Morphologic changes in the lymphocytes of persons exposed to ionizing radiations

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Title:
Morphologic changes in the lymphocytes of persons exposed to ionizing radiations
Series Title:
United States. Atomic Energy Commission. MDDC ;
Physical Description:
12 p. : ill. ; 27 cm.
Language:
English
Creator:
Dickie, Annamae
Hempelmann, L. H ( Louis )
Los Alamos Scientific Laboratory
U.S. Atomic Energy Commission
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Technical Information Branch, Atomic Energy Commission
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Oak Ridge, Tenn
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Lymphocytes   ( lcsh )
Ionizing radiation   ( lcsh )
Radiation -- Physiological effect   ( lcsh )
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bibliography   ( marcgt )
technical report   ( marcgt )
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Notes

Bibliography:
Bibliography: p. 10.
Restriction:
"Date Declassified: July 22, 1948"
Statement of Responsibility:
by Annamae Dickie and L.H. Hempelmann.
General Note:
Manhattan District Declassified Code

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University of Florida
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oclc - 277773809
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MDDC 1102




UNITED STATES ATOMIC ENERGY COMMISSION









MORPHOLOGIC CHANGES IN THE LYMPHOCYTES OF PERSONS
EXPOSED TO IONIZING RADIATIONS




by

Annamae Dickie
L. H. Hempelmann, M. D.


Los Alamos Scientific Laboratory


Date Declassified:


July 22, 1948


This document is for official use.
Its issuance does not constitute authority
for declassification of classified copies
of the same or similar content and title
and by the same authors. '




Technical Information Branch, Oak Ridge, Tennessee
AEC, Oak Ridge, Tenn., 6-20-49--750-A378


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Digitized by the Internet Archive
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MORPHOLOGIC CHANGES IN THE LYMPHOCYTES OF PERSONS
EXPOSED TO IONIZING RADIATIONS


By Annamae Dickie and Louis H. Hempelmann, M.D.


Examination of supravital preparations of the blood of persons exposed to ionizing radiations and
toxic chemicals at the Los Alamos Scientific Laboratory has revealed the presence of an unusually
large number of refractive neutral red bodies in the cytoplasm of the circulating lymphocytes. These
bodies are also present in smaller numbers in lymphocytes of unexposed persons. They can be dis-
tinguished from intracellular vacuoles which also stain with neutral red. The increase in neutral red
bodies is evident even when exposure to radiation is within the tolerance range, i.e., not greater than
0.1 roentgen of x-rays or gamma rays per day delivered to the entire body. The neutral red bodies are
found in increased numbers after acute exposure to doses of ionizing radiation which do not affect the
total white blood cell count, the total lymphocyte count, or the differential blood cell picture.


METHODS AND MATERIALS

This study covers a two-year period from 1944 to 1946. All of the sul-'cts have been employed
at the Los Alamos Scientific Laboratory. Except for those in the control group, all persons have been
engaged in work which has resulted in exposure to ionizing radiation or toxic chemicals. With rare ex-
ceptions, the exposure was limited to one type of ionizing radiation or to one kind of chemical. The sub-
jects included in this report were carefully selected for uniformity of sex and age, and the conditions
under which the study was carried out were kept as uniform as possible. The subjects were healthy
adult males, the great majority of which were between twenty and thirty years of age. All were in res-
idence at Los Alamos project. All subjects were essentially of the same economic status. Each blood
sample was taken in the morning between 8:30 and 10:00 o'clock. Excluded from this series was any
subject showing evidence of illness (except for a group of ambulatory controls specifically chosen for
upper respiratory infections), extreme fatigue, or blood abnormalities of known or unknown etiology
(except radiation).
As far as can be ascertained, dissimilarities between the control and exposed subjects were limited
to occupation and number of blood counts per subject. All exposed persons were engaged in laboratory
work of some sort, while the occupations of the control group ranged from physical work performed out of
doors to clerical work. Relatively few persons in the control series worked under conditions comparable
to those under which the exposed subjects worked. As far as the number of blood counts per subject is
concerned, not more than one or at the most two blood counts were obtained for each control, while
monthly or bimonthly blood counts were done on the exposed personnel. In many cases, this was the only
blood count which a control subject had experienced. The emotional response in some of these indivi-
duals cannot be ruled out as a factor which may have modified their blood pictures.
Every practical means was used to minimize technical errors and to reduce the statistical variation
of individual counts. All pipettes and hemocytometer chambers used in this study were standardized
by the National Bureau of Standards. The red blood cell count was determined by examining two sides
of a hemocytometer chamber filled with well-mixed diluted blood from a single pipette. Both sides of
two chambers containing mixed diluted blood from two pipettes were counted to determine the white blood
cell count. The hemoglobin content of the blood was estimated by means of the oxyhemoglobin method
of Evelyn. The differential leucocyte picture was obtained by examining three hundred cells two hun-
dred on a dried film of blood stained with Wright's stain and one hundred in supravital preparations. In
most cases, the number of refractive neutral red bodies in 50 lymphocytes was counted and recorded.
Since the morphologic change in the lymphocytes reported in this article can be visualized only in
supravital preparations, a detailed account of this technique as used in the Los Alamos Medical Lab-
oratory will be given.


