*
We may say that this reaction is more sensitive in the de
tection of certain globulins than the Kahn or Wasseraaim reactions.
This is brought out clearly by a study of the results under the
class IX fluids* It may be that a group of globulins is generated by
syphilitic infection and that we arc testing for any one of this group.
Thus the specific globulin responsible for the Kahn test may dis
appear first, in which ease we are getting results because of non
specific globulin* It Is also possible that there is seme change in
the lipoid content of the spinal fluid which makes globulins more
easily prcclpitatable* The main part of the evidence points to the
fact that there are specific globulins, however*
The logical conclusion Is that the use of vanadium tetrachloride
in the following of treatment is indicated. Once nsurosyphills has
been diagnosed by a standard, proven diagnostic teat, the vanadium

99
solution rill follow the globulins formed. The vanadium reaction may
maain peaitlvo longer than the Kahn or iiaeserwarm reaction*, and nay
indicate isuoh concerning the treatment course* The reaction is maeh
simpler to carry out than either of the standard methods commonly
used*
The diseases of hyperalbuninosis are not apt to gire false re
actions* the albumin would be soluble in the eater which is added to
the precipitate. The reaction may hate some value for this* but it
is doubtful. For too great a eg* percent of albumin would necessarily
haw to be present* any diseases which camonly produce hyperglebu-
littosis may Interfere* Two false positive reactions are found in the
122 Kahn and %eeerjaann negative spinal fluids received from the State
Hospital* These were all frota insane patients* Some forms of
insanity are known to produce excess globulin* notable is dementia
praeeox* This increase in globulin Is not in any way specific, only
occurring in como oases* The taro false positives may be due to an
excess globulin produced by a fora of insanity*
The first of these false positives was found with fluid number 32
and may have been a fault in technique. The second gave globulin at the
proper pH and was a true positive vanadium reaction. A mistake in the
handling of the fluids prior to shipment is hardly possible* but may
also be considered. The solubility of the first precipitate (number £2)
in water was not determined and it may have been as excess albumin as
well as excess globulin* or specific globulin.

100
the relativo agreement of the venadltsa reaction with the Kahn
reaction (based en olese I aat III fluids onjy) is 99*2^. The abso
lute agreement is lie propose that this is a sensitive test
for certain globulins found In syphilitic spinal fluids*
With regard to other conditions producing excess globulin in
the spinal fluid, little has been determined. The work of Borland
(167) shows that vua&dio acid ie more or less specif!e for certain
globulins* the work of eri (268) goes even further In showing there
is a tendency for -vanadio old to be specific for syphilis globulins,
but without aay sensitivity whatsoever*
We can stake definitely that every ease which sheers globulin in
excess of the normal will not give a positive vanadium reaction* It
is Impossible for us to state tbs offset of various diseases on the
test* The samples examined at the State Board of Health have no hist
ory attached* The attending physician rarely inferas ti laboratory
of the diagnosis*
Koore* Eagle and Mohr (172) point out clearly the facts on falsa
diagnosis of syphilis Very little critical work has been done in the
field and a wide opening for further research exists*

101
SUiaiARY OF CQSCLUSIOHS
1 The reaction of potass!xm dichromate and nitric acid solu
tions on syphilitic and non-syphilitic spinal fluids has boon investi
gated* It 1b judged that this reaction is too unpredictable to be of
use in tbs diagnosis of syphilis or tbs following of syphilitic
treatment*
2* The reaction of an aqueous solution of vanadium tetra
chloride with syphilitic and non-syphilitic spinal fluids has bom
investigated* It has shown definite value for the following of
syphilitic treatment* The action of vanadium solutions prepared is
the manner given is specific in a great degree for the globulins of
syphilis* tittle Is known, or can be found out with regard to the
effect of other diseases on the vanadium test* It is reasonable to
assume that any condition causing a great exeess of globulin would yield
a positive reaction* This would occur with almost any metal salt one
happened to choose*
3f* The reaction Is that of a reproducible solution and is ready
for evaluation In actual hospital practice* It may be a much surer
test for syphllltio globulin than any proposed to date*
4. We may say that this is not intended for a diagnostic test*
but that if the blood test is positive in the secondary stages* this
will surely shew neurosyphilis as scon as the Kahn test* Perhaps the
VCljft reaction will show turnroayphi 11s sooner* since non-specific

102
globulina appear before the 8peai.fio ones. The solution should be
of certain use in suoh eases*

