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E
A. PRELIMINARY STUDY OF MOPOIASIS IN

YOUNG FLEXED TAILED MICE.

Thesis

Suhittod to the Faculty of the Michigan State College in
Partial Fulfillment of the Roquirenonta for the Degree of

Master of Science.

By
Dorothy M. Andoroon

May ’ 1933.

THESIS

 

ACKNOWLEDGEMENT

'me author wishee to «press her appreciation to all thoee
tho vere or assistance in this iork. Gratitude in particularly
extended to Dr. H. R. Hunt of the Zoology Department, at IhOIO
auggeation and under I110” guidance this work was done; to
Dr. E. T. Hellman of the Pathology Department, who rendered
valuable technical assistance; and to ur. o. F. Edwards er the

hoteriology Department, who took the mierophotographe unwed.

93804

TABLE OF CONTENTS

Page

I - The History of the Flexed Tailed Mutation...............1

II ’ The M086 of the PI‘esent St‘ldYoeooeeoeeeeeeeeeeeeeeeee

III - Normal HGMOPOiOSiSeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee

1e

Conflicting Theories of Hem0p019313eeeeeeeeeeeels

IV'- Materials and Methods usedeeeeeeeeeeeeeeeeeeeeeeeeeeeezo

10 materialseoeeeeeeo.............................20

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3.
4e

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KillingeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeZl

Fixation , Dehydration, Rubedding , etc. . . . . . . . . .83
seetioningeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee25

Stainingeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee25

6.1M8th0d Of Obtaining the Dataeeeeeeeeeeeeeeeeeeezg

'7.

D1ff1°u1ti 33 or Cell Identification. . e e e e e e e e e .33

V ' Presentation 0: the Dataeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeesa

VI ' Discussion Of the Dataeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee40

VII ' Canclusionseeee....eeeeeeeeee......e.................46

VIII - Tables

1e

2e

5.

4.

5e

6e

'7.

Table I - Course of the Lnamia..................47
Table II - Distribution of Sizes of the

Different Cells of the Erythrocytio LineaheeAB
Tables III to VII - Individual Counts of

Nomal MicBeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee49'55
Tables VIII to XII - Individual Counts of

Anemia Mice...............................56-62
Table XIII - Summary of all Normal Counts......63
Table XIV - Summary of all Anemic Counts.......64
Table IV - Table of Differences Between

Normal and Anam103eeeeeeeeeeeeeeeeeeeeeeeeeee65

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TABLE OF CONTENTS (Continued)
3352
X - Plates
1. kplanation of Plates. ete...........o.........68
3. Key to Abbreviations used on Plates............69
3. Microphotographs:
Plate I to IV - Liver of Normal Mouse
at Birth...................i-ii
Plates V to VII - Liver of Anemia Mouse
at Birth.................iii-iv
Plates VIII and II - Bone Marrow of
Normal Mouse..................v
Plate X - Spleen of Anemia Mouse...o.........vi
Plate XI - Cross-section of Blood Vessel
of Normal Lionse..............vi
Plate XII - Liver of 17-day-old Mouse.......vii
4. Color Plates.
Plate XIII - Normoblasts...................viii
Plate XIV - Erythroblasts..u................ix
Plate IV - Proerythroblaste...................x
Plate XVI - Hemocytoblaste...................xi

Plate XVII ’ A Megakaryocyte....u........uxii

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THE HISTORY OF THE FLEXED TLILED MUTATION

The mutation in the mouse, Mus museulus, which has been named
"flexed tail” was found in J'anuary. 1927, by Dr. H. R. Hunt in his
albino stock. The gross appearance of the flexed tailed animal has
been well described by Mixter who says of it: "In appearance it
varies from a tail which is stiffened by the fusing of a few
vertebrae to a stiff tail bent into several angles in different
planes. Not only is the tail stiffened and bent but it may also
be shorter than the normal length which is about the same as the
length of the body.......At birth the flexed mouse is paler and
smaller in size than a nonnal new born mouse".1

Breeding experiments by Hunt, Palmer. and Mixter have shown
that the factor for flexed tailness is probably a simple MeaeIian
reeessive, but that there seem to be modifying factors at work
which somewhat affect the expression of this recessive gene.

