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 ~~FPNNZ)TDR-62-95 SWC
~ TDR
62-95

CLASSIFICATION OF CIRCULATING LEUKOCYTES

. ,,IN THE NORMAL MEXICAN BURRO

by

Ronald E. Engel
Captain USAF (VC)

Sadie Cartwright F. A. Spurrell

TECHNICAL DOCUMENTARY REPORT NO. AFSWC-TDR-62-95

EJanuary 1963

Research Directorate
*AIR FORCE SPECIAL WEAPONS CENTER
Air Force Systems Command
Kirtland Air Force Base
New Mexico

Project No. 7801

 HEADQUARTERS
AIR FORCE SPECIAL WEAPONS CENTER
Air Force Systems Command
Kirtland Air Force Base
New Mexico

When Government drawings, specifications, or other data are used for

any purpose other than in connection with a definitely related Government
procurement operation, the United States Government thereby incurs no
responsibility nor any obligation whatsoever; and the fact that the Government
may have formulated, furnished, or in any way supplied the said drawings,
specifications, or other data, is not to be regarded by implication or other-
wise as in any manner licensing the holder or any other person or corporation,
or conveying any rights or permission to manufacture, use, or sell any
patented invention that may in any way be related thereto.

This report is made available for study upon the understanding that the
Government's proprietary interests in and relating thereto shall not be im-
paired. In case of apparent conflict between the Government's proprietary
interests and those of others, notify the Staff Judge Advocate, Air Force
Systems Command, Andiews AF Base, Washington 25, DC.

This report is published for the exchange and stimulation of ideas; it does
not necessarily express the intent or policy of any higher headquarters.

Qualified requesters may obtain copies of this report from ASTIA.

Orders will be expedited if placed through the librarian or other staff member
designated to request and receive documents from ASTIA.
TDR-6Z-95

ABSTRACT

The morphology of circulating leukocytes of the normal burro is reported.

The granulocytic and agranulocytic series are described and compared in part
to those of the horse. Lymphocytes are divided into small, medium, large
transitional, and plasma cellular types. Occasional cell types found in blood
smears from the normal burro are described.

PUBLICATION REVIEW

This report has been reviewed and is approved.

SDON D I. PRICKETT JOHN J. DISHUCK

Colon I USAF Colonel USAF
Director, Research Directorate DCS/Plans & Operations
TDR-62-95

1. INTRODUCTION.

Series of tests using the burro (Equus asinus asinus) were designed to
measure hematological effects produced by bilateral cobalt-60 irradiation to
the region of the brain. The burro was chosen as the experimental animal
because it is docile, larger than man, has a body volume near that of man,
carries vital organs relatively the same distance from the ground as man, has
a relatively long life span, is adaptable to variable climates, is easily handled,
is easily maintained, and is excellent for repeated blood sampling. l, Later
experiments at the Nevada Proving grounds (AEC) and the work of Trum and
Rust and their co-workers have shown that the burro manifests neurological
alterations at lower doses than other species; therefore, this animal appeared
to be ideally suited for studying neurological changes attributable to ionizing
radiation injury since these changes occur below the LD 50/30. Reversible or
irreversible brain changes producing neurological symptoms might be associ-
ated with peripheral blood alterations; thus correlative values may exist in
peripheral blood changes and neurological symptoms as noticed in the burro
irradiated to the brain with small dosages of gamma radiation.

The burro has been used extensively in hematological studies following

whole-body ionizing radiation exposures. 1-7 The normal hematological
parameters of the Southwestern burro have been reported. 8 The detailed
cellular morphology of the peripheral blood of the normal burro has not been
described, although color charts have been published.

This preliminary report describes the cellular morphology of the leukocytes

found in the peripheral blood of 46 normal adult male burros ranging in age
from 31/z years to 8 years. It is submitted because of an increased interest
in use of large animals in radiation research and so that it may serve as a
source of data for such studies in which comparisons of normal data are
desirable.

2. MATERIAL AND METHODS.

Blood samples obtained from the jugular vein were used for smearing two
slides. The edge of a 75x25mm polished-edge No. 1 microslide was used to
streak the blood. The slides were air dried; although in damp, cold air a hair
dryer was used. The slides were stored in a standard slide box with separated

compartments. The unstained slide appearing to have the best smear was

I
TDR-62-95

10
stained with modified Wright's stain.