103
BIBLIOGRAPHY
1. 0. Morgan Moran, J.A.K.A., 40, 937 (1903)
2. Bbssarmaan, Malaaar and Brook, Boot. aed. Wochsebr., 2, no, 1$ (1906)
3. Wasoenaann and Plaut, Bant. taod. ^eehaehr., 31* n0* 44 (1906)
4. L* fftefcaelle, Borl. kUn, Woefeaehr., 44, 1477 (1907)
5. L* Mlciaaolio, Ibid, pagan 1103-7
6. Saaaamwm, Bari* kiln. iSochaehr., <4, 1599 (190?)
7., Flaiaotaaann, Bari* kiln* Itocbachr., 4£9 490 (1908)
8* Varney, (discussion), J.A.li.A,, 1, I994 (1908)
9. Plant, Keuck and Roaai, J.A.M.A., &, 13 (1908)
10* Vsrnoy and Morae, J. Cut* Die., 3g, 624-9
11, J. Bronfenbrenraor, Blochea. Boll., 87-9 (1915)
12* E, Klsuener, tain, klin. aochsehr,, 21, 213-46 (I908)
13* E, Elausner, ibid*, pages 345-78
14. Krcweke, Red. Kllnik, BarUn, 7&t 210 (1923)
15* w* Schuraan, Bout. mi. Wochachr., 35, 6l6 (1909)
16. Rax Honra, J.A.R.A., 33, 289-96 (1909)
17. Roguehl, (editorial), J.A.K.A,, 33, 1931-2 (1909)
18* L. Beltz, Dautsch Z Korvenhoilk, 43, 63-80
19. E. Jones, Rev, ileur. & Faychiat*, 2, 379-91 (1908)
20* Breiger and Hans, Dent, mi. Wochechr,, 33, 2203 (W)
21* Broiger and Bans, ibid*, 36 78 (1910)
22* K* Jacobathal, Biol. Abt*, Artxl* Vereina, Hanburg, 3, Id (1909)

ioU
23. X* fccobathal, s. XamunltAt*, £, 107-X2S (1910)
24* Brock and ild&ka 8* lamnitat*, £ 476*82 (X$ll)
25* Stvwi* X jfcftgit* Frcncalaej 3cb#-t* wch#ehr.* *j&, 395*6.
26. Str# SfttiMfo, ised. foebcebr., 51. 624*5 (17*1)
27. Sabbatini, SI renslere md.t 1911, no* 3*5*
28. aoguchi, J. lap. lied., 557-68 (1711)
29. tma*m*l, Alexandre* cad FVSedMtttft* Kuneh. sjed, Soohochr,, £1
1271-4 (1714)
30* 1*. Fopoff, 2* Sraramltat#. M 218*54 (1?U)
31, f. Veil 4 v, Kafka* lied, mau, 2* 13X4*8 (1911)
32* ffcuwa Serte# Beutech Z* Henrenlaatlk, £2 169-93
33* Cferl Unge, 2* Choswtherojsie, £ 44*77 (1912)
34* 7* Sarria,* nerratnl, '^echamtohr. ^r@e*tinghft, ££, 52
35. Kryja* Brandt end aras Wei*. kUn. feiMwfer 33, 1-6 (1920)
36. Glaeeoe and Senses* J. Sab. Clin* Ked*t £g* 534 (1940)
37. Sjset-in and Paul, Kolleid 2*, |£, 310*4 (19)
38. J* Sadek, Berl, kli, Veefcccbr.* S, §93*5 (1915)
39* *# tlletti5# Sentr. Bleebe, Biej&ys. 4, 813
40. Vile and Moke# J. Oat. Di,t ££, 607-23
41. J. A. Kolaer, J.A.H.A.* j&, 1461*3 (1915)
42. d* Brenfetibrenaar and * Seeks*, Meehea. Bull* £, 377*380
43. Alfred Garden, fi.Y* M#d. d.* §£, 343*5 (1915)
44. H. 4* Krebe, Bent. ned. ^ochecbr., £1, 1771 (1925)
45. v* 3ttO£ra, Bunch* mcd. VeefaMhr** 6£* 1212*3 (1915)