Work on this phase of the genetics of the animal is now in progress.

Environmental conditions such as diet, tanperature, litter size.
etc.. have not been shown, to date, to have any affect on the
appearance of the flexed tail. Diet, caging. and temperature have
been constant for all mice. Nor has the flexed condition been
restricted to any particular color-type of mouse. but is found
among albinos, agoutis, blacks, browns, and piebalds.

It was noticed early in the study of flexed tailed animals that
at birth they were not only smaller in size than their straight
tailed litter mates (in F2 or backcross litters), but that they

were markedly lighter in color. Mice are naked at birth, so the

color of the skin and hence of the blood is easily seen. A normal

-2-

newbborn mouse is a vivid rosybred. whereas these flexed anhmals

are a pale pink, this being especially noticeable on the top of

head and around the thighs and shoulders where the skin is drawn
closely over the skeleton. This pale color strongly suggested

that an anemic condition was associated with the flexed tailed
character. To determine if this supposition were true, Mr. Russell
Mixter, in the research already referred to. carried on an extensive
study of the circulating blood of these ananic animals. Erythrocytic
and leucocytic counts were made. and the hemoglobin content of the
blood determined at different age levels beginning at birth and
continuing at regular intervals up to adulthood, i.e.. six months

and on. The same determinations were made on a control group which
was made up of two genetic types of normal animals: the heterozygous
normal mice which were litter mates of the flexed animals studied,
and hanosygous nomals, the offspring of normal I normal parents. For
he reader's convenience I have sumnarized Mixter's data and included
it herein.for reference. (See Table I).

This table shows at a glance that the supposed anania at birth
proves to be real. The flexed animals have an average red blood cell
count that is less than that of the heterozygous straights by
1,560,000 11151.600 cells per cubiczmillhmeter. This difference is
10.29 times the magnitude of its probable error, so it is undoubtedly
significant statistically. The count for the hanozygous straights
exceeds that of the flexed by 600,00011;152,000. This difference is
£.5 times the size of its probable error, not as marked a difference

as the first one is; but it is statistically significant nevertheless.

-3...

The same is true of the hmoglobin content which Mixter expressed
as the percentage of the normal hemoglobin content of hunan blood.
The average hemoglobin content for the flexed at birth is 39.‘%,
and that for the heterozygous straights is 60.7%. The difference
is 81.3 it .93 which is 22.9 times the size of its probable error.
so that the difference as between flexed and normal is even more
marked in.hemoglobin content than is that between the erythrocytic
counts of the two. As may be seen from the table the leucocytic
count for flexed mice is normal at all ages. so no further
consideration will be given it in this discussion.

Incidentally it should perhaps be pointed out here that all
three types of animals show a much lower erythrocytic count at.
birth than at subsequent ages. There is a rapid rise in the number
of erythrleytes and leucocytes during the first three postnatal
weeks. This increase continues up to about eight weeks. but at a
slower rate. Kindred and Coryz, in a series of studies of the blood
of the fetal albino rat, have shown that the number of erythrocytes
and leucocytes drops quite markedly just after birth, so that the
blood count then is considerably lower than during the fiast prenatal
period. Their work also shows that a rapid rise in the number of
blood cells begins about three days after birth and continues in a
rather spasmodic fashion up to six weeks. after which the rise
continues slowly to 5% months when the counts are the same as in
the adult. They attribute this decrease at birth to three possible
causes:

1. Loss of the blood corpuscles circulating in the placenta.
z. Hemorrhage via the umbilical vessels.
3. An increase in the percentage of earlier forms of blood

cells at this period.