The differential cell counts were made in areas of the smears that contained
all cell types, free from rouleau formation, and smeared to a one-cell thick-
ness within a 20X field. These areas were not easily found because of the
tendency of burro blood to undergo rouleau formation. For the most part,
this area was posterior to the tongue of the smear and in the area that colored
light was reflected. On each of 2, 990 slides, 100 white cells were counted in
determining their type.

3. RESULTS AND DISCUSSION.

To reduce technical error or poor distribution, careful attention to the

technique of smearing and staining the blood film was of paramount import-
10
ance. When poor smearing resulted, it was necessary to stain both slides.
The inherent tendency of the lymphocytes to fall out in the middle, and the
granulocytes to fall out on the outer edge of a smear, was apparent even in
slides obtained with good technique. The eosinophils and the heavier cells
had a tendency to fall off first in the smear.

Poor smearing and staining techniques having been avoided as much as

possible, a consistent method of counting cells had to be devised. In the
attempts to get a differential cell count for a true uniform distribution, the
battlement, edge count, and the cross-sectional methods were -tried. These
methods had been proposed as helpful, but did not eliminate the errors due to
nonuniform distribution of the cell types.

At least 400 cells must be counted before the results of,a differential cell
12
count may be considered reliable within 7. 5 percent. A series of 100 cell
differentials observed under the given criteria presented a similar variance.

The metamyelocyte and less mature cells of the granulocytic developmental

series of the burro are identical to those seen in the horse. These cells in
the peripheral blood of the burro are not as rare as in the horse.

The cytoplasm of the band neutrophil is similar to that of the mature

neutrophil. The nuclear chromatin is finely dispersed. The nucleus varies
from long oval to horseshoe shape. If the constriction of the nucleus is more
than three-fourths of its diameter, the cell is classified as a mature neutrophil.
(It is necessary to put this restriction on the nucleus, because over one-half

2
TDR-6Z-95

of the neutrophils would be band neutrophils, if the classical description of

band neutrophils is followed. ) Some nuclei are twisted on themselves, giving
the impression of segmentation. Because of the fine chromatin pattern, these
cells are classified as band neutrophils.

The segmented neutrophil of the burro is quite similar to that in the horse.
Numerous minute acidophilic granules are observed in the cytoplasm. Light
green to blue-gray irregularly shaped fragr-ents are found in the cytoplasm.
The color, shape, and location of the fragments are consistent with "Dohle
bodies." Their significance is unknown. 13

Large triangular clumps of chromatin are attached to the indistinct nuclear

membrane. These clumps are set off by clear, almost linear areas of
parachromatin. Smeared chromatin occupies the remaining portions of the
nucleus. Usually three to five lobes are present. These may be clearly
connected by a condensed thread of chromatin; or if gathered on one another,
indistinct separations occur, and a piled appearance results.

The eosinophil of the burro is similar to the eosinophil of the horse. The
cytoplasmic granules are usually tightly packed, nonuniform in size and
brightly eosinophilic. The cell has a red raspberry-like appearance because
the cytoplasmic membrane conforms to the outpocketing of the granules.
These granules obscure other organelles that may be present in the cytoplasm,
but occasionally a light blue cytoplasm can be observed between them.

The nucleus usually has two lobes, but there may be only ore or as many
as four. The nuclear detail is obscured by the granules for the most part.
Slight precipitation of chromatin occurs along the nuclear membrane. The
chromatin-parachromatin separation is indistinct, thus giving the impression
of smearing.

The basophil is identical to that in the horse. The cytoplasm contains

either tightly packed or loosely scattered, irregularly shaped, strongly
basophilic granules which usually partially obscure the nucleus which is fre-
quently bilobed and appears smudged.

The small lymphocyte is the smallest of the lymphocytes, and seldom, if

14
ever, divides. The cell membrane tightly encircles at least two-thirds of
the nucleus which is usually round and characterized by heavy, chromatin

3
TDR-62-95

clumping. The typical nuclear halo is not usually observed in the small
lymphocyte. The cytoplasm may have a mottled, hyaline, or homogeneous
basophilia or it may be quite clear with azurophilic granules.