105
46. Kgo Heeht, 2. Iswmitate., 4, 258-66 (19X5)
4?. Gradwohl <&, 240 (1914)
48. W J, Bruce, . Lab. Clin, liad., i, 2X5 (1918-19)
49. Sijaaanuei, BerX. fclin. Tochachr. 0, no. 30 (19X5)
50. J. A. Cutting, J.A.U.A., 1810 (1917)
51. Hoeonfeld, Ked, Kliaik, 26, 553-5 (1930)
52. . Brandt, J. Lab. Clin, Ued., 5, 1077-84 (1940)
53. Pfeiffer, Kofeer and Field, Pros. Soc. Ixp. Biol. Sed. 1, 153-7 (1915)
*
54. Hoffwm and Schwarts, Arch, Int, fcfed., , 293-302 (1916)
55* 0* Genoese, Pollelin, Rom, 26, 97-100 (1919)
56. Beverl, Folielinlco, Jg|, 450 (1921)
57. Guillain and Ubert, Ann. de Med., 771 (1921)
58. L, Lowery, Boston Ited, Surg. J 121, U5-7 (1917)
59. Sache and Georgl, fed* Xlinik, 805 (1918)
60. E. Meinioko, Z. ImamlUt*., 350-63 (1918)
61. B* Uolnlcko, ibid., page 51>7
62* Fr. Reichert, Cent, Bakt. Bamelteak., I. Abt. 90. 328 (1923)
63. E. Uainicke, Deut. md. Wochachr., 48, 384-5 (1922)
64. B. feinlcke, Klin. Wochachr., 8, 112-3 (1929)
65* ?. Klliagar, Berl. kilo, Vochschr., 8, 1001-3 (1918)
66. P. Ellinger, 2. Phyeiol. Cheau, 16. 245-65 (1921)
67. Alekeeovokli, ?* Rueo, Fhye, Cheta. Soe## 53 1. 331 (1922)
68. Rubellng, Klii, Wecfeechr,, 12, 501-4 (1938)
69. Hermann, Kolleid 2., 8$, 297-301 (1939)

106
70. SteiafioU, J. Leb, Clin. lied., & 445 (1919)
71. Oulllaln, Laroche and Lechelle, Bull. neo. One. S3ed. Bop. Baris
M W99 (1920)
72. tt. Wright and Koraack, Bloch. J.# 1 635 (1923)
73. 0. Quart!, Boll. soc. ital. Biol. Spar., 1145 (1926)
74. 4, Vernee, Compt, rend. Acad, da Sc* 165. 769 (1917)
75. 4. femes, ibid., 66, 575 (1918)
76. A. Vemos, Ibid., jLJ, 500 (1918)
77* A. Verms, Compt* read. sec. Hoi*# 23, 1425 (1925)
78. Cornwall and Aronson J.A.M.!,, 25 1697 (1920)
79. Cornwall, J. Icb. Clin, Sed. 0* 831-52 (1924-25)
80. H. Bold, Had. Klin., 2, 940 (1921)
81. Strenpel, Munch, sed. Wochschr,, 2, 120 (1922)
82. H* Bold, Beut. sed* Woehachr. jt|, 247-8 (1922)
83. D. A* Johnston, J. Lab. Clin. Med £, 277 (1923)
84. Dreyer and Ward, Lancet, 1921 I. 956
85- Spsroni, J.A.&.A., 22, 1056 (1921)
86. Houghton, J*AB,88 719 (1927)
87. J. B* Moore, The Mft4sr.S %£*&%£ £ .MilU Charles C. Thorns,
BoltlaoreTl933 5
66. Kasahora, Intern. Med. News, (Jap.), no. 987 (1921)| Abst.i Che.
Abete,, M. 438 (1922)
89. Ayer and Foster, Repert phar., 24, 114 (1922)
90. Kahn, Arch. Bormt. A Syph,, 5, 570 (1922)
91. C* Y* Wang, Lancet, 1922 I. 274-6

107
92. Herrold, J.A.H.A., 22, 957 (1922)
93. Herrold, ibid,, 8, 203 (1923)
94. C* Bmck, Munch, saed. Woehachr., 69,, 569 (1922)
95. Teichnann, J.A.K.A., JO, 286 (1923)
96. Becker, J.A.K.A., §g, 1525 (19*4)
97. Schwarz and Orunewald, Klin. Wochschr., 3. 1026 (1924)
98. Gustav Kleiner, Klin. Wochschr., 1, 811 (1932)
99. 0, Beba, Hunch. mad, Wochschr,, 21# 1793 (1924)
100. Jervell, Korsk Magazin Laegavidonskaben, 86, 757 (1925)| Abat,*
Che. Abete., go, 949 (1926)
101. Kahn, Abstracta of Bact., 17 (1921)
102. Kfthn>t |erg^i^^^|^l^e. & William* A
103. Epstein and Bawl, Beat, md, ochschr., 48, 89 (1922)
104. C, Brack, Klin, Wochschr., 1, 1656 (1922)
1Q5. Kahn, Tissue Xnaunlty. Chas, C, Thoms Co,, Baltisora (1936)
106. Kadlach, Med, Klin., 1649 (1926)
107. Bosenfeld, Blochs. 35., 16£, 343 (1926)
108. Walker and Sleeper, J. Lab, Clin. Hod., 1£, 1048 (1926-27)
109. Taceone, Podiatrist Beviata, 131-41 (1926)
110. Taecone, ibid., 3£, 1097 (1927)
111. Prune11, Prensa Med. Argentina, 232 (1919)
112. Prunell, Compt. rend. aoc. blol,, 2l, 1315 (1928)
113. Takata and Ara, A, Trap. Med. Tr. sixth congress, I, 667 (1925)
114. Statib and dealer, Klin, Woehschr., 14, 1638 (1935)