 

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.4-

Tnis condition may then be considered as a so~called
"secondary anenia'. i.e. an anemia characterized by a reduction
in the umber of circulating red blood cells attributable to a
fairly definite cause.3 This should not be confused with the
mania associated with the flexed tail. which is entirely different.
Since Hixter made no fetal blood counts we do not know definitely
if this drop occurs in these mice at birth. but it is quite possible
that conditions in the mouse are similar to those in the rat.

one of the interesting features of this particular anemia is
that the mice. if they live, recover from it rather rapidly after
birth. it the age of one week the erythrocytic count for the
flexed animals has practically risen to that for the heterozygous
normals and has sanewhat surpassed that of the homozygous normals,
showing that there is an intense hmopoietic activity in the flexed
animal during its first week of postnatal life. it one week. the
hemoglobin content of the flexed is still low. although it has
improved since birth. it two weeks the hemoglobin cmtent is
practically equal to, and at three weeks it surpasses that of the
heterozygous straights. It may be said. then, that by three weeks
of age all signs of the anemia have vanished. The animal is normal.
Once the normal condition is reached. it appears to be mintained
for the duration of life, for the comparative counts of the three
types at an adult age show no very great differences.

It is interesting to note in this connection that Mixter's
data show that the red blood cell counts for the heterozygous straights
are higher at all ages at which counts were made than for the hmosygous
straights. 131s difference between the erythrocytic counts for these

two types at birth is 870,000 1 159 .000. a difference which is 5.5 times

--5-

its probable error. This is not at all what one would expect to find.
Any difference between these two types of'normals ought, logically.
to be in favor of the homozygous straights since they are carrying
none of the mutant genes. It has been suggested that the better
condition of the heterozygous animals is due to their heterozygosity.
This theory has not been tested out however.

In summary then. we seem here to be dealing with two very closely
associated genetic conditions. a bone ananly. and an anemia. These
must be caused either by one recessive mutant gene or by two or more
very closely linked genes. The present paper will be concerned with

a study of this ancnia only.

THE PURPOSE OF 1113 PRESEIT STUDY

Anemia is defined as that condition characterized by a deficiency
in the oxygen-carrying substance of the‘blood. hemoglobin. It may be
manifested by (l) a diminution of the mnount of hemoglobin in each
erythrocyte, or (8) a decrease in the number of red blood cells.
inanias are camnonly classified as being of two general types:

(1) primary - an intrinsic anemic condition of unknown cause. and

(8) secondary - those produced by definite causes such as hemorrhage,
acute infectious diseases. acute infections, tumors, etc.3 Evans4
has pointed out that such a classification is unsatisfactory since
probably all enemies are secondary to some definite cause. if that

cause could but be discovered. He suggests that although none of

the several classifications of anemia in vogue are entirely satisfactory,
perhaps the one by Morawits canes sanewhat closer to the root of the
matter. Morawits divides enemies into those due to blood loss or
increased destruction of red blood cells. and those due to decreased
blood formation. but this does not complete the clinical picture as

it does not include changes in hemoglobin content of individual red
blood cells, i.e.. changes in the color index. These classifications
must at best be sanewhat arbitrary for clinical use.

It seemed.that further studies of this anemia would prove both
valuable and interesting. It was hoped that a histological examination
of the subject might render. if'not proof. at least valuable clues as
to the actual causes of this anemia. It will be noticed that no
definite diagnosis has been made concerning this anemia. It would
be impossible to give it its clinical classification until sanething

was known concerning its cause. If the cause or causes could be

-7-

discovered, we might also know more about how the flexed gene actually
gets in its work.

As will be shown later this anania is particularly interesting,
since it differs in its characteristics fran any other anemia of mice
known to date. Mr. Mixter established the fact of its existence. but
his data fail to tell the whole story, for they do not show, for
instance, whether this is the type of anemia caused by blood loss or
destruction of red blood cells, or by decreased blood formation.

So with a view to gaining information concerning the histological
nature of this anemia. the investigation reported in this paper was
begun in January 1951. and has been continued to the present time.