The medium lymphocyte approximates the size of the mature neutrophil.

The cytoplasm can be basophilic and coarse, almost granular, or light blue,
having a nonheterogeneous appearance. Azurophilic granules may be found.
A clear nuclear halo is usually observed. The nucleus is generally round to
oval in shape, but may be bean shaped, rectangular, or may have definite
fissures. The nuclear margin is well defined. The chromatin and para-
chromatin are not clearly separated. There may be large chromatin clumps
dispersed in a smudged chromatin background. Nucleoli are not observed,
except in cells that have been spread out excessively. The nucleus is defi-
nitely mature.

The cell membrane of the large lymphocyte is delicate. This cell is larger
than the mature neutrophil and assumes an oval or circular shape on the
smear. The cytoplasm is light blue-green, and appears granular although no
granules or azure dust can be identified. There may be small circular clear
areas in the cytoplasm. In some cells, the cytoplasm appears to be divided
into two zones. The outer zone is homogeneous and light blue-green; the inner
zone is more heterogeneous and basophilic. The nucleus is large, round to
oval and has a distinct nuclear membrane. it has a medium coarse chromatin
structure. If the cell has a fine chromatin nuclear structure and basophilic
cytoplasm, it is comparable to the prolymphocyte of Ferrata, 15 and the pro-
lymphocyte described by the Committee on Nomenclature. 11

The hematopoietic reticular cells and reticular lymphocytes are placed in

the immature lymphocyte group. This group carries no criteria other than
cells that are precursors of lymphocytes.

The monocyte is sometimes difficult to distinguish from the large lympho-

cyte. 14 The cytoplasm varies from a light slate-gray to a light blue. Typical
azure dust, noted in monocytes of other species, is observed. The nuclear
membrane is delicate. The chromatin and parachromatin are quite distinct
from each other. The fine chromatin structure is preserved. Small, discrete,
delicate clumps of chromatin are scattered through the nucleoplasm.

4
TDR-6Z-95

The plasma cell is identical in morphology to those of other species.

The plasma cellular lymphocyte varies in size between the medium and
the small lymphocyte. The cytoplasm is strongly basophilic. There is the
impression that small acidophilic granules are present. The typical plasma
cell "hof" is not seen, although the lymphocytic nuclear halo is usually dis-
cernible. The cell membrane is in the association with one-third of the
eccentrically placed nucleus. The plasma-.cellular lymphocyte nucleus has a
radkern appearance, but does not appear as coarse as the smaller, more
36
compact plasma cell nucleus. Downey found a similar cell in nine cases
of benign lymphocytosis accompanied by adrenal hyperplasia. These cells
showed plasma-cell characteristics and were described as "abortive plasma
cells. 11

The plasma cell precursor attains a size comparable to the large

lymphocyte. The narrow rim of cytoplasm has a strong basophilia and a
granular appearance. There may or may not be a "hof," The large round
nucleus is somewhat eccentrically located. Its membrane is extremely deli-
cate. Discernible nucleoli are covered with finely dispersed chromatin.

Immature plasma cells are not always easily distinguished from the more
basophilic precursors of lymphocytes. Studies indicate there are lymphatic
and myeloid types of plasma cell. 15 The transformation of lymphocytes to
large, basophilic cells resembling hematopoietic reticular cells has been
acknowledged. This multipotentiality of the lymphocyte has been demon-
strated. 1721 Sundberg states,

Plasmoblasts, hematopoietic reticular cells, and reticular

lymphocytes are morphologically distinguishable from one
another, but often their similarities are greater than their
differences. The most remarkable differences are the inten-
sity and opacity of cytoplasmic basophilia, the assumption
and retention of an eccentric nucleus, and the relatively dis-
crete clumping of the nuclear chromatin of the immature
plasma cell.

5
TDR-62-95

Somewhat similar cells were found by Schulze22 in a case of agranulo-

cytosis in a horse. These cells had intensive blue staining cytoplasm with
vacuoles and a fine azurophilic granulation. The intensely staining oval
nucleus had agglomerations of clumps of chromatin.

