BONE MARROW AS A PRACTICAL
CLENECAL DIAGNOSTHC AGENT IN
CANINE PRACTICE

Thesis for #he Degree of M. S.
MlCHTGAN STATE COLLEGE

Frederick Wifliam Meier
1954

ou.‘.v.§.,.'c’..oo.'fioo:b

:°.“?‘n

«may

 

This is to certify that the
thesis entitled

Bone Marrow as a Practical Clinical Diagnostic l
Agent in Canine Practice

presented by

Frederick William Meier

has been accepted towards fulfillment
of the requirements for

J3;— degree in Matholog

fimmml< The/L

Major professor

 

Date November 23, l95h

0-169 1

   

BONE MARROW AS A PRACTICAL CLINICAL DIAGNOSTIC

AGENT IN CANINE PRACTICE

BY

FREDERICK WILLIAM MEIER

A THESIS

Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Animal Pathology

1954

THESIS

db

ACKNOWLEDGMENTS

The author wishes to thank Dr. Frank Thorp, Jr., under whose
supervision this investigation was undertaken. Dr. Thorp's genuine
interest in the problem, together with his spontaneous enthusiasm
toward research, were a constant inspiration.

To Dr. R. A. Runnells he wishes to express his appreciation
for the many helpful suggestions given so generously.

Grateful acknowledgment is due Drs. R. D. Earner, E. S.
Beneke, Charles H. Cunningham, and R. F. Langham for their ever—
ready willingness to assist in acquiring a thorough understanding of
their subjects in the classroom as well as in the laboratory.

To Dr. M. Lois Calhoun go my sincere thanks and profound
gratitude for the careful manner in which she read the manuscript,
and for the constructive criticism she offered so graciously.

For having made available the facilities of the clinical labora-
tory, sincere thanks go to Dr. Lloyd B. Sholl.

To all others on the teaching staff and otherwise concerned
with the fulfillment of the requirements of Michigan State College

the author wishes to thank for their assistance in one way or another.

ii

343487

The acquisition of material proved to be a much more diffi-
cult task than had been anticipated. The investigator extends his
sincere thanks to Drs. S. R. Elko, M. P. L'Heureux, and M. Lipson,
of Detroit, Michigan, for the part they played in securing material to
carry out this work.

To Mrs. Lillian George, secretary in the author's animal
hospital, many thanks are due for her assistance in typing the manu-

script.

iii

ABSTRACT

The principal problem in this investigation was to find a site
in the body of the dog which could furnish marrow substance of good
quality and in adequate amount to make bone marrow biopsy a prac-
tical clinical procedure which could be used as an aid in diagnosis.

Reports of marrow biopsies in dogs were very scant. The
procedures described were copied from the human field: puncture
of sternum, iliac crest and dorsal processes of vertebrae. The
author presented reasons why these sites were unacceptable. The
small size of the bones, together with their anatomical position--
being relatively deeply situated and well covered--made them ex-
tremely difficult to approach and to use as a practical clinical pro-
cedure.

In the study of the topography of whole bone marrow sections
another problem was encountered. After fixation of a marrow pencil
the myeloid substance of the dog becomes very crumbly. To obtain
a marrow Specimen which would furnish entire cross and longitudinal
sections it was decided to process the whole marrow pencil intact

up to and including the paraffin bath.

The modus operandi of obtaining marrow from the bone by
suction also had to be modified from the procedure used in human
medicine. Here again size and shape of the instruments were not
directly applicable to use in canine practice. A biopsy cannula was
designed and made to fit the specific requirements demanded by the
smallness of the marrow cavity. The author introduced a curved
biopsy cannula which could be entered into the lumen of the bone for
a limited distance.

Femoral puncture has been proposed as a preferred practical
clinical procedure for doing bone marrow biopsies on the dog. It
involves a minor operation capable of being performed either under
local or general anesthesia. The surgery is absolutely safe: there
is no danger of penetrating a body cavity. The proximal portion of
the femur is a reliable source of good quality marrow inasmuch as
it responds very early to any stimuli in the adult.

The locus of the biopsy is easily accessible and causes a
minimum of discomfort to the patient.

Normal bone marrow has been described grossly and micro-
scopically. A scheme outlining the gross and microscopic appraisal

of myeloid tissue has been presented.

It has been shown that qualitative bone marrow findings were
often invaluable when correlated with those of peripheral blood; in

fact, bone marrow findings may furnish the only clew on which to

base a diagnosis.

The photomicrographs were taken by the author of Specimens

which were used as a part of this investigation.

v i (f

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TABLE OF CONTENTS

INTRODUCTION ...............................
HISTORICAL REVIEW ...........................
MATERIAL AND METHODS .......................
Distribution of Bone Marrow ....................
The Normal Bone Marrow Picture ................
Technic for Fixing Bone Marrow .................
A New Biopsy Procedure . . . .5 ..................
Description of the Biopsy Procedure ..............
ANATOMY AND HISTOLOGY OF BONE MARROW .......
Early Development ...........................
The Five Different Types of Marrow ..............
Marrow Stroma and Vascular System ..............
The Free Cells .............................
Mature and Immature Elements ..................
Heteroplastic Hemopoiesis ......................

The New Formation of Free Cells from Fixed Cells

How the Myeloid Elements Enter the Blood ..........

vii

Functions of the Myeloid Tissue .................
Regarding the Question of Lymphatics of Bone Marrow . .
PRESENTATION AND EVALUATION OF FINDINGS ......
General Considerafions ........................
Appraisal of Marrow Structure ..................
Description of the Hemopoietic Cells of Bone Marrow
The Myelograms ............................

DISCUSSION ..................................

viii

Page
53
54
56
56
60
65
77
81
88
9O

91

TABLE

II.

III.

IV.

LIST OF TABLES

Signalment of Dogs Used ....................
Clinical Tests of Ten Dogs ..................

Nomenclature Recommended for Cells of
Blood and Blood—Forming Organs ..............

Percentage of Distribution of the Marrow
Cells from the Femurs of Ten Dogs ...........

Comparison of Dog Data with Another
Femoral Myelogram .......................

ix

Page
11

13

66

78

80

Figure

10.
11.

12.

13.

LIST OF FIGURES

Shaft of femur with a portion of cortex removed

showing marrow intact before fixation .........

Shaft of femur with a portion of cortex removed

showing marrow intact after fixation ..........

A cross section of the fourth sternal segment

from a twenty-pound dog ...................

A section from the highest portion of the wing

of the ilium (crest) from a twenty-pound dog . . . .

A section of a dorsal process of a thoracic

vertebra from a twenty-pound dog ............

Instruments used in performing femoral

marrow biopsie s ........................

Normoblasts follow the path of the vessels

which occupy the interstices between fat cells . . . .

A wide capillary between fat cells connecting

sinusoids ..............................

No rmal ma rrow .........................

Normal marrow ............... . .........

Cross section of a humerus from a dog one

year old ..............................

Central artery and vein ...................

Page

18

18

22

22

22.

25

34

34
36
36

38

39

4O

Figure Page

14. Entire cross section of the marrow pencil
from a seven months old dog ................ 42
15. Plasmacyte ............................. 75

xi

INTRODUCTION

A correct diagnosis is the most important aspect of the prac-
tice of veterinary medicine. Obviously it would be only a remote
coincidence if the therapy prescribed were to help the sick patient
to recover if the diagnosis had been incorrect. Twenty years in
the practice of veterinary medicine have shown that far too many
mistakes are being made in individual private practice. However,
the records of large clinics, human as well as veterinary, reveal
that they too have erred in their diagnoses altogether too frequently.
The inference of the foregoing is: a diagnosis is less likely to be
incorrect when four to eight doctors examine a patient than when the
entire examination is carried out by one man.

Any clinical procedure therefore which will aid in arriving
at more correct diagnoses and/or point to the origin and nature of
the patient's ailment at the earliest possible moment is a contribu-
tion to, and a step forward in the practice of veterinary medicine.

It was thought that examination of bone marrow could be used
clinically to this end. It is not intended here to give the impression

that the study of bone marrow is a new idea in veterinary medicine;

instead, the object was to find a practical method of bone marrow
biopsy which could be used as a routine clinical procedure.

The literature contained very little information on practical
clinical marrow biopsy procedures as applied to small animal prac—
tice. Indeed, nearly all of the material reported on has been from
dogs which had been used in some other experimental work in the
human field and not from apparently healthy animals. The questions
asked were:

1. What is the pattern of bone marrow of healthy dogs?

2. What is the differential picture of a bone marrow biopsy
smear of presumably normal dogs?

3. Where can biopsy material be obtained if this is to be a
routine clinical procedure?

4. Where is the site most representative of bone marrow
activity in health as well as in disease?

5. What are some of the diseases in which bone marrow
may give a clue or even be the only tissue to give an answer?

6. Are bone marrow biopsies dangerous to the patient if
properly executed?

7. In which branch of veterinary medicine does bone marrow

examination belong ?

The answers to the above questions will be discussed in the
following sections, except the last question, which properly should
be discussed here.

The science dealing with the study of blood is known as hema-
tology. The thought occurred that the study of myeloid tissue, which
is bone marrow, might appropriately be called myelology. These
two sciences have much in common and may therefore be studied
by the same group of medical men.

Karl Rohr, in his book "Das Menschliche Knochenmark,” ex-
presses the opinion that the pathologic anatomist might think it arro-
gant for the clinician to have made the study of marrow-pathology
the latter's problem. Contrary to this belief, it may be stated that
the study of bone marrow as viewed here properly belongs in clinical
pathology.

Hematology is as old as medicine itself. In fact, no authen-
tic record has been found which would claim when blood was first
used as a diagnostic agent. On the other hand, the study of bone
marrow as a diagnostic agent is relatively recent. What has been
the reason for the delay? It was shown by Neumann (1869) that
bone marrow is the sole source of red blood cells of the normal

adult individual. This pronouncement has never been successfully

challenged. It has also been shown by numerous other investigators
that granulocytes are produced exclusively by bone marrow under
normal conditions. Bone marrow is also capable of giving rise to
lymphocytes and cells of the reticulo-endothelial system. Thus it
may be said that bone marrow is the most important hemopoietic
organ of the normal adult.

Evidently the reason for the delay of the use of bone marrow
as a diagnostic agent lay in that no practical way had been found as
yet to use it clinically. The development of marrow biopsy will be
presented in the historical review of this thesis.

Bone marrow biopsies have furnished not only information
about the progress of disease, its prognosis and response to treat-
ment, but also have given some important clues of other functions.
Prior to this time marrow functions were based on deductions made
from peripheral blood pictures, embryological and experimental re-
search conducted on laboratory animals.

In comparing blood and bone marrow it will become apparent
that blood is merely a vehicle for products of metabolism and some
formed elements, and does not produce anything, while bone marrow
is an active manufacturing center. One must not, however, minimize

the importance of blood. One only needs to recall the words of the

famous German poet, Johann Wolfgang von Goethe, who said, ”Blut
ist ein besonderer Saft."

No satisfactory substitute for blood has been found as yet.
To assure the greatest success in arriving at a correct diagnosis
the clinical pathologist should always be mindful not to divorce
pathologic anatomy, hematology, and bone marrow study, but evaluate
the findings comparatively.

Cytogenesis will be discussed only briefly because the view-
points of investigators are still too divergent. Furthermore, it is
questionable whether the mode of genesis of the cells per se is as
important from the standpoint of diagnosis of disease as it is aca-

demically.

HISTORICAL REVIEW

During the first part of the nineteenth century students and
research workers in the field of biological sciences thought that the
usefulness of bone marrow appeared to be ended with the part it
played in the development and growth of the skeleton, except for a
few minor functions such as to reduce the weight of bones, decrease
their brittleness, and act as a shock—absorber or cushion for intra—
medullary vessels.