It is to be regretted that more progress has not been made;
however, the problem proved to be a genuine Pandora's box out of
which sprang an unexpected host of surprising elements the moment
the lid was raised. Most of these elements, as will be shown later,
have been a hindrance rather than a help to the investigator in her
attempt to find out Just what else was in the box. However, a start

has been made, and it is hOped in the right direction.

-3-
iNORMAL HEMOPOIESIS

Before the methods used in attacking this problem can be
fully understood or the data obtained can be discussed, the
salient facts concerning blood formation should be reviewed.
Before studying the abnormal condition we must familiarize
ourselves with the normal. At this point I shall digress at some
length to describe the process of the manufacture of blood tissue.

Blood is not the simple liquid.material it would seem to be
to the casual observer. Canplexity - morphological, chanical,
physiological, and genetic - is its most outstanding and constant
characteristic. In brief, blood may be considered as a tissue,
the constituents of which are several kinds of cellular elements
(corpuscles) , sane anal]. irregular cell-like elements (the platelets),
and a liquid.medium.lplasma). Or as one textbook of histology
states it: ”It may be considered as a form.of connective tissue
in which the cells have no fixed spatial relationships and the
intercellular substance 18 a fluid”.5 The corpuscles are of two
main types: the erythrocytes or red blood cells, and the
leucocytes or white blood cells.

As has been shown in the historical account of this anemia.
this problem is apparently concerned only with the erythrocytic
elements of the blood. Therefore, throughout this paper attention
will be focused on the red blood cells practically to the exclusion
of the white blood cells, which though of great importance to the
organism.do not seem to have any direct bearing on the particular

condition with which we are dealing.

.9-

As compared with the other tissues of the vertebrate body,
the blood cells are very short lived. The exact life span of a
human red blood corpuscle is not known exactly, but experimental
work has furnished estimates varying from fifteen to forty or
more dsys. Whatever this span may be, we do know that cells are
constantly being worn out and destroyed and new ones manufactured
to take their place, so that under normal conditions their number
is kept at a constant level. This process of the manufacture of
blood cells is called hanopoiesis, and the tissues in which it
specifically occurs are designated as hemopoietic tissues and in
the adult are located outside of the vascular system proper. The
hanOpoietic tissues may be subdivided into two types not always
sharply differentiated from.each other: the lymphoid tissue
(lymph nodes, spleen, etc.) wherein the lymphocytes are produced,
and the myeloid tissue wherein the erythrocytes and granular
leucocytes are formed. In the normal adult mammal, the myeloid
tissue is more diffuse and is found in several parts of the body,
such as the yolk vessels, liver, spleen, and bone marrow.

During foetal life, the first blood cells are formed from
the endothelium of the walls of the yolk vessels. These blood
cells are nucleated, basOphilic cells, and do not much resemble
the erythrocyte as we know it. They are very temporary and are
soon replaced by blood cells of a more differentiated nature
produced within the body of the embryo itself. These new cells,
although nucleated, elaborate henoglobin, and serve to carry oxygen
to the tissues of the embryo. After the formation of the liver,
the thin strands of:mesenohymal cells (which occupy the tiny

interstices between the liver cords and their surrounding

vascular plexi) begin to differentiate into blood-forming cells.

-10.

For the rest of the foetal life and for a very short period after
birth, the liver is the center for much blood manufacture. The
spleen may also be a center for red cell formation at this period.
The bone marrow begins its hanopoietic function in the later stages
of foetal life and as birth approaches it more and more takes over
the blood-forming functions which are gradually being relinquished
by the other organs mentioned. Thus at birth the animal is still
in a transitional state showing some of the processes characteristic
of the embryo, and some characteristic of the adult. ”The transition
fran the embryonic to postnatal life is not marked by any sudden
special changes in the blood or connective tissue. The processes
of development and differentiation we have followed in the description
.......continue without interruption".6 Regardless of the place
where hcnopoiesis occurs. the cells involved are of the same type
and the process is the same.