The transitional lymphocyte resembles the true monocyte very closely.

It differs, however, in several aspects. The cytoplasm is more reddish and
gives the impression of much larger granulation, or it may be strongly baso-
philic with no demonstrable azure dust. The nucleus may be lobulated, bean
shaped, or may show increased irregularities. It has large clumps of
chromatin and the distinction between the parachromatin and chromatin is
not as sharp as in the monocyte. Various degrees of cytoplasmic and nuclear
changes are observed. The term "transitional" is used because the cell
resembles a number of cell types. Lymphocytes are known to transform into
histocytes, macrophages, 24 and plasma cells. 15,19, 21 There is no
attempt to follow this series of cells further than classifying them as transi-
tional cell types.

The following description of cells is intended to show cell types occasion-

ally found in the normal smears during this study. They were not found in
every animal examined. However, they were observed often enough to be
mentioned.

The binucleate lymphocyte is identical to the medium lymphocyte with the

exception of the nuclear structure. Binucleate lymphocytes are said to have
undergone amitotic division, but this might be due to deranged mitotic
25
division. These cells are nonspecific and may occur during antibiotic
therapy and in damaged lymphoid tissue. 25, 26

Occasionally, cells are found in the stained smears resembling leukocytoid

19
lymphocytes or Downey type II lymphocyte. 16 Dougherty2 7 found that the
type 11 lymphocyte was not specific for infectious mononucleosis and that it
was produced by nonadrenocortically mediated response to stress. He states,
"The numbers and types of circulating lymphocytes reflect the degree of bal-
ance existing between adrenocortically mediated and nonadrenocortically
mediated responses to stress stimuli."

Only on very rare occasions are megakaryocytes found in the peripheral

6
TDR-62-95

blood smears. Just as rare were eosinophilic cytoplasmic inclusions in the

medium lymphocyte and the monocyte.

Cytoplasmic budding and pyknosis or karyorrhexis are observed in the

lymphocytes. Frank and Dougherty assume these are due to ACTH and
cortisone susceptibility of the mouse lymphocytes. Neither pyknosis nor
karyorrhexis is associated with the number of smudged leukocytes. Such
cells found in stained human blood smears have little or no direct relation-
ship to dead cells. 29 A small and constant number of dead leukocytes are
29
found in normal blood.

4. SUMMARY.

One hundred differential cell counts were made from 2, 990 different
peripheral blood smears from 46 normal adult male burros. The granulocytic
series is found to be similar to the horse. The mature agranulocytic series,
composed of small, medium and large lymphocytes, does not differ from that
of the horse. Hematopoietic reticular cells and reticular lymphocytes are
categorized in the immature lymphocytic group. The plasma cellular lympho-
cyte, plasma cell precursor, and transitional lymphocyte are considered to
be expressions of the multipotentiality of the lymphocyte. Binucleated
lymphocyte, Downey type II lymphocytes, and megakaryocytes are occasion-
ally observed in peripheral blood smears.

7
 TDR-62-95

REFERENCES

I. Rust, J. H., Wilding, J. L., Trum, B. F., Simons, C. S., Kimball,

A. W., Jr., and Comar, C. L. The lethal dose of whole-body tantalum-
182 gamma irradiation for the burro (Euus asinus asinus). Radiology
60:579, 1953.

2. Trum, B. F., Haley, T. J., Bassin, M., Heglin, J. and Rust, J. H.

Effect of 400 fractional whole body gamma-irradiation in the burro
(Equus asinus asinus . Am. J. of Physiology; 174:57, 1953.

3. Kuhn, U.S.G., III. Response of the burro exposed to an experimental

nuclear excursion. U.S. Atom. Energy Comm. ORINS-10. 1, 1959.

4. Thomas, R. E. and Brown, D. G. Response of burros to neutron-gamma-

radiation. Pergamon Printed in Northern Ireland. Health Physics 6:19,
1961.

5. Rust, J. H., Trum, B. F., Wilding, J. L., and Lane, J. J. Hematologi-

cal response of the burro (Equus asinus asinus) to total body tantalum-18Z
irradiation. Acta haemat. 12:327, 1954.