That vital functions of bone marrow began to be appreciated
and can be realized from the writing of Koelliker in Germany and
Beale and Wharton Jones in England about the year 1846 as quoted
by Neumann (1869).

Neumann (1868, 1869) of Germany was the first to show that
bone marrow was the only place in the body of normal adult animals
where red blood cells were produced. He also recognized the nu-
cleated, granular variety of blood cells originating in this newly
discovered hemopoietic organ. Almost simultaneously and entirely
independently came the announcement of bone marrow as a hemo-
poietic center by Bizzozero of Italy (1868). His findings were al-

most identical with those of Neumann.

The introduction of special staining methods in hematology
by Ehrlich about 1882 gave that science the impetus it needed to
come into its own. The newly discovered staining methods were ap-
plied to bone marrow studies, and much work was done in various
parts of Europe.

Pianese (190 3) punctured the lower femur to prove a diagnosis
of a parasitic disease. The same year Wolff (1903) in Germany rec-
ommended tibial puncture of experimental animals. Ghedini (1908)
described in detail trephining of the proximal third of the tibia for
bone marrow. Twenty-six cases convinced him of the diagnostic
value of this new procedure and showed the difference between the
composition of blood and bone marrow.

This work received very little attention outside of Italy.
Three reasons were given for this disinterest:

1. A knowledge of dependable differential blood morphology
was still lacking.

2. Quality of tibial marrow was very variable and uncertain.

3. Trephination was too much of a surgical procedure.

About fifteen years elapsed before any important biopsy pro-
cedure was reported. Seyfarth (1923) was the first one to do sternal

and costal trephination on living human patients. Since this was a

surgical procedure like that of Ghedini's it was not generally accepted.
However, Rohr (1949) stated it was used by Schilling in Germany,
Dameshek, Custer, and Peabody in the United States of America.

It was Arinkin (1929) who introduced a simple and practical
method for doing marrow biopsies on living persons.

Many technical improvements of biopsy instruments, methods,
and sites followed in the next few years.

The first reports of marrow biopsies in the United States of
America appeared about 1933 (Rohr, 1949).

The use of bone marrow biopsy is now a firmly established,

standard procedure in most of the modern hospitals and clinics of

the world.

MA TERIAL AND METHODS

Distribution of Bone Marrow

The preliminary work involved the investigation of the normal
distribution of myeloid tissue in the skeleton of the dog. For this
purpose use was made of necropsy material as it came to the post-
mortem room. Dogs of small to medium size were selected because
the purpose of this work was to find a practical biopsy procedure
capable of being used in routine small animal practice.

It was learned that thoracic and lumbar vertebrae, sternum,
iliac crest, ribs, and diploes contained only red marrow. The long
bones contained a widely variable mixture of red and yellow marrow.
The proximal end of the tibia was rich in red marrow in dogs less
than six to nine months old. After one year of age one would find
a considerable admixture of yellow and red marrow. The femur and
humerus have an ample supply of red marrow in the proximal por-
tions and it persists in adequate quantity even in advancing age. As
the animals advance in age a peculiar distribution of red marrow
was noted: red marrow was more noticeable at the periphery of

the marrow pencil throughout the length of these bones. However,

10
there was a greater density of red marrow at the proximal end of
the femur and humerus.

The marrow cavity of the ribs was so small that it was not
considered further as a biopsy site.

Vertebrae, sternum, and tuber coxae do not have a marrow
cavity per se. The interior of these bones is entirely cancellous.
The minute spaces between the relatively thick cancelli are filled

with red marrow.

The Normal Bone Marrow Picture

Ten dogs were used to obtain entire cross and longitudinal
sections of bone marrow in order to establish the normal topography
and differential picture of myeloid substance. The ages of the dogs
varied from seven months to twelve years. There were five males
and five females in this group. The breeds included some pure-
breds as well as mixed breeds. Sizes of dogs varied from small
to medium. All animals were in good physical condition; there was
no history of any illness for the past several weeks, and up to sev-
eral years in some instances. See Table I.

A thorough physical examination was given each animal at the

time of acquisition. The dogs were then held for a ten-day observation

 

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I

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TABLE I

SIGNALMENT OF DOGS USED

11

 

 

 

Case Age Sex Breed Weight
I 12 years female Fox Terrier 15 lbs.

II 7 months female German Shepherd (mixed) 25 lbs.
III 4 years female Cocker Spaniel 40 lbs.
IV 9 months male Terrier (mixed) 18 lbs.
V 2% years female Collie 43 lbs.
VI 1 year male Collie & Shepherd 40 lbs.
VII 1 year male Collie 8: Shepherd 52 lbs.
VIII 6 years male Terrier (mixed) 40 lbs.
IX 7 months male Terrier (mixed) 18 lbs.
X 11 years female Terrier (mixed) 12 lbs.

 

 

12
period. Following this a final checkup was made which usually re-
quired several days. This included the following:

Fecal examination for parasites
Urine analysis

Hemoglobin determination

Red blood cell count

White blood cell count
Differential blood cell count
Nonprotein nitrogen determination
Creatinine determination

Blood sugar determination

The results are tabulated in Table II.

Bone marrow biopsy for differential count was performed under
complete nembutal anesthesia. The animals were then sacrificed while
still under complete anesthesia by inhalation of chloroform. Whole
bone marrow substance was obtained immediately following death and
fixed in a solution consisting of 15 per cent formalin, 1 per cent acetic
acid, and 5 per cent zinc chloride. After twenty-four hours in the
above solution the specimen was dehydrated and embedded in tissue

1
matt. The mounted sections were stained with eosin and hematoxylin.

—-——

 

 

1
Fisher Scientific Company, New York, New York.

L a fi.. -__.’-.z$]‘

13

TABLE II

CLINICAL TESTS OF TEN DOGS

 

 

Case Number

 

 

 

 

 

Test - _
I II III IV
Blood count:
Differential:
Neutrophils ........ 80.0 59.0 61.0 79.0
Lymphocytes ....... 14.0 33.0 34.0 17.0
Monocytes ......... 4.0 2.0 2.0 4.0
Eosinophils ........ 2.0 6.0 3,0 0.0
Basophils ......... 0.0 0.0 0.0 0.0
2
W.B.C. 15.6 13.05 16.05 11.80
3
R.B.C. ............. 5.40 5.30 4.70 5.90
, 4
Hemoglobin ......... 107.0 87.7 87.7 83.0
Blood chemistry:
NPN5 .............. 42.7 37.5 34.5 41.2
Creatinine 1.20 0.70 0.60 0.70
7 0
Sugar ............. 67.0 65.0 63.0 72.
Numbers indicate per cent of total number of cells counted-
3
2 Numbers indicate thousands/mm White Blood Cells.
3
3 Numbers indicate millions/mm Red Blood Cells.
4 Numbers indicate per cent of normal. Hemoglobin of 14-5
mg per 100 m1 of blood = 100 per cent of normal.
Numbers indicate mg % Nonprotein Nitrogen.
Numbers indicate mg %
Numbers indicate mg %

 

 

 

14

TABLE II (Continue d)

 

 

Case Number

 

 

Average
V VI VII VIII IX X
59.0 84.0 50.0 76.0 82.0 79.0 70.9
13.0 10.0 25.0 15.0 10.0 19.0 19.0
8.0 2.0 10.0 5.0 5.0 2.0 4.40
20.0 4.0 15.0 4.0 3.0 0.0 5.7
0.0 0.0 0.0 0.0 0.0 0.0 0.0
9.55 9.15 6.70 9.40 13.5 7.25 11.20
5.40 5.60 5.30 5.90 5.30 5.46 5.42
76.0 97.0 91.0 103.0 90.7 97.0 92.01
39.0 33.7 39.0 45.0 42.7 47.2 40.3
0.70 1.50 1.50 1.60 0.70 1.10 1.03

67.0 74.0 60.0 70.0 80.0 72.0 69.0

 

 

15

Technic for Fixing Bone Marrow

In the study of bone marrow of the dog as a hemopoietic
organ the conclusion was reached that the humerus and the femur
play an important role. Sternum, dorsal processes of thoracic and
lumbar vertebrae, and the iliac crest do not appear a satisfactory
source for obtaining bone marrow as a biopsy procedure. In the
experimental laboratory one usually finds dogs of medium size or
often of a large medium size. However, in the practice of small
animal medicine the small breeds numerically predominate. It is
very difficult, if not impossible, to do satisfactory and practical
bone marrow biopsies in the sites last mentioned.

Advances in methods of reduction of fractures of the long
bones have given much valuable information as to procedures which
may be used in performing biopsies involving bone marrow.

Obviously the tibia of the dog will be brought to. one's mind
because of its ease of accessibility for bone marrow biopsy. Un—
fortunately tibial marrow is just as unsatisfactory in the dog as it
is in the human being except in the very young or in those cases
where there is extraordinary hemopoietic activity. In dogs just past

one year old red marrow in the tibia is prominent by its paucity.

16

If necropsies are performed, bone marrow for imprints,
smears, or small sections can be obtained by squeezing marrow
from a rib with a suitable plier, and from the femur or humerus
with a curette.

To study entire cross sections as well as longitudinal speci-
mens one should follow a different technique. It was learned early
that bone marrow of the long bones of dogs cannot be extruded from
its bed intact as is possible in some laboratory animals; e.g., the
rabbit (Steinberg and Martin, 1946). Many bone papillary projections
extend from the wall of the lumen of the marrow cavity. Further-
more, the marrow pencil is delicate and becomes very crumbly after
fixation. To obtain entire and intact sections of marrow of any por-
tion of the long bones the author used the following procedure, which
is a modification of a method described by Mayer gLal. (1945) for
staining femoral marrow of rats and guinea pigs:

Cut the long bone transversely at both metaphyses. Hold the
diaphysis in a small bench vise with the cut ends against the jaws
of the vise. Then proceed to cut a groove or slit lengthwise from
one extreme end to within about one-eighth to three-sixteenths inch
of the opposite end just through the cortex of the bone. Do the same

about one-fourth around the circumference of the bone. Follow this

17
by carrying out the same procedure on the opposite side of the bone.
Now cut transversely through the cortex of the bone at the end and
between the two slits on each side. The author used a Dremel Moto-
tool No. 21 with a steel cutting rotary saw, one inch in diameter and
about 0.025 inch thick. It is now possible to lift the portion of bony
cortex away from the marrow. Two small periostum-lifters are very
useful for this. As the. portion of bone to be removed is raised
gently, inspect the junction of cortex and marrow, and free any por-
tions of marrow adhering to wall of lumen of bone. The marrow
pencil is now held between the two remaining strips of cortex, the
latter being held firmly in a normal position by the annular ring of
bone allowed to remain (Figures 1 and 2). The entire specimen can
now be fixed and embedded in paraffin. In cases where the marrow
substance is more than four millimeters in diameter, additional slits
may be cut through the cortex of bone to facilitate penetration of
fixing and embedding solutions. The remaining portions of bone
can now be removed carefully and the marrow will be held intact
by the paraffin. This latter step is very important, as otherwise
the marrow pencil may fall apart. Portions for embedding in prep-

aration for sectioning can now be selected from the marrow pencil.

 

 

Dremel Manufacturing Co., 14th and Clark Streets, Racine,

Wisconsin.

 

 

marrow intact before fixation. The light and dark areas
are indicative of the variation of the thickness of the

peripheral layer of red marrow. The central portion is
all fatty marrow. X 2%. Compare with Figure 2, below.

'- -. . a ‘-

 

Figure 2. Shaft of femur wit prton of cortex removed showing
marrow intact after fixation. X 221-. Compare with Fig-
ure 1, above.