One can scarcely enter any sort of a discussion of this
process without at once becaning involved in the more or less
conflicting theories and certainly confusing teminology of the
hmtologists. I shall outline the most outstanding of these
theories later on, but to present a relatively uncanplicatcd
picture of this process, I shall at present follow Maximow's
description.6'7

The red blood cell as fmmd in the circulating blood, is not
produced directly from other cells like itself, but is the end
product of a series of cell mitoses and accompanying metamorphoses.
The first cell entering into the genetic lineage of the erythrocyte

is a rather large, free. mboid, basophilic cell of mesenchymal

origin, resembling the large Inphocytes of the adult, and was

l O

 

named ”bemocytoblast" which means "a cell producing blood cells".
Ihen they were first observed by Maximow in 1907 he called them
"large lymphocytes". and it is his belief that they are "the common
stun cells of all the other blood elements in the anbryo and in
the adult"?. These cells are large in size (up to léfbin humans),
have a usually pale basOphilic cytoplasm which forms a broad ring
around the nucleus. There may be vacuoles present in the cytopla-e
The nucleus is large, sometimes kidney-shaped. and is characterized
by clumps of chromatin scattered loosely about, giving it a
so-called "basket weave" appearance. The nuclear manbrane is
coarse and shows up quite conspicuously in stained specimens.
The hemocytoblasts may undergo several successive mitoses
producing daughter cells like themselves. Finally a significant
change occurs.) Maximow describes it thus: ".....a peculiar
unstable equilibrium of the cell develops at the knight of the
mitotic process. All of the various potencies of development
which are present in the cells are in a latent condition; one
of them.is suddenly followed and both of the daughter cells which
originate frcn such a mitosis at once show new properties.
Their destimr apparently has been fixed during the mitotic
processes and they have become either a pair of erythroblasts
or a pair of myelocytes of one of the three types".§ The
hanocytoblast is also the stem cell of another cell type, the
megakaryocyte which will be referred to further on.

The next several generations in the erythrocytic series
are termed collectively erythroblasts. The youngest cell of
this group, that is, the cell directly produced from the last
henocytoblastic mitosis, Maximow calls the proerythroblast.

The oroerythroblasts. or basonhil erythroblasts. differ frail

.004.

 

-12-

the hanocytoblast in several ways. They have not its multipotencies.
They can only form erythroblasts. They have lost the ameboid
movanent of the hanocytoblast, and are fixed. The nucleus is
regularly spherical and maller, and its chranatin shows a more

even distribution than does that of the hemocytoblast. 'me
cytoplasm is intensely basic and very homogeneous in appearance.
They frequently have the shape of a triangle with well rounded
corners.

The next youngest generation of erythroblasts is sanetimes
called the megaloblast. This cell is always larger than any of
its descendants. Maximow does not give its exact dimensions
but I assume from his figures6 that it is somewhat smaller than
either the proerythroblast or the hemocytoblast. his rather
conflicts with other descriptions of it, e.g., one group of
hematologists say: ”This (the megaloblast)is representative
of the largest red blood-cell we have observed in the
erythropoietic series".8 It shows a more canpact nucleus than
its predecessors, a less conspicuous nuclear manbrane, and
cytoplasm containing a small amount of hanoglobin, which,
however, may not show up in a stained cell. In cells stained
with the Rananowsky mixture (eosin and methylene blue), the
cytoplasm may give a polychromatic effect - that is, it may
vary in color from purplish-blue, to lilac, to gray.

The succeeding several generations of erythroblasts show
progressive tendencies to increase the amount of hemoglobin in
the cytOplasm, to condense the nucleus (and thus show a wider
ring of cytoplasm around it), and to become somewhat smaller in

total size. In contrast, in the earlier generations of this type

-m-

the nucleus shows a rather characteristic checkerboard
distribution of the chranatin, and a thin ring of protoplamn
around it. This means then that a considerable amount cf
morphological variation is to be observed among the different
cells of this group as a whole. The last generation of this
erythroblastic series is scarcely to be distinguished from.the
cells of the next stage, the normoblasts or nucleated erythrocytes.