108
115. Sacho and Witebsky, Klin. Wochechr., £, 1233 (1928)
116. Hinton, J. Lab. CUn. Bad., 4, 621 (1928-29)
117. Ifeyser, Klin* Wochsehr., £, 792 (1928)
118. Treuter, Boot, sod* Wochochr., 54, 994 (1928)
119. H* Bailor, Klin. Woeheehr., % 1405 (1930)
120. Buller and Kandula, £, Irsaunltate., §g, 183*92 (1934)
121. Flinn, J, Ub. CUs, Bed., 682-6 (1930)
122* Sauer, I* lenonitato, £g, 51*2 (1930)
123. L* Fnpi. Urol. Catan. Rev,, XI* 188-94 (1931)
124. J&rehioni and Alinatela, Klin, Wochochr,, 3, 1345 (1932)
125. Marchioni and Allenstein, Ibid,, pages 1424-6
126 B, S. KUne, %icrqacpj>ifi Sllge Lrecls&fifelSg Teeta £or ho Diftgnc-
ia m gxclugjon S Williana and Wilkins Co
Baltimore, lid., (1932)
127. Kiss, 2. Iraaunitots., XL* 195-241 (1932)
128. Sehttbe and Harrsa, J. I*b. Clin. Bed,, 8, 65 (1932-33)
129. Looney, J, Biol. Che., Ji, 63 (1927)
130. Schube, J. Lab. CUn. Med., 2£, 314 (1934)
131. Deadcen, KlUot and Sadth, Ass. J. Clin. Path., 2, 246-50 (1937)
132. Osante, Buraok and Kreisler, T, Lab. Clin. Bed., 26, 1349 (1940-41)
133* agle, J. Lab. CUn. Bed., 1,8, 684 (1932-33)
134. Siiapaon, Lab. J* Australasia, 2, 18 (1940)
135. Oruskin, Ara. J. CUn, Rath., 2, 441-7 (1931)
136. flruskin, ibid., 5, 162-4 (1935)
137* Kopaeseeeki, Coapt. rend, soc, biol., 11,6. 932 (1934)

109
138. Bauer and Woeaeek, Had. Klin., &, 107$ (1935)
139. Laughlen, Cenad. K. A. J., 3, W U935)
140. Moor*, J. Lab. Clin. Med., £4, 973 (1938)
v
141. Robinson end Stroud, 10|, 1170 (1937)
142. Thomason, Jord. Med., 8, 1972 (1940)j Abstr.i Chora. Abate., 21*
3710 (1941)
*
143. A. Vernos, Coajjrfc, rend., 203. $84 (193$)
144. Tasn and Takagi, T* Orient. Med*, 34 329 (1941)* Abetr.t Chea.
Abete. 21* 7440 (1941)
145* F. Ryt*, J, Lab. Clin, Med., gg, 82 (193$)
14$. P. Dahr, Beut. raed, Tochachr,, 3O, 94 (1934)
147. P* Dahr, Munch, raed, Woehsohr., &, 1723 (1934)
148. J. 3chuck, Arch. Gymkcl., JO, $16-25 (1940)
149. Porto and de Britto 0 Silva, Rev. Assoc* fhulista Med. (Sao Paulo),
1$* 87-98 (1940)
150. S. Ide and T. Ida, Fr, 814,052, June 14, 1937
151. van Kverdingen, Chora, hentr,, 1940 I. 2354
152. Rosegger, Klin, Woehochr., J, 501 (1938)
153* Kaehucharov, 2, Ian&aiiate*, 22. 410 (1938)
154. A. M&xxini, to. J. Clin. Path., J, 163 (1939)
155* Frohlieh, Arch, Deraratel. Syphilis, 180, 109 (1940)
15$. Beet and Tfeylor, Jh& &Z£*k9tei&S&l fijflU g£ Pmctlcj^ SUe
lian and Wilkins, Baltimore, Md (1940)
157. Sorason Wright, Applied Physiology. 7th Ed., Oxford Chiversity Press,
Hew York, 0940T
158. H, Iscoveaeo, Compt, rend. eoc. biol., 6g, 181-2 (190$)