19
Sections of five microns thickness are preferred for topograph-
ical studies as well as the determination of the myeloid-erythroid
ratio. The latter will be dealt with in the section under ”Discussion."
The regressive staining method with hematoxylin and eosin was

used throughout this work on paraffin sections. It was found that
more satisfactory differentiated staining for making the myeloid-
erythroid ratio count was obtained if the sections were definitely
overstained with hematoxylin, and then decolorized to a point where
the leukopoietic cells appeared decidedly vesicular. At this stage
of decolorization the cells of the erythropoietic system still_retain
much of the basic stain. It seems that only the chromatin structure
of the white cells stains with hematoxylin. Parachromatin, specific
granules, and azurophil granules either do not take this basic stain
at all or do not retain it. The author noticed that if the staining
with eosin was allowed to remain more intense than usual the con-
trast was enhanced still further. A Whipple disc was used to indi-
cate the area being counted; it also served as a measuring device.
The nuclei of the normoblasts in sections measure two to four microns
in diameter and stain so intensely that no structure within them can

be discerned. Often a small, lighter, bluish staining portion of the

cytoplasm was visible .

20

A New Biopsy Procedure

The human clinical pathologist obtains marrow for biopsy
from the sternum, iliac crest, and dorsal processes of vertebrae of
adults and older children. In infants the tibia is used also because
the marrow of this long bone is still sufficiently active to be of value
in obtaining a reasonably reliable myelogram.

Veterinary literature contains very few accounts of biopsy
procedures in dogs. Bloom (1945) stated that sternum and iliac
crest have been used to obtain marrow by needle-puncture method.
Horn gLal. (1953) in Germany described sternal puncture in the dog
and claimed to have obtained marrow specimens which produced good
smears for satisfactory myelograms.

At the outset of this investigation the task of finding the best
location for doing bone marrow biopsies was pursued. Since the ob-
ject of this work was to find a way to use bone marrow as a prac—
tical clinical diagnostic agent, dogs of small, medium, and medium
large size were examined for favorable biopsy sites. The workers
previously mentioned do not state the size of dogs used. In this
work no dogs of the so-called large breeds were used because rela-

tively few of these animals are seen in the average small animal

practice.

. rmal

 

21

As can be seen from Figures 3, 4, and 5, there is a paucity
of marrow in sternal, iliac, and vertebral bones. To this must be
added the difficulty of executing punctures of these small and well—
covered bones.

The femur has presented the best quality of marrow, and can
be made accessible without too great a discomfort to the patient.
The most advantageous site on the femur is the lateral surface of
the junction of the proximal and middle thirds.

Biopsies have been done during this investigation under local
anesthesia without apparent pain to the patient. The only time that
any signs of discomfort were noted was when suction was applied to
draw marrow substance into the syringe. The latter sensation is
experienced by human patients also according to information obtained
from human clinicians.

General anesthesia is such a simple and safe procedure now
that it is recommended in all cases unless contraindicated. In ner-

vous and apprehensive animals general anesthesia is to be preferred.
Description of the Biopsy Procedure

1
For local anesthesia, codrenin has been used.

“u._-_~

Parke, Davis 8: Company, .105. Campau Ave. at the River,
Detroit 32, Michigan.

22

 

Figure 3. A cross section of the fourth sternal segment from a
twenty-pound dog. The entire bone is finely cancellous

and well covered. The larger vacuoles are artifacts.
x 4%.

 

Figure 4. A section from the highest portion of the wing of the
ilium (crest) from a twenty-pound dog. The part is en-
tirely cancellous. The cancelli are relatively hard and
thick. X 4%.

 

 

Figure 5. A section of adrsal process of a thoracic vertebra from
a twenty-pound dog. The portion where marrow puncture
would have to be made is one millimeter thick. X 4%.

23

Pentathol sodium or some of the other short-term general
anesthetic substances were used by the intravenous route.

The lateral surface of the hind leg was clipped very closely
with a No. 40 Oster clipper blade from the knee to just above the
trochanter major. The clipped area was then scrubbed with germi-
cidal detergent.1 The detergent was removed with water and sponge.
Ether was applied, followed by 70 per cent alcohol with a final ap-
plication of tinted phemerol.Z The field of operation was then draped
with a sterile shroud provided with a slit to permit making an incision
through the skin over the femur at the junction of its proximal and
middle thirds. The skin incision was generally about one and one-
half inches long for a medium-size dog. After passing through skin,
subcutaneous fascia, and fat, the tensor fascia latae and biceps fe-
moris muscles were severed. The posterior portion of the vastus
lateralis was now in plain sight. No incision was made into this
muscle. It covers about two-thirds of the anterior portion of the
lateral surface of the shaft of the femur. By spreading the previously

incised muscles somewhat and then retracting the vastus lateralis

 

Ibid.

Ibid.

24

cephalically the whitish glistening surface of the femur comes into
plain view. A specially designed spreader (Figure 6) has given satis—
factory results for dilating the opening. The work can be done without
an assistant if necessary. A hole one-sixteenth or three thirty-seconds
inch in diameter was drilled through the cortex of the bone. A safety
stop (Figure 6) prevented the drill from plunging into the marrow sub-
stance. At the start of the drilling operation caution must be taken
to avoid letting the drill slide off to the sides of the bone into soft
tissues. A closely fitting cannula can be inserted into the previously
drilled hole, and with the aid of a conventional—type syringe marrow
may be aspirated. Inasmuch as the marrow cavity of smaller breeds
of dogs is very small, biopsy with the usual straight needle or can-
nula has not given the desired results in some instances. It has
been found that bending a hypodermic needle so that the included
angle was about 120 degrees will permit the passage of the biopsy
needle through the previously drilled hole. The needle was then
thrust into the marrow substance for a distance of about one-fourth
inch in a direction longitudinal to the lumen of the bone and the
aspiration was made.

If the bending was made over a round shaft about three—eighths

inch in diameter the curvature was not too abrupt, and allowed insertion

 

Figure 6.

 

3 ‘7

Instruments used in performing femoral marrow biopsies:
(1) drill chuck with drill and safety stop in place; (2) re-
tractor; (3) drill chuck wrench; (4) drill with safety stop;
(5, 6, 7, 8, 9) a3pirating needles. Approx. X 2/3.

25

26

and withdrawal of a stilet. The free end of the biopsy needle or
cannula should be honed and polished smoothly to prevent tearing the
stroma and blood vessels of the bone marrow. To have a little
blood mixed with the marrow substance is not altogether undesirable.
The blood serum appears to free the cells from the matrix of the
myeloid tissue, giving a better smear. If there was no bleeding--
and frequently there was not--a drop or two of plasma or serum on
a watch glass was mixed with the aspirated marrow (Isaacs, 1937).
From this mixture a smear was made exactly as in the case of
peripheral blood films. After air drying, the slides were stained
with Wright's stain.

The following size aspirating needles should be available:
fourteen, sixteen, and eighteen gauge.

All instruments were sterilized by autoclaving.

The wound was closed with interrupted sutures using No. 000
plain catgut to approximate the muscle tissue, whereas cytor was
used on the skin.

The most surprising observation following the femoral marrow
biopsy as described here was that the animals did not go lame or

1
Bauer & Black, Division of The Kendall Company, Chicago,

Illinois .

27
show any signs of discomfort. There was little swelling, and healing
was by first intention in all cases.

Other methods of processing biopsy material preparatory to
making smears and stains:

The method of adding a little homologous serum to the marrow
aspirate as suggested by Isaacs (1937) prompted the author to try
commercial homologous serum (canine). The red cells were cre-
nated, and the staining quality was poor. Homologous serum is readily
available in veterinary hospitals, and would have simplified the biopsy
procedure further. It is presumed that the preservative in the com-
mercial product was responsible for the poor results.

Physiological saline solution was tried as a diluent. This re—
sulted in smears in which the nuclei were much more distinct than
in any other method used, but most cytoplasms became very fragile.
Nuclear pattern is the most important single factor in the differen-
tiation of blood and marrow cells. Some modification of a saline
solution might have some merit.

Addition of anticoagulants to the marrow aspirate also re-
sulted in inferior staining quality of the cells.

The better-stained smears were obtained from direct smears
of the orange—red liquid portion of the aspirate before coagulation,

or by adding a little autogenous serum or plasma.

28
It is advisable to make six to eight smears. One has a better

choice of selecting a superior quality smear as well as having pre-
pared slides available if special staining procedures should be desired.
Marrow smears were made like blood smears, air dried, and stained
with Wright's stain as soon as they were dry. Staining time was two
minutes with stock solution to cover the slide and four minutes more
after addition of an equal volume of distilled water. A new batch of

stain should be timed until satisfactory staining quality is obtained.
The slides were rinsed with tap water while in a horizontal position

and rapidly air dried at room temperature.

ANATOMY AND HISTOLOGY OF BONE MARROW

Early De velopment

Before proceeding with a more detailed description of the
various types of marrow it is well to discuss briefly the three pe-
riods through which the development of the hemopoietic organs pass
in the ontogenesis of mammals.

The mesoblastic period is initiated by differentiation of the
mesenchyme from mesodern in the wall of the yolk sac of the embryo
in the first few days of development of the individual. The so-called
blood islands Spread to other extraembryonic tissues. A primitive
blood vascular system is built to supply a rapidly expanding embryo
with blood. Beginning about the second month of life in the human
embryo the hepatic period begins with formation of blood cells in
the liver. The medullary period starts approximately the fifth
month of prenatal human life (Neumann, 1869). The common origin
of bone and bone marrow lies in the deeper layers of periosteum
and perichondrium, respectively (Maximow, 1910).

In postnatal life bone marrow is the only source of blood cells
in the mature and normal individual, except such cells as are derived

from lymphoid tissue (Neumann, 1869).
29

30
It is to be noted that the perivascular mesenchyme is ubiqui-
tous in distribution and has retained its primitive potencies of hemo-

poiesis (Best and Taylor, 1950).

Of the five kinds of bone marrow which differ macroscopically
and microscopically (Jackson, 1904), the two most important varieties

are the red and the yellow or fatty bone marrow Only the red mar-

row plays a role in hemopiesis, producing the red blood cells and

leukocytes. In the embryo and newborn, red marrow only is found

in the bone cavities. With progressing age red marrow in the long

bones is gradually replaced by yellow marrow with its fat cells. In

the normal adult, red marrow is found in the vertebrae, the ribs,

the sternum, the diploes of the bones of the skull, and in the prox-
imal epiphyses of the femur and humerus. In the dog red marrow is

present at the periphery of the marrow pencil of the last—named

bones. Bone marrow forms 2 or 3 per cent of the body weight

(Wetzel, 1920; Fairman and Whipple, 1933).
The Five Different Types of Marrow

l. Embryonal marrow. This consists of red marrow only
but differs from adult red marrow as will be subsequently shown.

The latter is also known as lymphoid marrow (Neumann, 1869).

31

Waldeyer (1865) stated that this primary marrow substance be-
longs to a group of connective tissues of a more undifferentiated character
which may develop in various directions, and may assume a permanent
morphology when required. It consists of spindle-shaped and stellate
cells which connect freely with each other through long processes. The
protoplasm has been changed to a very fine fibrillar inte rcellular sub-
stance which is particularly prominent near embedded blood vessels.

The entire structure is permeated with an albuminous and mucous liquid.
He claimed to have observed a similar embryonal connective tissue in

the deeper layers of primitive periosteum which later changed to bone

and bone marrow. This occurs at the onset of bone formation in the em-

bryo; more specifically at the beginning of ossification.

2. Lymphoid or red marrow. The reticular structure is much

less prominent while the leukopoietic and erythropoietic cells pre-

dominate. Normally myelocytes, metamyelocytes, band cells, and

polymorphonuclear cells make up the bulk of the cellular elements,

decreasing in number in the order named. Myeloblasts are rare and

progranulocytes are present in only moderate numbers. The ratio
of erythroid to granulocytic elements is approximately one to three.
Another cell type seen regularly in normal red marrow is the mega-

karyocyte. Some fat cells are always present. The ratio of fat to

32
lymphoid cells varies greatly in different animals and at different
times in the same animal, depending on the physiological activities.