MDDC 1102








MDDC 1102


Microscopic slides and cover slips are carefully cleaned with potassium dichromate-sulphuric acid
solution. They are then rinsed with distilled water and ethyl alcohol and stored in a dust-free box. A
small drop of blood from a fresh stab incision on the finger or lobe of the ear is picked up with a clean
cover slip (No. 00). The glass is inverted and allowed to fall on a microscopic slide on which an alco-
holic solution of neutral red and Janus green has dried. When the blood has spread between the two
surfaces to form a thin film, the cover slip is rimmed with petroleum jelly. The preparation is kept in
an ice box until just before examination, when it is allowed to come to room temperature.
In order to obtain adequate uptake of the dyes by the leucocytes, it has been necessary to use much
stronger solutions of stain than those recommended in the literature.' The dye solution used in this
laboratory consists of 40 cc of a saturated alcoholic solution of Janus green and 16.4 cc of a saturated
alcoholic solution of neutral red dissolved in 100 cc of absolute ethyl alcohol freshly prepared by the
sodium phthalate method. The alcoholic solution is allowed to flow onto clean microscopic slides, and
the excess is wiped off immediately. The slides are dried and stored in light-proof, dust-free con-
tainers. Freshly stained slides are prepared every 48 hours. Presumably, the intensity of scattered
sunlight at this altitude (7300 feet above sea level) modifies the dyes in an unknown manner which makes
necessary the use of higher concentrations of staining solutions. The appearance of the cells and up-
take of dye by leucocytes resulting from the use of slides prepared with these strong solutions do not
differ in any way from that observed in other laboratories where more dilute solutions of supravital
dyes can be employed.


RESULTS

It was observed that the lymphocytes of persons exposed to ionizing radiations and toxic chemicals
contained more refractive neutral red bodies than did the cells in the blood of unexposed subjects.
These bodies are irregularly placed throughout the cytoplasm. They are irregular in shape, vary con-
siderably in size, and have a more decided brick-red color than do the neutral red-staining vacuoles
(Figures 1 and 2). There is no increase in number or size on standing twelve hours at ice-box tem-
perature. The number of refractive bodies per cell varies from zero to more than thirty. The refrac-
tiveness of these bodies resembles that of the granules in cells of the granulocytic series and distin-
guishes them from vacuoles and nonrefractive bodies which also stain with neutral red dye.
Examination of the same blood samples stained with Wright's, Giemsa and peroxidose stains re-
vealed no morphologic abnormalities of the lymphocytes similar to that described above.
By using contrast microscopy, additional information can be gained about the structure of the re-
fractive neutral red bodies. If individual cells in a supravital preparation are examined first through
an ordinary microscope and then through a phase microscope, it is observed that the refractive neutral
red bodies show a high phase contrast while the nonrefractive and vacuolar neutral red staining bodies
show a low phase contrast. This indicates that the density of the neutral red bodies is high and that
they probably exist in the solid or granular phase. The mitochondria is the only other structure in the
lymphocytes that shows a high phase contrast. These bodies are not easily confused with the neutral
red bodies under the contrast microscope, as they are smaller, and their green color is poorly trans-
mitted through the phase optical system, while the red color of the refractive bodies is well transmitted.
The increase of the neutral red bodies in the lymphocytes of the exposed groups represents essen-
tially a quantitative rather than a qualitative change. The modification in the number and distribution
of neutral red bodies in lymphocytes for subjects variously exposed to radiation is shown in Figure 3.
In order to facilitate the demonstration of lymphocytic changes of this nature, it was decided to differen-
tiate between cells which contain an abnormally large number of refractive neutral red bodies and
those which do not. Therefore, it was agreed, on a completely arbitrary basis, to designate any lympho-
cyte with five or less neutral red bodies as "normal" and to consider any cell with six or more bodies
as "abnormal". This terminology will be used in the remainder of this article.
One thousand and sixty four hematologic studies on 364 subjects have been analyzed. Figure 5 is
a scatter diagram showing the per cent of abnormal lymphocytes in all subjects, divided into control
and exposure groups. The explanation of the type and degree of exposure of each group is given in