6. Trum, B. F., and Rust, J. H. Radiation injury. Adv. Vet. Sci. IV ed. by
Bradly, C. A. and Jungherr, E. L., New York Academic Press, 1958

7. Lane, J. J., Kuhn, U.S.G. and Rust, J. H. The mortality response of

the burro (Equus asinus asinus) to a single total-body exposure of gamma
radiations from Zr-95/Nb-95. Rad. Res. 11:314, 1959.

8. Wilding, J. L., Kimbal, A. W., Whitaker, M. W., Trum, B. F., and

Rust, J. H. Some blood values of the southwestern burro (Euus asinus
asinus). Am. J. Vet. Res. 13:509, 1952.

9. Nikitin, V. N. Haematological atlas of farm and laboratory animals.

(Translated Titles of Plates in Volume II.) Moscow, Sel'khozgiz, 1956.

10. Schalm, 0. W. Veterinary Hematology. Philadelphia, Lea and Febiger,

196 1.

11. Benson, E. S. Errors of some common hematologic measurements.

Post Grad.Med. J. 27:A-24, 1960.

12. Barnett, C. W. The unavoidable error in the differential count of the

leukocytes of the blood. J. of Clin. Invest. 12:77, 1933.

13. Wintrobe, M. M. Clinical Hematology 5th Ed., Philadelphia, Lea and

Febiger, 1961.

14. Gowons, J. L. The life-history of lymphocytes. Brit. Med. Bull. 15:50,

1959.

8
 TDR-6 2-95

RE FE RE NCE S (cont'd)

15. Sundberg, R. D. Lymphocytes and plasma cells. Ann. N.Y. Acad. Sc.,
59:671, 1955.

16. Downey, H. and McKinlay, C. A. Acute lymphadenosis compared with

acute lymphatic leukemia. Arch. int. Med. 32:82, 1923.

17. Sundberg, R. D. Lymphocytes: Origin, structure and inter-relationships.

The Lymphocyte and Lymphocytic Tissue Vol. 1, ed. by Rebuck, John W.,
New York, Paul B. Hoeber, Inc., 1960.

18. Daugherty, T. F., and White, A. An evaluation of alterations produced

in lymphoid tissue by pituitary-adrenal cortical secretion. J. of Lab.
and Clin. Med. 32:584, 1947.

19. Rebuck, J. W. Structure of the lymphocytic series of cells in relation

to disease. The Lymphocyte and Lymphocytic Tissue, ed. by Rebuck,
John W. , New York, Paul B. Hoeber, Inc., 1960.

20. Hill, M. Re-utilization of lymphocyte remnants by reticular cells.

Nature 183:1059, 1959.

21. Roberts, J. C., Jr. Role of the lymphocyte in antibody formation. The
Lymphocyte and Lymphocytic Tissue, ed. by Rebuck, John W., New York,
Paul B. Hoeber, Inc., 1960.

22. Schulze, J. Agronulozytose beim Pferd. Mh. Vet. Med. 12:435, 1957.

23. Downey, H. Development of histocytes and macrophages from lymphocytes.

J. Lab. and Clin. Med. 45:499, 1955.

24. Rebuck, J. W., and Crowley, J. H. A method of studying leukocytic

functions in Vivo, Ann. N. Y. Acad. Sc., 59:757, 1955.

25. Ingram, M. The occurrence and significance of binucleate lymphocytes

in peripheral blood after small radiation exposures. Immediate and low
level effects of ionizing radiation. Taylor and Francis, Ltd. , 1959

26. Dobson, R. L. Binucleated lymphocytes and low-level radiation exposure.

Immediate and low level effects of ionizing radiations. Taylor and Francis,
Ltd., 1959.

27. Dougherty, T. F. and Frank, J. A. The quantitative and qualitative

responses of blood lymphocytes to stress stimuli. J. Lab. and Clin. Med.
42:530, 1953.

28. Frank, J. A. and Dougherty, T. F. Evaluation of susceptibility to stress

stimuli determined by 'stress' lymphocytes. Fed. Proc. 12:1953.