The principal method of replacement of the myeloid elements
of the normal blood is by homoplasia. Under unusual conditions
more primitive structures may be stimulated to activity. When this

occurs heteroplasia aids in hematopoiesis.

3. Fibroid marrow. This type of marrow is characterized
by the presence of a fibrillar tissue in the medullary spaces and
the larger Haversian canals of the long bones. It is particularly
noticeable in the terminal phalanges of man. Generally speaking it

resembles ordinary connective tissue.

4. Fatty or yellow marrow. No sharp line of demarcation
can be drawn between red and yellow marrow. In this tissue lym-
phoid marrow cells are prominent by their paucity. The proportion
of fat cells to lymphoid cells is reversed from that in red marrow.
The amount of yellow marrow in the body varies inversely as the
hematopoietic activity. Neumann (1869) observed that the fat cells
in the long bones recede from the proximal to the distal end, and
from the periphery to the center of the marrow pencil during

increased hematopoiesis. This process is reversed during decreasing

33
hematopoietic activity. This change is known as Neumann's law

(Ne umann, 1882).

The interstices between the fat cells are taken up by a few

lymphoid cells, b_l_ood capillaries and reticular cells and fibers. The

general appearance of this tissue on casual inspection microscopically
reminds one of lung tissue where the respiratory alveoli are occu-

pied by fat cells and the interalveolar spaces are the interstices

described above. Macroscopically it is of yellowish color, and

greasy to the touch (Figures 7 and 8).

5. Gelatinous marrow. In the long bones of starving animals

or some suffering from debilitating diseases gelatinous marrow has

a reddish, translucent and jelly-like appearance. The fat cells have

atrophied and assume a stellate appearance. The intercellular spaces

are now filled with a homogeneous groundsubstance of slightly dif-

ferent shades of red. Fixed preparations bear some resemblance

to cartilage. Blood vessels and red marrow cells are extremely

rare. Virchow (1859) believed this to be a degenerative process of

fatty connective tissue. He called it a mucoid degeneration because

he allegedly was able to demonstrate the presence of mucin.

 

Figure 7.

   
 

Figure 8.

34

thblsts-olow’h—T thepthohe vesels which occupy
the interstices between fat cells. See Figure 8 (below),
which is a section through a capillary. X 1125.

 

 

A wide capillary between fat cells connecting sinusoids.
Note littoral cells in the capillary wall and myeloid cells
outside of vascular channel. X 1125.

35
The characteristics of the different types of marrow are of
great importance from a diagnostic standpoint. It is obvious that
embryonal marrow and gelatinous marrow are in no way identical.
Lymphoid marrow differs from the two just mentioned in
that it contains much less intercellular ground substance and has a
considerably greater number of lymphoid marrow cells. Its red
color is due to the many blood vessels present. It must be empha-
sized that all kinds of transitional forms of the marrow types dis-

cussed may occur.

Marrow Stroma and Vascular System

As in lymphatic tissue, the stroma consists of primitive
and phagocytic reticular cells. The network of cells and fibers is
looser and its meshes are larger than in the lymphatic tissue (Max-
imow and Bloom, 1949).

The circulation in bone marrow is a closed system (Drinker
$22., 1922), and is characterized by the presence of many large
vessels, called sinusoids, through the walls of which innumerable
cells pass into the blood stream. The sinusoids are lined by flat-
tened, fixed macrophages (littoral cells) like those forming the walls

of lymph node sinuses (Figures 9 and 10).

36

 

  
   

l ..

Figure 9.‘~N’ote “the littoral cells in—the walls at a sinu‘sfioid. Also
observe the extravascular position of the newly formed
blood cells (left), as well as the intermingling of normo-
blasts, band cells, and polymorphs (right). X 1125.

~90

 

     
    

a... R x‘ —

~-

I

O
_I
.o 4

Figure 10. Erythrocytes are resent in the simsid. Note the wall
of the vascular structure with a littoral cell in the wall
and the variety of myeloid cells outside the vessel wall
above. X1125.

37

The medullary artery, after passing obliquely through the nu—
trient foramen of the bone, usually divides into two main divisions,
one running towards each end of the bone, and gradually assumes a
more or less central position in the marrow (Figure 11). This cen-
tral artery gives off smaller branches which pass out radially, oc-
casionally almost at right angles to the parent trunk, sometimes
more obliquely. It may also divide into two or more primary
branches, especially as it approaches the more cancellated parts
towards the end of the bone. Evidently some branches of the medul-
lary artery pass directly into the cortex of the bone. The author
has seen cross sections of small arteries in the bone substance and
in Haversian canals. Many islands of myeloid tissue within the cor-
tex are not an infrequent occurrence in younger dogs. The intra-
osseous arteries apparently furnish the blood supply for these little
islands of red marrow. No blood vessels were observed in the
cellular islands (Figure 12).

The central artery, for the greater part of its course through
the marrow, is usually accompanied by a large, very thin-walled,
venous channel, which receives innumerable smaller tributaries

running more or less radially from the periphery towards the center

of the marrow (Figure 13).

38

 

 

 

 

 

 

Figure 11. Note the three concentric zones: left middle, central ar-
tery; to right, myeloid tissue proper; extreme right, pe-
ripheral or endosteal zone. X 40. (see formula, p. 58)

39

   

\ __-

'_———7

'e 12. Cross section of a humerus from a dog one year old.
There is relatively little yellow marrow present. Note
the many islands of marrow substance in the cortex of
the bone. The numerous small arteries within the osseous
substance are difficult to discern at this magnification.

X 15.

40

    
 

c. ' V "I ‘ ..

, . xx. 44.... . 1'. \ " _ .

Figure 13. Central artery and vein. Note the large size and the ex-
tremely thin wall of the vein. Several nerves (arrows)
are seen adjacent to the venous wall and in the periar—

terial connective tissue. X 125. See close-up of nerve

   
 

in Figure 13a, below.

  

l-_.-

Figure 13a. Close—up of nerve. We a portion of arterial wall in
upper right-hand corner. X 1125. Compare with Fig-
ure 13.

41

The sinusoids are connected by relatively wide capillaries

(Sabin, 1928), making the whole vasuclar structure a closed system

as stated above .

In transverse section, the marrow may, for convenience in

description, be mapped out into three concentric zones:

Central or Vascular Areas, containing the thick-walled

medullary or central artery of the marrow, together with the accom-

panying venous sinus, nerves, and connective tissue. It appears that

anatomy and histology of nerve tissue in bone marrow have not re—

ceived attention by investigators. The photograph presented by the

author shows several nerves embedded in perivascular connective
tissue. The nerve fibers appear to be of the myelinated type (Figures

13 and 13a). Amyelinated nerve fibers were not seen.

2. Intermediate zone, or marrow tissue proper, lying between

the central and peripheral zones, and consisting of a delicate reticu-
lum composed of branching and anastomosing connective tissue cells,

and fine connective tissue fibrils, in the meshes of which lie the

marrow cells to be described in some detail later. In this layer

are also found the fat cells which may constitute a considerable part

of the whole (Figures 11 and 14).

Figure 1 4 .

 

42

Entire ' cross section of the marrow pencil from a seven
months old dog. Note the spreading of fat cells from the
center toward the periphery. There is a very large branch
of the central artery near the periphery in the upper left-

hand corner. X 40.

43
3.

Peripheral zone, or endosteum, composed of a condensation
of the connective tissue framework of the marrow-ma delicate, mem-
branous structure to which allusion has already been made in describ-

ing the removal of the marrow from the interior of the bone.

The stroma of the myeloid tissue is distinguished by the con-

stant presence of fat cells. These are scattered singly in the red

marrow, but in the yellow bone marrow they have crowded out prac-

tically all of the other cells. Between them remain, besides the

blood vessels and reticular fibers, only scattered fixed macrophages

and primitive reticular cells. The latter are probably the main

source of the new blood cells when the yellow bone marrow is trans-

formed into red marrow (Figure 14).
The Free Cells

In contrast to the free cells of the lymphoid tissue, those of

re myeloid tissue are extremely varied in form and are scattered

regularly throughout the tissue. The vast majority of them are

:ng myeloid elements (erythroblasts and myelocytes).

.41. a..- - A

_.
“'7'
*4

 

44

Mature and Immature Elements

Mature myel_o_id__ele_r_n_e_n_t§. A few mature, nonnucleated ery—

 

throcytes and the three types of granular leukocytes as they occur
in the circulating blood are found between other cells. Thus, the
tissue which produces these elements always contains a ready supply

of them, and in case of need can forward large quantities at once

into the blood. §

Immature myeloid elements. The other free cells in the bone

 

marrow are: (1) hemocytoblasts (free stem cells); (2) erythroblasts
(the precursors or the red blood corpuscles); (3) myelocytes (the
precursors of the granular leukocytes; (4) megakaryocytes; and (5)

some lymphocyte s.

(1) Hemocytoblasts. The myeloid tissue of all adult mammals

 

contains ameboid, nongranular, basophil cells of lymphoid nature.
They vary in size, the largest measuring fifteen microns, and are
scattered singly or in groups of two or four. Their structure cor-
responds exactly to that of the lymphocytes (Maximow, 1909). These
elements are the free stem cells of all the other myeloid elements.
From the unitarian‘point of view, they are morphologically identical

with and have the same prospective developmental potencies as the

large r lymphocyte s.

45

(2) Erythroblasts. The young forms of the red blood corpuscles

 

are spherical cells with spherical nuclei and are called erythroblasts.
In living cells the cytoplasm is homogeneous, and of a yellow color
which intensifies as the cells develop into erythrocytes. The round
nucleus of the erythroblasts always presents a checkerboard distribu-
tion of angular particles of chromatin. The nucleoli gradually invo-
lute. The number of mitotic divisions in the cell lineage is not
known. The changes in the erythroblasts as they develop into
erythrocytes are clearly shown in marrow smears.

The youngest erythroblasts (those closest to the stem cell)
are called basophil erythroblasts, because of the intense basophilia
of the protoplasm.

The erythroblasts of the next youngest generation have a very
small amount of hemoglobin. After fixation and staining with the
Romanowsky mixture (eosin-methylene-azure) the cytoplasm varied
from a purplish-blue to lilac or gray. These erythroblasts are,
therefore, called polychromatophils. This staining reaction is due
to the appearance of the pink-staining hemoglobin in the basophil
cytoplasm of the erythroblast, which stains blue with eosin-methylene-

azure .

46

The polychromatophil erythroblasts divide mitotically. Some
of them remain in the tissue in a resting condition for future use.
In the others the amount of hemoglobin increases while the basophilia
of the cytoplasm diminishes. In this way normoblasts arise in which
the cytoplasm stains a bright pink with the Romanowsky mixture.
Normoblasts are smaller than polychromatophil erythroblasts and .
only slightly larger than mature erythrocytes. The small round ;
nucleus contains a dense accumulation of angular chromatin par-
ticles and stains very dark. After an unknown number of mitotic
divisions, the normoblasts lose their capacity for proliferation, and
the nucleus is condensed to a dark-staining body. Each mature
normoblast loses its pycnotic nucleus and is transformed into a red
blood corpuscle. Some investigators hold that the nucleus is lost
by karyolysis, while others believe this occurs by extrusion.