MDDC 1102


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Figure 1. Microphotograph (1200 x) of supravital prepara-
tion of blood cells from a subject recently exposed to
small doses of gamma radiation. Arrows point to the two
refractile neutral red bodies in the cytoplasm of the
lymphocyte in the center of the field. The other cyto-
plasmic shadows are mitachondria.







MDDC 1102


ti



























Figure 2. Microphotograph (1200 x) of a supravital preparation
of blood from a subject recently exposed to small doses of gamma
radiation for a period of two months. There are two lymphocytes
in this field. The upper one has three large refractile neutral
red bodies. The lower lymphocyte has fifteen of these bodies,
some of which are out of focus in this photograph. The large
shadow at 3:00 o'clock in the lower cell is formed by two neutral
red bodies. The other shadows in the cytoplasm of both cells are
mitachondria which stain with Janus green.








MDDC 1102


Chart 1. This exposure refers to the period 1943 to 1946 and does not take into consideration the
previous exposure record of the subject. In practically all cases except those in Radiation Groups
U and m, previous exposure to toxic chemicals or radiation is negligible. Each point in the scatter
diagram represents the per cent of abnormal lymphocytes for a single subject. In the case of most of
the controls, this point is based on one examination; in the case of the exposed subjects, the point rep-
resents the average of many (in one instance, thirty) examinations. Where radiation exposure was
intermittent, it is impossible to correlate the time of the hematologic examinations with the time of
the exposure. For purposes of comparison, Figures 4, 6, 7, and 8 show scatter diagrams of the total
white blood count, the total abnormal lymphocyte count, the total lymphocyte count, and the per cent
lymphocytes in the differential white blood cell count for each exposure group. As in the case of Fig-
ure 5, each point represents an average figure for all examinations on a single individual.
Figures 9 and 10 show the increase in per cent abnormal lymphocytes in persons accidentally
exposed to single largeinstantaneous bursts of general body radiation. It is unfortunate that previous
determinations of per cent abnormal lymphocytes had not been made on these subjects. A complete
hematologic report of these cases will be presented in-a forthcoming article. Single exposures to
general body gamma radiation which did not exceed 5 roentgens and which were delivered over a period
of several hours produced no increase in the percentage of abnormal lymphocytes.
Similar increases in abnormal lymphocytes have been observed in rabbits and cows exposed to
large doses of ionizing radiation. Morphologic changes of this type in the lymphocytes of mice and rats
exposed to radiation could not be detected because the large amounts of neutral red dye normally
taken up by the cytoplasm of the lymphocytes interferes with the identification of the refractive bodies.