no
159 flama, last. Path. Lund*, Acta Bath, et Microbiol, Stand*, 5,
16-24 (1928)
160. Heufeld, Krankheitsfrsch,, g, 63-9 (1925)
161. August and Tarboureieh, Bull* 00c franc* dernatol. syphilog*, 45.
368-72 (1938)
162. Malloy, Kahn and Westell, J, Infectious Diseases, *8, 203-11 (1931)
163. F.ntmer, Momtaachr, Kinderbeilk, 1, 387-93 (1932)
164. Warren, J. Lab, Clin. Med., &, 1146-50 (1936)
165. Victor Meyer, J. Biol. Chora, j>, 115-31
166. Havout and Laignel-Lavanatine, Bull, era. see. raed* hop., 3* H51
(192C)
167. Boy land, Moche, 2., 26, 1383-90 (1932)
168* A* Keri, Rnssongn di studi psichiatriei, 21 639-5 (2uly and August,
1935)
169. Levinson, Ass, J* Bis. Qilldren, 1§, 568-76 (1919)
170. k. Pfeiffer, i^indUcJa* gia 22S
l2£MSMm£tSB 4s Mm* desden (1938)
171. Bouris, Corapt. rend, 00c. biol, gi, 106-8 (1927)
172. Moore, Eegle and Mohr, J.A.M.A., ll£, 1602 (1940)

BIOGRAPHY
Robert Somera Leopold es born In Dayton, Ohio,
the 21st* of June, 1915* He attended granger and
high schools in several sections of the country,
finally graduating from high school in take Charles,
Louisiana, In 1932.
Ur, Leopold graduated frota Mississippi Stats
College with a Batchelor of Science degree in Zoology in
193?. The year following graduation ho assisted in
chemistry at his eisaa enter.
In 193, Hr* Leopold entered the graduate school
of th University of Berth Carolina, and graduated from
this institution with the degree of Master of Arts in
Chemistry in 1940,
Since the fall of 1940, Mr* Leopold has been
enrolled in the gradante school of the University of
Florida* During the second semester of the 1940-41
school year he held a Graduate Council Scholarship and
continued on this scholarship the following year.
Hr, Leopold is a member of the teoricen Chemical
Society and the Bets Beta Beta and Kappa Hu Epsilon
national fraternities.
i
\

fid* dissertation ms prepared under the direction of the Chain**
of the candidate*# Supervisory Committee end has been approved by all
member* of the Ccenltteo It tea submitted to the Graduate Council
and me approved ae partial fulfilment of the requirements for the
degree of Doctor of Philosophy*
Date
S5SH
SUPERVISORY COSKITTSB*
Chairman



13
In 1917 Cutting (50) noted that the salt solution (spinel fluid) in
a negative reaction did not allow the gum mastic to precipitate. In
order for the blank to remin a colloidal solution, he found that the
efficacy of the test was not altered by the addition of 1 co, of 0.5$
KgCCh solution to each 99 cc, of the mastic made up In alcohol. The
KgCO^ did, however, keep the blank from precipitating.
In 1930, Hoaenfeld (51) proposed a change in the mastic test in
order that curves similar to these of the gold sol test would result.
He prepared t colloidal solution fro buffered &Cl solution, pR 7*5
and an alcoholic bass solution of mastic. In this manner lie claimed to
have a reproducible solution and to get curves which wars of more use
than those in the Lange test, Brandt (52) was still not satisfied with
the sensitivity of the mastic test. He found that the speed of mixing
had mors te do with sensitivity and reproduction of the test than was
usually assumed. His test is run in the following manner. A series of
tubes containing the etnas amount of mastis is set up, ami as rapidly as
possible graduated amounts of water are added. After setting for a
while each tube is nade to the same volume with more water. Once the
particle else is determined by the original addition of water, more water
does not change it. In this way a reproducible colloid of the same sen
sitivity is always obtainable. In a period of 14 years some 70,000 tests
were run by this method. Since the normal and syphilitic curve both
depend on the globuliryfobumin ratio, there can never be a colloid reaction
which differentiates syphilitic from non-syphilitic, if the globulin goes