(3) Myelocytes. Besides the erythroblasts, the young forms

 

of the three types of leukocytes (heterophil or neutrophil, eosinophil,
and basophil) are common cell types of the myeloid tissue. The
myelocytes of each of the three types are provided with the charac-
teristic granulation and cannot be transformed into myelocytes of
another type or into elements of another kind. They have a compact

round or oval-shaped nucleus, and proliferate intensely by mitotic

 

47
division. Some of the progeny remain unchanged while others undergo
progressive maturation. Finally, each cell is transformed individually
into a mature polymorphonuclear granular leukocyte.

Myelocytes with heterophil granules (neutrophil myeclocytes of
man) are larger than the mature heterophil leukocytes.

The youngest generation is that of the promelocytes. The
oval or round nucleus contains a loose chromatin network and sev-
eral nucleoli. The ameboid cytoplasm is slightly basophil, although
it often shows acidophil areas. The specific granules are scarce
and usually are confined to the periphery of the cytocentrum and to
the acidophil areas in the cell body. In dry smears the promelo-
cytes may contain, in addition to the heterophil granules, azurophil
granules.

The promyclocytes often show mitosis. In the following gen-
eration, which may be called heterophil myelocytes proper, the
protoplasm becomes diffusely acidophil while the specific granules
increase in number and fill the whole cell body, except for the cyto—
centrum. The chromatin network of the nucleus becomes coarser
and stains darker, and the nucleoli become indistinct. Mitoses are
common. During division the granules are evenly distributed among

the daughter cells and continue to increase in numbers as the latter

grow.

48

As the result of an unknown number of mitoses, a generation
of heterophil myelocytes appears which has lost its capacity for di-
vision. The nucleus in these cells, as soon as it is reconstructed
after the last mitosis, shows a beginning polymorphism and has the
shape of a kidney or a horseshoe. Such cells are called metamyelo-
cytes and band cells, respectively; each of them maturates without
division and is transformed by progressive constriction of the
horseshoe-shaped nucleus into a mature heterophil leukocyte.

Myelocytes with eosinophil granules are less numerous than
the heterophil myelocytes, but in general undergo the same changes.
These appear more numerous in the dog. Among them also different
generations can be distinguished. They all have a slightly basophil
protoplasm. The eosinophil promyelocytes contain a small number
of Specific granules which do not all stain alike. The youngest
among them show a distinct basophilia and stain bluish with eosin—
azure. From these there are all transitions to mature, purely
eosinophilic, red granules. Mitoses are common in the eosinophil
myelocytes, especially in the large ones. The horseshoe-shaped
nucleus of the metamelocytes becomes constricted into two lobes

in the mature leukocytes.

I1. I?! 1.1 . .
'1'... HID-LIA ..1 11.. . Tin

49

Myelocytes with basophil granules are much scarcer than the
heterophil myelocytes and are difficult to study because the granules
in man are easily soluble in water. This difficulty induced some
investigators to declare that cells with basophil granules in the bone
marrow are degenerated eosinophil myelocytes. For the most part
the basophil myelocytes are small, with a paler nucleus than other
types of myelocytes. The protoplasm contains a widely varying
number of specific, basophil, metachromatic granules of unequal
size. Mitoses have been found, but are very rare.

(4) Megakaryocltgi. These giant cells with a polymorphous

 

nucleus are characteristic of the mammalian bone marrow, where
they are scattered evenly among the other elements. Some of them
have a diameter as large as seventy microns. 'The form of the cell
body is spherical, but its surface is often provided with irregularly
shaped processes.

The nucleus is deeply constricted in many places; the lobes
bulge at the periphery, while their central parts are all intercon-
nected by short, branched stalks. The interior of the nucleus shows
a chromatin network and indistinct nucleoli. In the living cell the
abundant cytoplasm is homogeneous. The presumed role of the

megakaryocytes is the production of platelets.

50
In every normal bone marrow many megakaryocytes are found
degenerating while there are frequent signs of their formation from
hemacytoblasts.

(5) Monocytes. Many hematologists believe that monocytes

 

are formed in the myeloid tissue. Others claim they are formed
extramedullarily; e.g., lymphoid tissue. Under normal conditions
they are found only in sparing numbers. The general appearance is
like those of peripheral blood. Earlier forms are extremely difficult

to differentiate from large lymphocytes.

Hete roplastic Hemopoiesis

Under physiologic conditions, the needs of the adult organism
for myeloid blood elements are usually supplied by homoplastic
hemopoiesis-—the production of mature cells by young elements of
the same type. But not all of the young forms reach maturity;
some of them remain unused in the tissue. Whenever the require-
ments of the body are increased--in the very young organism or in
the adult under pathologic conditions-~homoplastic hemopoiesis does
not suffice, and new erythroblasts and myelocytes develop from stem

cells. This is called heteroplastic hemopoiesis.

51
When a hemocytoblast (stem cell) divides, one of its various
latent potencies suddenly develops and both of the daughter cells
which originate from such a mitosis show new properties. Some
hemocytoblasts always remain in the tissue as the source of future
hemopoietic processes when demand arises. Of the progeny of the
hemocytoblasts, some become basophil erythroblasts. These divide
mitotically and are transformed in the succeeding generations into
polychromatophil erythroblasts. When hemocytoblasts develop into
myelocytes the two daughter cells, immediately after the reconstruc-
tion of the nuclei, accumulate the characteristic granules in their
protoplasm. Transitional cells between the hemocytoblasts and early
myelocytes have been described under the name of leukoblasts and
more recently as progranulocytes (Committee for Clarification of the

Nomenclature of Cells and Diseases of the Blood and Blood Forming

Organs , 1949).

The New Formation of Free Cells from Fixed Cells

In the embryo as stated before the hemocytoblasts of the bone
marrow which produce the young myeloid forms originate from un-
differentiated fixed mesenchymal cells. In the adult the same process

may occur. It has been explained that some of the cellular elements

52
of the reticulum always remain undifferentiated. But under physio-
logic conditions it is rare for primitive reticular cells to become
free, basophil hemacytoblasts in the bone marrow. The mitoses of
the myelocytes and erythroblasts--and occasionally of hemocytoblasts
--usually are sufficient. Pathologic stimuli sometimes facilitate the
new formation of hemocytoblasts from the primitive reticular cells.

Heteroplastic development of lymphocytes may also occur. The author

TL- "

has not seen lymphoid follicles in the bone marrow of dogs. They

are frequently found in man (Rohr, 1949).

How the Myeloid Elements Enter the Blood

Because the myeloid elements arise outside the blood stream,
it is obvious that the newly formed, mature myeloid cells must pass
through the walls of the blood vessels to enter the circulation. The,
very thin-walled venous sinusoids make this possible. Through them
easily pass not only the ameboid mature granular leucocytes, but also
the nonmotile erythrocytes. When these are ready for circulation
they slip through the membrane into the blood stream in the lumen
of the sinusoid. The mechanism of this phenomenon is probably
regulated by changes in the permeability of the vessel walls, in the

surface energy or some other specialized mechanism.

53
The claim that red blood corpuscles in the adult, normal man
or other mammals are formed intravascularly is based on unconvinc-
ing evidence (Drinker e_t___a_tl., 1922; Sabin, 1928). In the embryonic
liver and bone marrow the red blood cells develop extravascularly,
while in the yolk-sac they are preponderantly of intravascular origin.
The latter statement is not entirely true because blood cells were
forming in the blood islands before a vascular structure had been

provided.

Functions of the Myeloid Tissue

The main function of the bone marrow seems to be the pro-
duction of myeloid elements for the blood. The macrophages of
the bone marrow also function like the macrophages in other tissues.

The cellular composition of the blood is intimately connected
with the condition of the bone marrow. Under physiologic conditions,
the relative numbers of the different cells in the bone marrow, as
in the blood, vary only a little. But all general pathologic processes
immediately affect the composition of the bone marrow. In most
general infections, and during suppuration, the heterophil granulo—
cytopoietic apparatus is stimulated and the percentage of myelocytes

of this type increases greatly. In certain other conditions, as in

= s: . -« -
IL“

54
typhoid fever and in a granulocytic angina of man, the heterophil
myelocytes decrease in number. Whenever there is an increased
need of erythrocytes or where the erythrocytes are destroyed in
large quantities, they and the precursors predominate in the myeloid
tissue. That temperature plays some role in the control of hemo-
poiesis is shown by the fact that the fatty marrow of the tail bones
of a rat becomes hematopoietic when the temperature of the bone is
raised to that of the body, as by placing it in the body (Maximow and
Bloom, 1949).

The production of erythrocytes depends in part on an antianemic
factor which is stored in the liver. This factor probably is the re-
sult of the interaction of a substance in the gastric juice (intrinsic
factor of Castle) with some substance in the diet (extrinsic factor).
The absence of the antianemic factor results in pernicious anemia
in man, which can be treated successfully by the administration of

liver, gastric extract, or B vitamin (Best and Taylor, 1950).

12

Regarding the Question of Lymphatics
of Bone Marrow

The author has not observed lymphatics in the marrow of

dogs.

55
The literature appears to ignore that part of the anatomical
structure almost entirely. Drinker et_al_. (1922) stated that they know
of no work upon the lymphatic system of the bone marrow.
Roger and Josue (1899) denied the existence of a lymphatic

system in bone marrow. No one has challenged them as far as has

been determined. I

PRESENTATION AND EVALUATION OF FINDINGS

General Conside rations

Up to this time no one had attempted to investigate the bone
marrow of the dog in a broader sense and with a view of using the
information thus found as a practical clinical diagnostic agent in
small animal practice. Only two groups of veterinarians have re-
ported bone marrow biopsies in the dog. Their work was restricted
to cytology of marrow smears (Meyer and Bloom, 1943; Bloom and
Meyer, 1944; Bloom, 1945; Horn, Jahn, and Wille, 1953).

Other reports of bone marrow studies on dogs came from
medical men engaged in research who were interested in the cytology
of the myeloid tissue of these animals used in other experimental
work.(Mulligan, 1941a, 1941b, 1945; Van Loon and Clark, 1943). No
reports of the preparation of whole marrow sections were published
by the preceding workers, except Mulligan (1941b). The information
obtained by their work was of much value. It was insufficient and
even unreliable to make diagnoses on the basis of cytological find-

ings from smears alone.

56

..- ”x “44 .

it; i:

57

It was exceedingly important that sections of marrow be made,
as will be seen from citation of the following:

Block (1950) reported an exhibit at the International Congress
of Hematology in Buffalo, New York, 1948, of twelve cases of meta-
static carcinoma which were incorrectly diagnosed as acute leukemia
on the basis of smears by experienced hematologists at the University
of Michigan.

It is not implied that sections should be made in all cases, as
will be shown in the discussion to follow later in this thesis.

The diagnostician should be fully conversant with the anatomy
and histology of normal bone marrow. With this in mind the author
investigated ten carefully selected dogs which were considered nor-
mal on the basis of the clinical examinations, as shown in Table I
in the chapter on Material and Methods.

It was probably the first time that whole cross sections of
myeloid tissue were prepared to study the topography of normal
marrow of the dog. The cellular elements of bone marrow vary
considerably quantitatively in different animals within the same age
group. Furthermore, they may be very similar in widely varying
age classes in apparently normal specimens (Figures 11 and 14).

This should indicate that not too much emphasis should be placed on

if
4

58
quantitative proportions between fat marrow and cellular marrow.
It appeared that the physiological activity of hemopoietic cellular
elements was more important. As long as the cellular elements
were in proper relative proportions qualitatively even though there
was an apparent wide difference quantitatively, the marrow specimen

should be pronounced as being normal by the clinician. It was ob-

IL u P-“-_1—
, .

vious that there would be a quantitative minimum. From the normal
specimens examined it may be concluded that a minimum amount of .-~.
cellular marrow well proportioned qualitatively could be set at ap-
proximately one-third of the total cross-sectional area. This applies
only to marrow of such bones and portions of such bones as are
known to be functionally active and responsive to normal and path—
ologic physiological demands.