Discussion

A not-too-thorough survey of the literature has failed to disclose previous descriptions of similar
morphologic changes in lymphocytes after in vivo exposure to small repeated doses of ionizing radi-
ation. Morphologic changes in living cells of various kinds have been reported following exposure to
single large doses of radiation. Prigosen" has reported the appearance of neutral red bodies in irra-
diated tumor cells. Recently, Shreks described an increase in cytoplasmic vacuoles in dark field prep-
arations of lymphocytes after in vitro and in vivo exposure.
Inspection of the scatter diagrams in this report indicates that there is an increase in abnormal
lymphocytes in the persons exposed to ionizing radiation. The per cent and total number of abnormal
cells can be correlated with the magnitude of chronic exposure. The significance of the data presented
in these diagrams is evident without further statistical treatment. There are several features of these
diagrams, however, which require further discussion.
A significant difference between controls (Group A, Class I) and the groups exposed to radiation
(Group B, Cla ;es I, II, I, and IV) is observed only for total leucocyte count, the proportion and abso-
lute numbers of abnormal lymphocytes. In the case of the total white blood cell count, the difference
is statistical and can be demonstrated best by comparing the average counts tor each group. Since
most of the total counts of all groups lie between 5000 and 9000 cells per cubic millimeter, the value
of any individual count has little significance in determining the exposure group into which the subject
falls. On the other hand, the difference in the per cent abnormal cells and absolute abnormal cell
counts between control and exposed subjects is reflected in the value for an individual subject. Most
of the controls have less than 20 per cent abnormal cells or 400 abnormal cells per cubic millimeter,
while all of the more consistently exposed subjects show percentages and total numbers of abnormal
cells above these levels. There is so little overlap of points for exposed and control groups that the
appearance of few or many abnormal cells has more than chance significance in determining the expo-
sure of any given individual.
A difference in percentage and absolute numbers of abnormal lymphocytes also exists between con-
trols and groups exposed to certain chemicals. In this respect, it should be emphasized that the biolog-
ical action of natural uranium is due to its chemical rather than its radioactive properties. Thus the
increase in per cent abnormal lymphocytes in group D is probably related to the chemical effect of
uranium. The increase in per cent abnormal cells in Group A, Class II (Figure 5) is added evidence








MDDC 1102


NUMBER OF
NEUTRAL RED BODIES IN LYMPHOCYTES

GROUP A -E* 0 1 2z 3 5
UNEXPOSED cASE 0 1 2 31
REPRESENTATIVE
CAS A C 0 1 i 2 j 3 4 +

GROUP B CASE A 0 I 2 i 3 + +
EXTERNAL RADIATK CASE B 0 1 2 3 +
CHRONIC EXPOSURE I i 3 51
REPRESENTATIVE CASE C 0 1 2 3 4 5 +


GROUP C CSE o 0i 2 13'4 +
ol AnVE TERIALA CAs i 0 141 +
CHRONIC EXPOSURE _____
REPRESEUTT C 0 1 2 34 t
ASrE O 2 13;4; +

ACUTE EXPOSURE sus 0 1 2 3 +
EXTERNAL RADIATION --
S-E CASE 16 DAYS0 1 21 3 415 +
I I I
PERCENTAGE 0 to 20 30o s5 o 10 a 10 90


Figure 3. This chart arranges the lymphocytes of representative individuals in various exposure
groups according to the number of refractive bodies which they contain. The bands extending across
the chart represent 100 per cent of the lymphocytes. The scale of lymphocytes in per cent is shown
at the bottom of the chart. The numerals in each segment of the band denote the number of refractive
bodies in that fraction of the lymphocytes. The lymphocytes containing more than five bodies are
lumped together in one group designated by the plus sign. Thus, in Case A Group A, 60 per cent of
the lymphocytes contained no neutral red bodies; 25 per cent, one body; 8 per cent, two bodies; 2
per cent, three bodies; and 5 per cent, five bodies.



I I TOTAL WHITE BLOOD COUNT I


EXPOSURE


GROUP A
UNEXPOSED


GROUP B
EXTERNAL RADIATION
GAMMA RAYS


GROUP C
RADIOACTIVE MATERIALS
c PARTICLE EMITTERS
MATERIAL A I MaTERL B


CLASS lI ,i I I n I | I I In I i I n
... . ..11 1 1 1 1 1 1 1 1 1 1 .1 1 1 1 .


"000
0000
9000


GROUP D
URANIUM


Figure 4. Scatter diagram showing the total leucocyte count for individuals in each exposure group.
The cross within the circle represents the average leucocyte count for the group.


---~,


* -,-











MDDC 1102


I ssi, n hl m I


GROUP A
UNEXPOSED


I


-- --- I -


flInflh isaaaI tVSLI[ ir*r


GROUP C
RADIOACTIVE MATERIALS
PARTICLE EMITTERS
-- IAL 5


I I


GROUP B
EXTERNAL RADIATION
GAMMA RAYS


TIw


Figure 5. Scarter diagram showing the per cent abnormal lymphocytes for persons in each exposure
group. The cross within the circle indicates the average value for the group.