29. Petrakis, N. L., Lieberman, E. and Fullerton, J. The dead leukocyte

content of the blood in normal and leukemic patients. Blood 12:367, 1957.

9
TDR-6 2-95

DISTRIBUTION

No. Cys

HEADQUARTERS USAF

1 Hq USAF (AFRD C-NE), Wash 25, DC

1 Hq USAF (AFCSG, Bio Del Br, Prey Med Div), Bldg T-D,
Wash 25, DC
1 AFOAR, Bldg T-D, Wash 25, DC
I AFOSR, Bldg T-D, Wash 25, DC

MAJOR AIR COMMANDS

AFSC, Andrews AFB, Wash 25, DC

1 (SC T)
1 (SCT-2)
I (SCGB)
SAC, Offutt AFB, Nebr
1 (SUP-4)
1 (SUP -5)
1 AUL, Maxwell AFB, Ala
1 USAFA, United States Air Force Academy, Colo

AFSC ORGANIZATIONS

1 ASD, (ASAPRL), Wright- Patterson AFB, Ohio

1 RTD, Bolling AFB, Wash 25, DC
1 AF Msl Dev Cen, (SRLTL), Holloman AFB, NM

KIRTLAND AFB ORGANIZATIONS

AFSWC, Kirtland AFB, NM

1 (SWEH)
25 (SWOI)

OTHER AIR FORCE AGENCIES

1 6570 AMRL, Wright- Patterson AFB, Ohio

1 6 571 ARL, Holloman AFB, NM.
1 SAM, Brooks AFB, Tex
1 USAF Aerospace Medical Center, (AMR), Brooks AFB, Tex

10
TDR-6 - 95

DISTRIBUTION (cont'd)
No. Cys

1 Medical Service School, (Radiobiology Br), USAF (ATC), Gunter

AFB, Ala

ARMY ACTIVITIES
I The Surgeon General, Department of the Army (Chief, Research
and Development Division), Wash 25, DC
1 Director, Walter Reed Army Institute of Research, Walter Reed
Medical Center, Wash 12, DC
1 Commanding Officer, Chemical Warfare Laboratories (Library),
Army Chemical Center, Md
1 Research Analysis Corp., ATTN: Document Control Office,
6935 Arlington Road, Bethesda, Md., Wash 14, DC
I Director, Army Research Office, Arlington Hall Sta, Arlington, Va

NAVY ACTIVITIES

1 Chief of Naval Research, Department of the Navy, Wash 25, DC

I Chief, Bureau of Medicine and Surgery (Special Weapons Defense
Division), Wash 25, DC
1 Commanding Officer, Naval Research Laboratory, Wash 25, DC
1 Commanding Officer, Naval Radiological Defense Laboratory
(Technical Info Div), San Francisco 24, Calif

OTHER DOD ACTIVITIES

1 Armed Forces Institute of Pathology, Wash, DC

1 Armed Forces Radiobiology Research Institute, Bethesda 14, Md
10 ASTIA (TIPDR), Arlington Hall Sta, Arlington 12, Va

AEC ACTIVITIES

1 University of California Lawrence Radiation Laboratory (Technical

Information Division), P. 0. Box 808, Livermore, Calif
1 Director, Los Alamos Scientific Laboratory, (Report Library),
P. 0. Box 1663, Los Alamos, NM
1 Brookhaven National Laboratory, Upton, Long Island, NY
1 US Atomic Energy Commission, Oak Ridge Operations Office,
(Tech Info Serv Ext), P. 0. Box E, Oak Ridge, Tenn

11
TDR-62-95

DISTRIBUTION (cont'd)

No. Cys

OTHER

1 Lovelace Foundation, 4800 Gibson Blvd, SE, Albuquerque, NM

1 National Academy of Sciences, National Research Council,
(Medical Records Section), Room 3Z5, Division of Medical
Sciences, 2101 Constitution Ave., NW, Wash 25, DC
1 American Medical Association, (Librarian, Quarterly Cumulative
Index Medicus),535 North Dearborn St., Chicago, Ill
1 University of Pennsylvania, (Biological Abstracts), 3815 Walnut St.,
Philadelphia 4, Pa
Official Record Copy, (SWRBM, Capt Engel)

12
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