The normoblast is the smallest cell of the series, with the
exception of the erythrocyte. It is either spherical or ovoid
in shape and is characterized in a properly stained condition.by
a clear pink cytoplasm.- due to an accumulation of a considerable
amount of hemoglobin - and a small, very dense, deep purple
nucleus in which scarcely any reticulation can be seen due to the
chromatin particles being so closely packed together. This cell
still retains the poser of undergoing mitosis, and may possibly
produce another generation or two of cells like itself. Gradually,
however, the nuclei of these cells become pyknotic and more and
more hanoglobin collects in the cyt0plasm. Eventually the pyknotic
nucleus is extruded from.the cell. The small, spherical,
enucleated red cells then pass through the permeable msmbranes
of the capillaries and are ready to begin.their oxygen-carrying
function in the blood stream.

Another derivative of the hemocytoblasts which will come
under consideration in this paper is the megakaryocyte, or so-called
giant cell. Concerning this cell, little was definitely known
until rather recently. It is now coming into more praninence,
and is believed to play an.important role in certain pathological

9
conditions . The theory has been held for some time and is still

10 14
accepted by some investigators ' , that these giant cells form

'14-

the blood platelets by freshentation of their cytoplasm, but
Maximow questions the truth of this thea'y. He says that both
the origin and the function of the platelets is mine—6. There
scans to be no doubt, however, that the megakaryocytes are
produced by the hanocytoblasts. In structure, these cells are
very large, some of them having a diameter as great as 49min
hmnans. They are formed from hanocytoblasts by the growing nucleus
of the latter undergoing a hypertrophy, and then beccming
constricted in several planes. Following this there are several
peculiar mitoses which involve only the nucleus. In these mitoses
the daughter nuclei fuse in the telophase stage forming a new
larger nucleus. Eventually a polymorphous nucleus is formed,
the individual lobes of which resenble the nucleus of the
hanocytoblast, i.e., they show the 'basket-type‘ of reticulation
due to the irregular grouping of chranatin granules, and the
outline of the nuclear membrane is quite cmspicuous. The
cytoplasm is clear, hmnogeneous, and faintly basic in its
staining reaction. It is very irregular in outline, frequently
showing pseudopod-like structures which make the cell sanewhat
resanble an snoeba. According to Jordanm, megakaryocytes are
found wherever hemopoiesis occurs.

It is rather interesting to note that Jordan attributes
to the megakaryocytes the potentiality of forming multiple
erythroblasts, at least in the yolk-sac. He describes this
transformation thus: "As polykaryocytes (multi-nucleated
megakaryocytes) they differentiate erythrocytes intracellularly
by a process involving the elaboration of hanoglobin about each

nucleus. Each perinuclear halo of hsnoglobin-containing

 

ls

 

’15-.

erythrocyte thus differentiated comes to lie free in a vacuole.
Subsequently, the giant-cell breaks up and liberates the several
erythrocytes.......8uch a process of intracellular giant-cell

11

production of erythrocytes has been described also by Foa in

8
and Denys“ have described both

bone-marrow. Pugliesel
erythrocytes and leucocytes originating in these giant cells".10

I can find no reference whatever to such a theory in the
work of the outstanding contanpcrary hanatologists, such as
Sabin and Maximow, but that does not necessarily disprove it.
If it is true that megakaryocytes are hemogenic cells, then
they should merit our attention in a study of this kind. For
this reason I have included than in m data.

Conflicting Theories of Homepoiesisz Dr. Florence Sabin
makes this statanent: "The history of the developnent of our
knowledge of the blood centers around two opposing theories,
and nowhere in medicine can there be made a more interesting
analysis of the value of constructive theories, whether right
or wrong, in the advance of science'.1‘ ‘

. The bones of contention over which the adherents of these
two theories have Opposed each other are, (l) the immediate
origin of the red blood stem cell, and (2) its relationship to
the stun cells of the other types of blood cells. The process
as I have described it above, showing a basophilic, lymphoid
cell (the hanocytoblast) which is the potential source of all
types of blood cells, red or white, is the explanation of
events as offered by the so-called unitarian, or monophyletic

theory or school. It is the most modern theory and much

progress in the knowledge of blood has been made under its

 

0.00...