99
solution rill follow the globulins formed. The vanadium reaction may
maain peaitlvo longer than the Kahn or iiaeserwarm reaction*, and nay
indicate isuoh concerning the treatment course* The reaction is maeh
simpler to carry out than either of the standard methods commonly
used*
The diseases of hyperalbuninosis are not apt to gire false re
actions* the albumin would be soluble in the eater which is added to
the precipitate. The reaction may hate some value for this* but it
is doubtful. For too great a eg* percent of albumin would necessarily
haw to be present* any diseases which camonly produce hyperglebu-
littosis may Interfere* Two false positive reactions are found in the
122 Kahn and %eeerjaann negative spinal fluids received from the State
Hospital* These were all frota insane patients* Some forms of
insanity are known to produce excess globulin* notable is dementia
praeeox* This increase in globulin Is not in any way specific, only
occurring in como oases* The taro false positives may be due to an
excess globulin produced by a fora of insanity*
The first of these false positives was found with fluid number 32
and may have been a fault in technique. The second gave globulin at the
proper pH and was a true positive vanadium reaction. A mistake in the
handling of the fluids prior to shipment is hardly possible* but may
also be considered. The solubility of the first precipitate (number £2)
in water was not determined and it may have been as excess albumin as
well as excess globulin* or specific globulin.


all four fluida selected.
The four selected fluida gave the proper raaction after a total
of Uo drops of old dilution had been added to the fresh dilution.
Maximum sensitivity was shows after the addition of 50 drops, and at
60 drops the ciaeseraaxm negative fluids gave positive reactions* When
the solution was at a fairly sensitive point (after the addition of
h5 drops of old dilution) all the samples In the box were tested* All
class X and III fluids agreed with their Vaeeeraan reports* Of the
four class 11 fluids in the shipment, two were positive and two
negative to VCl^.
these same fluids were then tested with the di chrome. te-HH0?$
method* All class X fluids oheoked. The class IX fluids gave two
negative results, one positive and one doubtful fro the four fluids*
Class III gave one doubtful result and the reminder negative* the
presence of all these doubtful reactions may be the degree of positiv
ity to which taoeonc refers, but careful examination under the
microscope failed to reveal any difference in structure of the
turbidity. At any rate, these reactions appear to be graded from
positive to negative by the mount of protein present*
Up to this time all fluids tested had been pooled samples. There
is a reaction on the addition of certain spinal fluids to others.
Plaut, Iteuoh and Rossi found this reaction unpredictable, and not
related to syphilis (9)* hile this fact was not known at the tas,
the fluids were known to be altered in some manner* they wer not the


b9
syphilis. The solution, especially the more dilute one, had changed
color In this length of time* At the timo they were made each had a
blue color. After ton hours tho more dilute one were blue-green* the
more the dilution, the sore green and less blue the solutions appeared,
At this time fourteen fresh samples of spinal fluid were received.
Through a misunderstanding those were all pooled. This misunderstand
ing was later cleared up, and individual samples received. Since a
sensitive solution was at hand, these fourteen samples were checked.
All save one gave a reaction which was in accord with its Wassormarm
reaction. The sensitive dilution of VC1}4 was mads by the addition of
20 drops of half-saturated VCljij solution to 12 ml. of distilled water.
The sensitisation was allowed to develop by letting the solution stand
for 10-12 hours.
This shipment of fourteen samples was divided into three groups,
or olasses. The tern class is used by the hospital to denote the aero
loga reaction and the presence or absence of syphilis. Thus class I
fluids are has semana or Kahn positive and syphilitic. Class II fluid
are Wassoraann or Kahn negative and derived from treated syphilitic
patients. Class IZ1 fluids are those tros, non-syphilitio patients and
are Kahn and hassermana negative. The one fluid which did not agrss
in its VCljj reaction (positive) and Wassermaim reaction (negative)
belonged to oaos II. A great deal of interest was taken in this find
ing, sines it indicated that the VCl^ test might be a precipitation
reaction which did not reverse ae soon as the /assoraann reaction.


table of Content (Continued)
Page
table X. Summary of tabulated Result on the pH
of the VClfj-Spinal Fluid Mixtures *..**.. 98
fable XI* Result of Analyses of the Various VCljt
Solutions..***. 99
Di semes! on of Results and Conclusions*.*.....................* 95
Summary of Conclusions**** 101
Bibliography**.105
iv