The degree of normalcy of marrow in long bones can be ap-
praised grossly or under very low magnification by applying the fol-
lowing formula:

2
- 100
R 52 x = ‘70 of red marrow

R

 

where (r) equals the inner radius and (R) the outer radius of the
concentric ring formed by the hematopoietically active myeloid tissue.

(See Figure 11, page 38.)

59

If the numerical value thus obtained is thirty or greater, the
marrow tissue may be considered to be within a normal range pro-
vided that the active cellular elements are well proportioned.

This was in close agreement with values given for human
marrow by Berman and Axelrod (1950).

For the dog the junction of the proximal and middle thirds of
the femur was such an area. Custer (1932) and Stasney and Higgins
(1937) found that the proximal end of the femur responds to changes
in physiological activity very early and quickly. This was in full
agreement with observations made during this investigation.

Meyer and Bloom (1943) described the technic for iliac punc-
ture in the dog. They admitted that one might meet with a great
deal of uncertainty in the execution of this procedure.

Horn eLal. (1953) suggested sternal puncture. They did not
state the size of dogs used, nor did they admit any failures. Iliac
crest and sternal puncture were rejected in this investigation for
the following reason: To make it a practical clinical procedure the
marrow puncture should be capable of being performed on dogs of
all sizes with absolute safety and certainty.

The practicing veterinarian would soon find out that puncture

of iliac crest and sternum would be an impractical task clinically

60
on the many small breeds of dogs which he is called upon to treat.
Furthermore, sternal puncture was not entirely without danger in
human practice where the cross section of the sternal marrow space
of the average adult measures approximately one-half to one inch.
Six deaths following sternal puncture have been reported (Fortner
and Moss, 1948).

No clinical procedure to aid in arriving at a correct diagnosis 3
is acceptable in practice if it is not absolutely safe. No owner brings
his pet to the veterinarian to have him killed or maimed in order to
make a diagnosis. Femoral puncture has been safe, reliable, and
without appreciable discomfort to the patient in all cases, as has
been described in Material and Methods.

Also, if material obtained by the puncture method was insuf-
ficient for preparing sections, should such be desired, more marrow
substance could be had by trephination at the same time. The latter

will rarely be necessary.

Appraisal of Marrow Structure

G ro s 5 evaluation.

 

(A) Necropsy material. The humerus and femur appear to be

first choice in order to judge the quality of marrow. In dogs less

61

than six months old the marrow was usually almost blood red, soft
jelly-like or nearly semiliquid throughout most of the length of the
bone. If any variation in the distribution of red and yellow marrow
occurred it followed Neumann's law as defined in the section on
Anatomy and Histology; i.e., more red at the proximal portion, with
increase of fat at the more distal region (Neumann, 1882),

Animals of age from six months to one year more often
show a marrow quality as just described, but some exhibit a def-
inite increase of yellow marrow as they approach one year.

After one year of age the transition from red marrow at the
proximal end to fatty marrow at the distal portion was more obvious.
As the age of the animals advances the central portion of the remain-
ing red marrow becomes fatty, leaving only a concentric ring of red
marrow at the periphery of the marrow pencil.

About this time a peculiar distribution of red marrow was
observed in several cases. A thin layer of red marrow covered the
full length of the entire periphery of the marrow pencil. This condi—
tion has not been described as far as was known. Cross sections
revealed yellow marrow in the more central area. In these cases
the transition from red to yellow marrow was usually very abrupt.

A clue to the true condition of the marrow in these cases was afforded

62

on closer inspection; namely, the color was of a somewhat lighter

red. Caution must be exercised not to confuse this condition with

the gelatinous-type marrow described in the section on Anatomy and

Histology. The latter was a lighter red throughout the marrow

pencil and would have to be differentiated microscopically (Figure 1).
(B) Biopsy material obtained by puncture method. The aspirated

material was deposited on a watch glass and quickly mixed. Bone

marrow substance had a light red color, was shiny, with a fatty sur-

face. If much fatty marrow was present a slightly yellowish sheen

could be detected. The entire consistency was that of a thick fluid

and sometimes somewhat stringy or like ropy milk.

Microscopic evaluation.

(A) Fixed whole marrow substance obtained at necropsy, tre—
phination, or puncture method. Prepared sections from above ma-
terial make it possible to distinguish three different marrow struc-
tures under low power:

1. Normal bone marrow. Cellular and fatty marrow from

the average young adult dog were about equal quantitatively.
As stated earlier in this chapter the proportions vary con—

siderably, and favor fatty marrow as age advances.

63

2. Hypoplastic marrow. This was generally designated as
fatty marrow. Its color was characteristically yellowish
white. The puncture material has a pronounced fatty
sheen when mixed with a little marrow blood. Caution
must be exercised not to designate every fatty appearing
biopsy a hypoplastic marrow. Peripheral blood and other
clinical findings must be evaluated as will be discussed
later.

3. Hyperplastic marrow. The characteristic feature of this
type of marrow was the paucity or almost complete ab-
sence of yellow marrow. It consisted almost entirely of
cellular marrow. If erythropoiesis was predominent an
intensely red coloration was noticeable macroscopically.
If leukopoiesis predominated a grayish red color was ob-
served.

Other conditions which can be recognized by a change in
marrow structure due to presence of an abnormal number of a cer-
tain cell type were:

1. Hemolytic, toxic, and deficiency anemias (Rohr, 1949;

Isaacs, 1937). Determination of the myeloid-erythroid
ratio was a simple and rapid method of evaluating normal

and abnormal marrows.

64
Z. Leukemias, agranulocytoses of infectious nature, acute
inflammation, chronic infections, and sometimes allergic

conditions (Custer, 1935).

3. Disturbance of the thrombopoietic system (increase of
megakaryocytes) . i,
4. Lymphoid marrow in lymphatic hyperplasia and granulo- I

cytopenia (Rohr, 1949).
5. Excessive reticular elements as in agranulocytosis :.
(Custer, 1935) and panmyelophthisis generally.
6. Pure fatty marrow as in aplastic conditions (Rohr, 1949).
7. Tumor cell marrow in carcinomata, sarcomata, myelo-
mata, and leukoses (Rubinstein, 1948; Rohr, 1949).
(B) Direct smears from puncture biopsies. Stained smears
should first be looked over under low magnification for density of
cellular elements and fat content. This permits a general evaluation
of the character of the marrow. A differential count was made under
oil immersion. The myelogram will be discussed at the end of the
section.
The color reactions in staining of smears were based on the
results with Wright's stain. Sections were stained with hematoxylin

and eosin as outlined in the section on Material and Methods.

65

A brief description will be given here covering the principal
features of the various cells as they appear in stained smears. For
more detailed description reference is made to Albritton (1952.),
Kohanawa (1928), and Rohr (1949).

Table 111 contains the more recently recommended nomencla-
ture for cells of blood and blood-forming organs. The table is ar-
ranged to list the cells of each series in the order of their develop-
ment starting with the youngest of each specific cell type. The older
names still in use are given in parenthesis in the section ”Descrip-
tion of Hemopoietic Cells."

Description of the Hemopoietic Cells
of Bone Marrow1

Erythrocytjc series. There are no cytoplasmic granules pres-

 

ent in this series.
. Z .
1. prglbLagi (pronormoblast, proerythroblast). Size, about

fifteen microns. Cytoplasm usually a narrow, deeply basophilic band

around a relatively large, round nucleus of delicate chromatin, more

 

Nomenclature recommended by the Committee for Clarifi-
cation of the Nomenclature of Cells and Diseases of the Blood and
Blood-Forming Organs (1949).

Names in parenthesis are still commonly used.

«I... u. WM”- 44

TABLE III

66

1
NOMENCLATURE RECOMMENDED FOR CELLS OF BLOOD
AND BLOOD-FORMING ORGANS

 

 

General Cell Type

Term for
Specific Cell Type

 

Erythrocytic se rie s ...................

Lymphocytic se rie s ...................

Monocytic se ries ....................

G ranulocytic se rie s ...................

Plasmacytic series ...................

Thrombocytic series ..................

Not identifiable ......................

Rubriblast

P ro rub ric yte
Rubricyte
Metarubricyte
Reticulocyte
Erythrocyte

Lymphoblast
Prolymphocyte
Lymphocyte

Monoblast
Promonocyte
Monocyte

hdyeloblast
Prog ranulocyte
Myelocyte
Metamyelocyte
Band cell
Segmented

Plasmablast
Proplasmacyte
Plasmacyte

Megakaryoblast

P romegakaryocyte
Megakaryocyte
Thrombocyte

Disintegrated cell

 

 

By the Committee for Clarification of the Nomenclature of
Cells and Diseases of the Blood and Blood-Forming Organs (1949).

67
dense and less translucent than that of myeloblast. Parachromatin
was distinct and pink. Nucleoli not too readily visible.

2. Prorubricytg (basophylic normoblast, basophil erythroblast).

 

Size, about like preceding or a little smaller. Cytoplasm basophilic.
Nucleus smaller and more dense than preceding, chromatin may clump
more, parachromatin as in preceding, nucleoli usually not visible.

3. M31913}: (polychromatophilic normoblast, polychromato-
philic erythroblast). Size, eight to twelve microns. Increase of
hemoglobin renders cytoplasm pastel blue to almost copper red.
Nucleus becomes still smaller (pycnotic), chromatin dense, and nu-
clear membrane coarse, less parachromatin, nucleoli not visible.
Note: Most mitotic divisions occurred in this group.

4. Metarubricyte (orthochromatic or acidophilic normoblast).

 

Size, seven to ten microns. Nucleus very small, practically no
parachromatin. Cytoplasm stains slightly less red than in mature

e rythro c yte .

Lymphogtic se rie s .

 

1. Lymphgblagt. Size, ten to eighteen microns. Cytoplasm
colorless to dark blue, no specific granules, usually a narrow band.
Nucleus round or oval, usually one to two nucleoli, fine chromatin

network, parachromatin pastel pink and distinct.

 

r._-__\_'

68

2. Lymphocyte (small and medium). Size, six to fourteen

 

microns. Small amount light blue cytoplasm. May have a few
azurophil granules, no specific granules. Nucleus round or oval,
slightly indented, chromatin in blocks which were indistinctly sep-
arated, appeared blurred. Parachromatin slight and difficult to

distinguish from chromatin.

Monoqtic se rie s .

 

1. Monoblast. Size, fourteen to eighteen microns. Cytoplasm

 

may or may not contain fine azurophil granules, moderate amount,
stained slightly basophilic, no Specific granules. Nucleus may be
round, oval, and slightly indented, one to five pale blue nucleoli,
chromatin pattern very fine strands, stained faintly, chromatin-
parachromatin relation distinct.

2.. mm (transitional or blood mononuclear). Size, four-
teen to twenty microns. Cytoplasm billowy, grayish blue, muddy
blue, Tauben blau, ground glass appearance. No specific granules.
Azurophil granules dust-like, hyaloplasm may stain pinkish. Nucleus
appears in many shapes. A rough contour, hill and valley effect.
Chromatin strands fine, network was lace-like, stained faintly, had

marked chromatin-parachromatin distinction. No nucleoli.

69

Granulocytic series.

 

l. Myeloblast (hemocytoblast, stem cell). Size, ten to eighteen

 

microns. Cytoplasm was basophilic, not as deep blue as rubriblast,
formed a narrow band around nucleus. There were no speeijig gran-
ules. May contain fine purple azurophil granules. The nucleus was
round or slightly oval, usually central, had up to five pale blue nu-
cleoli. The chromatin was in fine strands and was knitted into a
fishnet-like structure. The interstices between the thin chromatin
strands were distinct, often rectangular and regular; parachromatin
was scant, pink or bluish and very distinct from chromatin. Some-
times the nucleus had a stippled appearance almost like a red rasp-
berry.