 

-15-

influence by such adherents as Dominici, Pappenheim, Weidenreich,
Maximow, Danchakoff, and Ferrata.

The opposing theory is called the dualistic, or to be more
exact, the polyphyletic theory, and it is associated with the
work of Ehrlich, Naegeli, Schridde, Morawitz, and others. It is
based on the work of Ehrlich who is attributed with having laid
the foundation of our knowledge of blood.M According to this
theory there is no one cell type which serves as a canmon stun
cell for all blood cells, but instead ”there are different stem
cells for the red cells, for the leucocytes, and for the
lymphocytes, and they are located in specific places in the

14' Ehrlich classified the adult blood-forming organs into

adult".
the two types already mentioned - the bone marrow or meloid
tissue, giving rise to the erythrocytes and the myelocytes
(forerunners of the granulocytes) , and the lymphoid tissue

giving rise to the lymphocytes. The polyphyletic school claims,
furthermore, that the potencies of the cone of one type of tissue
are specific for their individual function, and are not inter-
changeable with those of the other type of tissue. In other words,
the lymphoid stem cells cannot produce red blood cells, nor can
the red blood stun cells over produce lymphocytes. This belief

is founded upon morphological differences observed by Ehrlich

and others in fixed and stained cells. The dualists have
interpreted these structural differences as being correlated

with physiological and genetic differences in the cells; but

the monOphyletic school in the work of Maximow and Danchakoff

has been able to demonstrate equally well that these morphological

differences are not intrinsic properties of the cells but are the

-17-

result of the type of technique used, and by means of newer staining
methods and extremely intricate technique has shown an essential
similarity between the cells of both tissues. Even these claims
are being met by the equally modern technique of the modern
adherents of the polyphyletic school. For instance, Dr. Sabin,
who tends to support this older theory, shits that "the primitive
stem cells of the different youps of blood cells cannot be
separated in fixed specimens even with the most perfect technique",
but she adds that, ".....these atoms can be analyzed with the
method of vital staining".14 Thus the battle wages between two
equally proficient groups of technicians.

Perhaps the theories of each group are based upon facts which,
though essentially the same, have lent thanselves to different
interpretations. It would seem that the real point of contention
is not so much concerned with the similarity between the early
cells of the different types of blood cells, as it is with how
long a demonstrable similarity exists. To develop this idea
further: Both groups believe undifferentiated mesenchymal cells
to be the ultimate source of all the blood cells, and recognize,
therefore, that blood and connective tissue are very closely
ellied, both embryologically and morphologically. According to
the dualists, some of this mesenchyme early in the embryonic
history of the individual, differentiates once and for all into
groups of pro-blood cells, sometimes referred to as angioblasts.
These angiollasts are of extraembryonic origin (in birds). They
proliferate outside the embryo, but later invade it as a consequence
of the increase in the cell masses which they form. In the body of

the embryo they becane the endothelium of the blood vessels, and

the erythroblasts; or, erythroblasts may develop fran the

 

C
9
O
O
C

 

~18-

endothelium of the blood vessels. So the line of development is:
mssenchyme, angioblast, erythroblast; or mesenchyme, angioblast,
endothelium of vessels, erythroblast, thus eliminating the
hamocytoblasts and proerythroblasts from the picture entirely.
The cells derived from.this angioblast would then be of two
sorts: one, the endothelium.and erythroblasts, the other, cells
which renain like the undifferentiated cells of the angioblast.
This latter group continues, by proliferation, into the postnatal
life, and eventually becomes localized in specific places,
particularly in the bone marrow. It may then becane transformed
into erythroblasts in answer to the body's continual need for new
erythrocytes, but always a group of undifferentiated cells are held
in readiness so that only part of each generation of cells becomes
erythroblasts, the other part remaining unchanged for future use.
While this school recognizes the presence of immature,
undifferentiated cells in the hematopoietic tissues of the adult,
yet they would assign to such cells the power to develop along
but a single line predestined from the embryonic period. These
cells, then,:morphologically indistinguishable from each other,
are potentially wide apart".16