I 1 n "iU*r *** n l-. s -


GROUP A
UNEXPOSED


I I I


. S MAL a.nvyrsus. o


GROUP B
EXTERNAL RADIATION
GAMMA RAYS


I I l x I I


GROUP C
RADIOACTIVE MATERIALS
-wPf wm-U~n


GROUP D
URANIUM


Figure 6. Scatter diagram showing the total abnormal lymphocytes per cubic millimeter for indi-
viduals in each exposure group. The cross within the circle indicates the average for the group.


EXPOSURE


S CLASS


GROUP D
URANIUM


EXPOSURE


CLASS
u ----


I r ~ ~~-r~-rrr r r


---- Y


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MDDC 1102


Figure 7. Scatter diagram showing the total lymphocyte count for persons in each exposure group.
The cross within the circle indicates the average for the group.


Figure 8. Scatter diagram showing the per cent lymphocytes in the differential count of individuals
in each exposure group. The cross within the circle indicates the average for the group.









MDDC 1102


INCREASE IN REFRACTIVE NEUTRAL RED BODIES
IN LYMPHOCYTES AFTER ACUTE EXPOSURE


100----l--------------- --------- -- ---- -- ---------------- -

90 -- ------------------ --
SI>




so



40

to -


GD--l -1 I 3 4 -t i- 16 2-__ : i +
LPOSUM -- 1 2 3 4 8 R. 16 20 24 7


Figure 9. Chart showing the change in per cent abnormal lymphocytes of four persons exposed
simultaneously to a burst of ionizing radiation. Only cases I and II sustained enough damage to
blood-form ng tissues to affect the blood count quantitatively.



INCREASE IN REFRACTIVE NEUTRAL RED BODIES
IN LYMPHOCYTES AFTER ACUTE EXPOSURE


DAvS AntR EIPOWAIu -


Figure 10. Chart showing the change in per cent abnormal lymphocytes of two persons exposed to
a single dose of ionizing radiation. Case V died on the twenty-fourth day following exposure.


I




a H





DAY SFTER ej








MDDC 1102


pointing to the fact that exposure to toxic chemicals changes the per cent and absolute number of abnor-
mal cells since nine of the fourteen points above the 20 per cent abnormal cell level represent plumbers
who are frequently exposed to lead fumes. These subjects have shown no clinical or laboratory evidence
of plumbism except for an occasional mild degree of basophilic stipling of the red blood cells. An in-
crease in abnormal lymphocytes is also found in persons working with industrial nonradioactive chem-
icals besides uranium and lead.
It is evident from Figures 9 and 10 that exposure to a single large dose of ionizing radiation in-
creases the proportion of abnormal cells in the circulating blood. The response of the lymphocytes of
the four subjects in Figure 9 is essentially the same although the radiation dosage of the subjects
differed by a factor of 10. It must be concluded, therefore, that the increase in abnormal lymphocytes
is not proportional to the dosage when administered as a single brief exposure.


SUMMARY

1. Analysis of the total leucocyte counts of persons chronically exposed to ionizing radiation and
toxic chemicals shows a significant statistical decrease in the exposed groups.
2. Analysis of the absolute number and per cent lymphocytes in the differential counts of the same
persons show no significant change.
3. Morphologic study of supravital preparations of blood cells of persons chronically and acutely
exposed to ionizing radiation indicates a striking increase in the number of refractive neutral red
bodies in the cytoplasm of the circulating lymphocytes. An increase in neutral red bodies is also found
in persons working with toxic chemicals.
4. It is shown that these neutral red bodies have high density and may be considered to be granules.
They have not been identified in fixed preparations.


REFERENCES


1. Fieser, Louis F., Experiments in Organic Chemistry, ed. 2, D.C. Heath & Co., Boston, 1941.
2. Prigosen, R.E., "Vital Staining of Tumor Cells After Roentgen Rays," J. Cancer Research 8:305
(1924).

3. Schrek, Robert, "Dark-field Observations on Lymphocytes Exposed to X-Rays and Other Injurious
Agents," Proc. Soc. Exper. Biol. & Med. 64:381 (1947).









MDDC 1102


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