The pattern of the myeloblast of the dog was not as distinct
as that of man. Of course not many of them have been viewed inas-
much as they are relatively rare. The author wishes to reserve fur-
ther comment on morphology of all myeloid cells because additional
study of these cells is now in progress.

2. Progranulocyte (leukoblast, promyelocyte). Size, fourteen

 

to twenty-one microns. Cytoplasm is less basophilic and more abun-
dant than that of the myeloblast. No specific granules. May contain

some azurophil granules. Nucleus round or oval, central or eccentric.

70
Nuclear pattern generally like the myeloblast, but less regular and
some slight clumping of chromatin at intersection of strands. Usually
not more than two but less distinct nucleoli. Good distinction of
chromatin-parachromatin.

3. Early myelocyte (promyelocyte). Although this cell does

 

not appear in Table II, it is included in nearly all myelograms. Three

types were recognized. Their outstanding characteristic was the pres-

ence of specific granules. May contain some azurophil granules. ;'
Size, twelve to eighteen microns. The abundant cytoplasm was of

varying shades of blue, generally pale blue, a little darker at the

periphery. The nucleus was round or oval, central or eccentric.

Nucleoli were not clearly visible if at all. The interstices of the

chromatin network were irregular; the chromatin strands were be-

coming coarse. The pink parachromatin was distinct.

(a) Neutrgnhilic. Specific granules stain weakly pink in the

 

dog, much less distinct than in man. A blurred appear-
ance.

(b) Eosinophilic. Round or spherical large specific granules,

 

like little oranges, uniform size, orange red. Not very

nume I'OLIS .

(c) Basophilic. A few large metachromatic specific granules,

 

uneven in size and distribution, dark color.

71

4. Late mjelocyte. These cells have their full complement

 

of specific granules. They have the appearance of the respective
mature forms except the shape of the nucleus, which was still round
or oval. The cytoplasm of the neutrophilic myelocytes was colorless
or clear, that of the other two may be pale blue. The thickened
chromatin strands form a coarse meshwork and stain deeply purple.
The small amount of parachromatin was distinct. Most mitoses
occurred in this stage.

5. Metamyelocyte (juvenile). Size, ten to sixteen microns.

 

The cytoplasm of the three types was colorless or slightly acido-
philic, relatively abundant. Specific granules were more numerous,
stain slightly more intensely. The nucleus was reniform, had a
thick nuclear membrane, a coarse meshwork of thickened strands
of deep-staining chromatin. Parachromatin was scant but distinct
from chromatin. Nucleoli were not visible.

6. B_a_r_1§__c_e_ll (stab, nonsegmented), neutrophilic. Size, ten to
fifteen microns. The abundant, colorless or slightly acidophilic
cytoplasm had a full complement of pale pinkish specific granules.
The nucleus was rod-like, deeply indented, with parallel sides.
Thickened chromatin strands form coarse masses with distinct inter-

stices. Scant parachromatin visible between chromatin masses. No

72

nucleoli visible. Cells with mere indentations from side to side were

still counted as band cells.

7.

*Segmented (polymorphonuclear, filamented). Size, nine to

 

fifteen microns.

(a)

Neutrophilic. The abundant cytoplasm was colorless or

 

slightly acidophilic. Specific granules were pale pinkish,
tan, buff, or beige, rather indistinct, of irregular size and
distributed haphazardly throughout the cytoplasm. The nu—
cleus had a distinctive pattern, a checkerboard appearance.
The chromatin formed large, irregular, deeply staining
blocks. Parachromatin is thin and of light rust color.
The sharp contrast between chromatin and parachromatin
gives it a characteristic pattern. Several blocks of
chromatin form lobes or bulbs. Normally there may ap-
pear from two to not more than five such bulbs in a
single cell. The bulbs were connected by a thin thread
or filament of chromatin. If the connections between
bulbs were more than a thin thread the cells were not
counted with segmented cells but were included with band

cells. No nucleoli were visible.

(b)

(C)

73

Eosinophilic. Size, nine to fifteen microns. There was

 

usually not much visible cytoplasm. In the dog many of
them have abundant cytoplasm. It was basophilic and had
specific granules scattered throughout. The latter were
large, spherical, or irregular and numerous. The spe-
cific granules of some of the mature cells stain dark tan,
and may be of irregular size. Some of them look like
Queen Anne cherries. The nuclear pattern was generally
like that of the segmented neutrophil but had less lobes,
mostly two but not more than four. Nucleoli not visible.
Parachromatin scant but distinct.

Basophilic. Size, nine to fifteen microns. The nucleus

 

occupied the major portion of the basophilic cytoplasm.
The metachromatic Specific granules varied in size,
shape, and distribution throughout the cytoplasm. The
nucleus was roughly Spherical, somewhat indented, lobes
were not discernible. The chromatin stained light blue
but looked faded. Parachromatin was indistinct. Nucleoli
not visible. The Specific granules were water soluble

which may be responsible for vacuoles sometimes seen.

74

8. Plasmacyte (Tiirk Cell, myeloma cell). Size, fifteen to

 

thirty microns. The very abundant cytoplasm was basophilic, the
nucleus round or oval, relatively small, eccentric, may contain one
or two nucleoli. The chromatin formed dense blocks arranged con-
centrically with distinct but somewhat irregular interstices. The
name "Radkern" has been applied to this type of nucleus. A small

amount of parachromatin, no specific granules, azurophilic granules

.n. <.‘..-.—.__._..

were seldom seen. When present the cytoplasm stained correspond-
ingly less basophilc (Rohr, 1949). The author found one of these in
a direct smear and is shown in Figure 15.

9. Megakaryocyte (megalokaryocyte). Size, thirty to seventy

 

microns. This was a unique nucleated cell. Each individual develops
from a stem cell. The cytoplasm was acidophilic and was the source
of blood platelets. Azurophilic granules aggregate in patches sepa-
rated by nongranular cytoplasm. The nucleus of the mature mega-
karyocyte was multilobular and complex. The chromatin strands
were so thickened that they formed heavy, dark- staining clumped
masses with little or no parachromatin visible. No nucleoli visible.

10. Reticulum cell (reticulo-endothelial cell, stem cell of

 

modern pathologists, mesenchymal cell, hemohistioblast). Aschoff
(1920), Ferrata (1910), Ferrata and Negreiros (1914), Maximow (1909).

Alexandrov (1930), Rohr (1940).

75

 

Figure 15. Plasmacyte. Note the relatively light color of the cyto-
plasm and the azurophil granules. X 1125.

A

 

76

This was probably the most primitive and least differentiated
of all cells of the mammalian body. Size, fifteen to twenty-five mi-
crons. The delicate abundant cytoplasm was usually of irregular
shape, pale to dark blue, may be mottled with considerable colorless
or slightly acidophilic hyaloplasm. It may contain some azurophil
granules. The nucleus was round or oval, the chromatin network
consisted of fine, loosely knitted strands, giving almost a vesicular
appearance. The color was reddish violet. Three to Six nucleoli
were usually present.

Parachromatin was dispersed in the irregular chromatin inter-
stices and well contrasted. In marrow films these cells may have
to be recognized by the nuclear characteristics; the cytoplasm was
so delicate that it frequently ruptured. Naked nuclei were not un-
common in marrow smears.

To aid in identification of cells the author used: (1) the color
atlas, Hamatologische Tafeln, by Sandoz (no date given); (2) Standard
Values in Blood, by Albritton (1952); and (3) oral communications
from Rebuck, in 1952, 1953, and 1954.

The method used in executing the marrow differential cell
count was as follows: An area near the feather-edge of stained

Smears was located under low-power magnification. The oil-immersion

,ii

F—q'Q—‘A - M -.—4~—.- ». . .
.. .r “i a! _.~:.| .

(3

.‘HI' - '.v-. ~

77
objective was used to count the cells, traversing the Slides cross-
wise until four hundred of the white cell series were counted. All

cells of the red cell series in the respective fields were counted.

The Myelog rams

All cells counted were tabulated as indicated in the individual
myelograms for ten dogs used in this investigation (Table IV).
Table V contains a comparison of ranges of this study with

Similar investigations carried on elsewhere.

'-_-.'_:'_-__-_~;- _ “I

78

TABLE IV

PERCENTAGE OF DISTRIBUTION OF THE MARROW CELLS
FROM THE FEMURS OF TEN DOGS

 

 

Cell Types

Case Number

 

 

 

 

 

 

 

 

 

I 11 III IV
Rubriblasts (pronormoblast) 0.00 0.50 0.18 0.33
Prorubricytes (basophilic
normoblast) ............. 4.55 4.97 3.86 4.84
Rubricytes (polychromatophilic
normoblast) ............. 14.00 16.92 16.15 16.23
Metarubricytes (orthochromatic
normoblast) ............. __2_._4_2 __fl_32 __3_._32 ”5.111.
Red series total ........ 21.00 26.67 23.50 26.55
Myeloblasts ............... 0.65 0.00 0.37 0.33
Progranulocytes (promyelocyte) . . 1.14 1.98 1.84 1.66
Myelocytes, neutrophilic ...... 6.69 4.15 4.04 4.97
Metamyelocytes, neutrophilic
(juvenile) ............... 7.67 9.64 8.45 12.78
Band cells, neutrophilic
(stab cell) .............. 26.60 29.83 27.02 27.10
Segmented cells
(polymorphonuclear) ....... 16.80 14.60 16.55 ”-25
Eosinophilic cells, all types . . . . 7.02 6.80 5.52 6-13
Basophilic cells, all types ..... 0.00 0.00 0.00 0.00
Lymphocytes .............. 10.02 5.48 6.98 4-97
Monocytes ................ 0.81 1.66 0.00 0-66
Megakaryocytes ............ 0.00 0.00 0.00 0'00
Plasmacytes (plasma cell) ..... 1.30 0.33 0.73 0'50
Reticulum cells ............ __Q;QQ ____0__1_6 0.55 ’93;
White series total ...... 78.70 73.00 74f?- 1,143
Myeloid-Erythroid
ratio ................. 3.76 2.74 3.16 2.76
J

 

 

 

79

TABLE IV (Continued)

 

 

Case Number

 

 

 

 

 

 

 

 

 

 

 

 

 

 

‘AL‘. .

 

 

 

 

 

 

 

 

“‘— Range
V VI VII VIII IX X
0.36 0.32 0.18 0.60 0.36 0.37 0.00 to 0.60
4.37 5.64 2.84 2.61 2.74 5.55 2.74 to 5.64
17.45 19.65 12.45 10.65 14.42 15.35 10.65 to 19.65
_ 2.18' 4.83 5.17 3._61 2.56 __ 4.82 2.18 to 5.17
24.40 30.49 26.50 17.48 20.15 26.10 17.48 to 30.49
0.73 0.16 0.53 0.20 0.36 0.00 0.00 to 0.73
1.45 1.12 1.78 1.00 1.28 1.66 1.00 to 1.98
9.47 10.08 14.42 13.88 12.98 9.62 4.04 to 14.42
6.55 14.80 18.15 12.28 12.61 11.29 6.55 to 18.15
25.48 21.90 17.25 23.75 19.20 22.75 19.20 to 29.83
15.29 11.25 16.20 16.30 17.92 16.84 11.25 to 17.92
5.10 3.55 5.52 3.01 5.31 3.29 3.01 to 7.02
0.00 0.00 0.18 0.00 0.18 0.18 0.00 to 0.18
8.74 5.32 3.56 10.45 7.50 5.74 3.56 to 10.45
1.45 0.80 1.06 1.00 1.10 1.48 0.00 to 1.66
0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.73 0.32 0.35 1.00 0.73 0.92 0.32 to 1.30
0.73 0.00 0.18 0.00 0.36 0.1__8_ 0.00 to 0.73
75.70 70.20 79.30 83.00 79.60 74.10 70.20 to 83.00
3.10 2.30 2.98 4.75 3.95 2.84 2.30 to 4.75

 

 

TABLE V

COMPARISON OF DOG

DATA WITH ANOTHER
FEMORAL MYELOGRAM

80

 

 

Van Loon

Author, from

 

 

 

 

 

 

 

Cell Types 31531., 1943 Table IV
(8 dogs) (10 dogs)

Rubriblasts (pronormoblast) 0.0 to 0.7 0.00 to 0.60
Prorubricytes (basophilic

normoblast) ..... . ...... 4.2 to 8.4 2.74 to 5.64
Rubricytes (polychromatophilic

normoblast) ............ 13.3 to 19.8 10.65 to 19.65
Metarubricytes (orthochromatic

normoblast) ............ 7.6 to 21.1 2118 to 5.17

Red series total ....... 17.48 to 30.49

Myeloblasts .............. 0.2 to 1.0 0.00 to 0.73
Progranulocytes (promyelocyte) . . 0.2 to 1.1 1.00 to 1.93
Myelocytes, neutrophilic ..... l 7 to 0 4.04 to 14.42
Metamyelocytes, neutrophilic

(juvenile) .............. 2 O to 4 3 6.55 to 18.15
Band cells, neutrophilic

(stab cell) ............. 10 3 to 18 7 19.20 to 29.83
Segmented cells

(polymorphonuclear) ...... 3O 1 to 39.7 11.25 to 17.92
Eosinophilic cells, all types . . 0.2 to 3.3 3.01 to 7.02.
Basophilic cells, all types . . . . 0.00 to 0.18
Lymphocytes ............. 0.0 to 3.4 3.56 to 10.45
Monocytes ............... 0.0 to 0.1 0.00 to 1.66
Megakaryocytes ........... O 0 to O 3
Plasmacytes (plasma cell) . . . . 0.32 to 1.30
Reticulum cells ........... 0.00 to 0.73

White series total ...... 70.20 to 83.00

Unclassified cells

(disintegrated cells) ...... 0 4 to 3 1
Myeloid-Erythroid ratio ...... 2.30 to 4.75

 

 

’1'!

A. _..... WALL
F. 4-’ ‘ f“ ’lt

DISCUSSION

Dogs are subject to a wide variety of diseases, which neces-
sitates that the veterinarian have at his disposal an adequate arma-
mentarium for making differential diagnoses as well as early prog-
noses. Blood examination, which has become an important adjuvant
to both diagnosis and prognosis, has always suffered from the weak—
ness of depending for its value on the inferences drawn from it.
Knowledge of the changes in the formative tissues and the interpre-
tations thereof have not been available to the veterinarian for prac-
tical application.

Marrow puncture is now used in human hospitals all over the
world. This procedure should be available to the veterinary prac-
titioner also so that he may correlate blood changes with those in
the marrow.

A safe and practical method for doing marrow puncture, and
trephination if necessary, must be provided.

Sternum, iliac crest, and dorsal processes of vertebrae are

used in human medicine.

81

82

In the canine Bloom and Meyer (1944) proposed the sternum
and iliac crest; Horn e_t_al. (1953) suggested the sternum as sites
for marrow puncture.

The author investigated the foregoing sites. While the quality
of marrow in these bones was good, the size and the difficulty of
executing the puncture on all sizes of dogs make them unacceptable
in his opinion. Added to this was an admitted uncertainty of obtain- 7
ing sufficient marrow plus a certain degree of danger of injury or _.
even death to the patient.

The proximal third of the femur has been found to be a suit-
able site for marrow puncture as described in the section on Materials
and Methods. Custer (1932) has stated repeatedly that the femur was
a source of a good quality of marrow and has the advantage of being
one of the first bones to respond to stimuli affecting bone marrow.
Menkin (1943) also observed the early response of femoral marrow
to stimuli. Femoral puncture was preferred because this site was
easily accessible; it was a minor surgical procedure which did not
cause the patient discomfort. There was absolutely no danger that
a body cavity might be punctured while executing a femoral marrow
biopsy. One can always be confident of obtaining a marrow sample

in sufficient amount to prepare six or eight smears and have some

83

material for making either paraffin or frozen sections. Preparation
of a few extra smears enhanced the chance of getting superior qual-
ity marrow films. Many of the marrow cells were very delicate,
which may result in films containing many cells with ruptured cell
membranes. 5
Sections were desirable and even indispensable in some cases. -.
g
In the section on Presentation and Evaluation of Findings a case was 5
g
cited where an opinion based on cytology alone resulted in a wrong ;,
diagnosis (Block, 1950). Hutchinson (1953) reported the advantage
of marrow sections in determining stainable iron by a histochemical
test using Prussian blue. He stated that not only does it indicate
when iron was required but also when enough has been given and so
can be used to obviate the danger of overdosage when iron was being
intravenously administered. This method could be used to differen-
tiate between iron deficiency and chronic intoxication anemias.
Another clinical procedure to which marrow substance lends
itself was a determination of phosphatase content if malignancy was
suspected (Kerppola, 1951).
Wolff and Limarzi (1945) stated that the most accurate cri—
terion for the diagnosis and treatment of the true anemia of pregnancy
was the study of bone marrow. The changes in the peripheral blood

were of a secondary nature, and in many instances were misleading.

84

Several cases of bacterial and protozoon diseases have been
diagnosed as a result of bone marrow findings (Anon., 1940).

It is only reasonable to hope that the practicing veterinarian

will take advantage of the many opportunities bone marrow offers in

...,._.r
....V

arriving at more correct diagnoses.
The importance of the reticulo-endothelial system of the mam—

malian body as a mechanism of defense against disease is well

’3 an‘I—‘J- A .‘n‘." -u- 1“.-

fAJ

known. Bone marrow offers an easy, safe, and sure way to exam-
ine this highly Specialized mechanism. The reticulo-endothelial
system of bone marrow is more widely distributed than that of any
other single organ. Bone marrow biopsy as a means of getting a
look into this mechanism is unquestionably much safer than the
highly dangerous liver and spleen biopsies. Thus marrow exami-
nation may be of inestimable value diagnostically and prognostically.
It may prevent more costly diagnostic examinations and sometimes
surgical expense.

The author realizes that the practical value of bone marrow
examination in veterinary medicine will have to be proven. At the
present time it appears that marrow biopsy evaluation will have to
be based on qualitative findings, myelogram and sections of small

portions of the total marrow substance.

85
Quantitative studies of bone marrow have been carried out
during the last twenty years. Isaacs (1937) reported a method of
counting the cells in one cubic millimeter of marrow, claiming good
results. Meyer and Bloom (1941) stated that their counts per cubic
millimeter of marrow varied from 56,000 to 317,000 cells, almost ::

600 per cent. They believed their method had diagnostic value. In

the opinion of the author the approach of Reich and Kolb (1942) to

n .. Si.4‘.f?'1h"!“i . l

the problem of quantitative study of bone marrow was truly scien- 4...-
tific in every way. Multiple sternal marrow samples were taken
from twenty—six patients. Statistical analysis indicated that quanti-
tative determinations on aspirated marrow samples were inaccurate.
They stated, however, that qualitative marrow studies were invalu-
able in establishing hematologic diagnoses.

The author has called attention to the variation of the quality
of marrow in dogs within areas of only a few millimeters. Custer
(1932) pointed out a similar variation in human bone marrow. Cal-
houn (1954) observed similar variation in equine and bovine bone
marrow. Quantitative counts do not appear to have any value diag-
nostically because of the nature of myeloid tissue. However, much

difference of opinion exists as has been pointed out by Calhoun (1954).

86
Determination of the ratio of the total number of myeloid
cells to the number of erythroid cells appeared to be a good cri-
terion of bone marrow activity. It should be a valuable clue to some
of the anemias, leukopenias, leukocytoses, and other blood dyscrasias.

A comparison could be made between the ratio of the count of the

v - ‘7."'- - -.-re~v7

smear and the picture obtained from sections.

- - H» . lav-Ar

Rohr (1949) has found the myeloid-erythroid ratio of the human
adult to be three to one. ___
Some discrepancy existed between the two cases on dogs re-
ported in the literature and the findings of the author.
It was appreciated that the number of cases examined during
this investigation was not sufficient to be conclusive. A comparison
might be permissible because the dogs used in this investigation
were known to be healthy animals as far as could be determined.
The object of the cytologic study of bone marrow of the dog
by Stasney and Higgens (1937) was to determine if any difference
existed in the quality of marrow from different sites. The dogs used
had been subjected to other experimental work, which could have been
responsible for the results: a myeloid ratio of 0.53:1.00 average.
The dogs of Meyer and Bloom (1943), as far as could be de-

termined, were out-patient cases apparently in good health. The

87
myeloid-erythroid ratio varied from 0.76:1.00 to 2.53:1.00 in ten
cases.

The author's cases showed a myeloid-erythroid ratio of ap-
proximately three to one. This agreed with the conditions reported
in human medicine. The life Span of the red blood cell is one E

(.-
hundred and twenty-seven days in the human subject (Shemin and i
Rittenberg, 1946); for the dog this has been found to be one hundred !
and twenty-four days (Hawkins and Whipple, 1938). It can be seen .2
that the blood picture of the dog is not too dissimilar to that of man.

Recently the author determined the myeloid-erythroid ratio of
a known frank case of anemia in a dog: it was two to one. The
animal had had melena for several months. An abdominal tumor
was shown to be a leiomyosarcoma originating in the muscular wall
of the jejunum. The melena and anemia were due to the hemorrhag-
ing tumor.

The values given by Stasney and Higgins and the lowest values
of Meyer and Bloom would indicate that the number of cells of the
erythropoietic system of bone marrow was greater than those of
the leukopoietic series. This appeared incredible inasmuch as no
observation had been made which could even approach such low

ratios in normal animals.

SUMMARY

A necessity for improving and expanding diagnostic facilities
in the field of veterinary practice has been realized, and a step in

that direction has been attempted.

Evidence has been presented to prove that diagnoses based

 

on cytology alone are apt to be unreliable. It is suggested that in-
formation derived from sections and smears should be correlated
with findings in the peripheral blood.

The clinician is cautioned to correlate anamnesis, Signalment,
present illness, peripheral blood, and bone marrow findings. He
should also take advantage of necropsy material whenever it is avail-
able.

Anatomy, histology, and cytology are described, pointing out
some features peculiar to the dog.

The latest nomenclature for cells of the blood and the blood-
forming organs is presented in Table III.

It is emphasized that a wide variation existed in the quality
and distribution of marrow in the same bone. Qualitative cell counts
of marrow were an invaluable aid to hematologic findings, whereas

quantitative counts were inaccurate.

88

89
A method was devised for making whole marrow sections for

microscopic study.

A new site for doing marrow punctures has been suggested,

and a method for evaluating gross and microscopic specimens has

been outlined.

An improved instrument has been made to facilitate biopsy

procedures in small animals.

- .2... -...._._ ._.._.,..A__.__.. . . 4,-
\ . - .‘ .. 71—“ r'KrF

l .

CONCLUSIONS

1. A technique of femoral puncture was described which can
be done with absolute safety and a minimum of discomfort to the
patient.

2. A series of conditions were reported which Show that
study of the bone marrow obtained in this way is of great assistance
in clinical diagnosis.

3. Five types of marrow were described to aid in differen-
tiation of normal and abnormal tissues.

4. Bone marrow biopsy gives the clinician an insight into
the fundamental processes underlying his findings in the peripheral
blood, and permits him to observe the direct effect of therapy on

the £0 rmative tis sue .

5. Finally the author sincerely hopes and expresses herewith
the genuine desire that the study of bone marrow may take the place

in veterinary diagnostic procedure it rightfully deserves.

90

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