The monOphyletic school likewise names the mesenchyms as the
first ancestor of the blood-forming cells, but it postulates that
the so-called angioblast and its products are but the temporary
blood-forming structures of the early embryo, and are soon
superceded by the hemocytoblasts which arise everywhere in the
anbryonic connective tissues by transformation of the small,
undifferentiated, fixed mesenchymal cells. These cells in the

adult, although found in the fixed connective tissues throughout

the body and believed to be able, theoretically at least, to

transform if necessary into hanocytoblasts, tend to centralize
their hmnopoietic functions within the red marrow of the bones
where they transform into hemoblasts as needed. So, according
to this theory, the immediate ancestors of the erythroblast are
but recently differentiated mesenchymal cells whose embryonic
multipotentialities have been maintained into and through adult
live.

Thus, according to one school, the similarity in the adult
between the stem cells of red and white blood cells is only
morphological, the physiological equality having been lost
during embryonic life; and according to the other school, the
similarity between then is continued into the adult life and

is both morphological and physiological.

 

 

 

Al.jall.v. . .. 1,: w ‘ (I,||.l.

 

MATERIAIS AND METHODS USED

Although a wealth of literature is available on blood
histogenesis and morphology, and on the many pathological
conditions of blood, there is a paucity of material in print
which has any direct bearing upon this particular anemic
condition or on the proper technique to use in studying it.

The only other hereditary anemia so far reported in mice is

that in Little's dominant whites. This anemia has been studied
and reported by deAberlem. Although anemia in these dominant
whites is entirely different frcxn that associated with flexed
tail, in that it is a severe aplastic anemia which is invariably
fatal, Dr. deAberle's work was the primary source of information
concerning the method of approach to this problmn.

Even this source was of quite limited value, however,
since her approach to her problan was of an essentially different
nature from mine; so from the first, the methods used were a
matter of trial and error in equal prOportions.

Materials: It was decided that the first thing to do was to
study the general histological picture of the normal animal at
birth and to compare this with enemies of the same age. Sane
time was devoted to raising a group of mice to be used for this
work. Homozygous normal females (2 to 6 months of age) were
crossed with flexed males. The F1 fanale offspring from these
crosses were then backcrossed with flexed males. These females
were isolated, one to a cage, as soon as pregnancy could be
observed, and were then examined daily until the young were

born, after whdnh the mother was returned to her mate and bred

again. Part of the animals in each litter were selected for this

 

 

 

work and the rest discarded. In order to get as much individual
variation as possible only two or four (8 normals and a anemics)
were used from a litter.

Selection of the new-born young to be used was based
entirely on a clear-cut differentiation between anemic (flexed)
young, and normal (straight) young. There are undoubtedly
borderline cases, both as to amount of tail flexure and as to
the enmia, which are hard to differentiate frm the normal.

In order to avoid errors in classification, only those animals
were used which, on the one hand, hhowed the pale pink color of
the anmics and a pronounced grade of tail flexure and, on the
other hand, those which had the ruddy color, straight tail and
larger size of the normals. Since in all cases (with one possible
exception) the pregnant mother had been examined daily, none of
the mice used could have been over 84 hours old when they were
killed for sectioning. They were, in all cases, old enough to
have had at least one feeding, for the stomachs were distended
with milk which can be seen clearly through the semi-transparent
skin. The exact age in hours was not determined.

Killing: Since it was thought that etherization might have
sme physiological effect on the blood cells before death
occurred and since hemorrhage was to be avoided as far as
possible, it was decided to kill these animals by strangulation.
A piece of ordinary cotton twine was tied about the neck and
suddenly pulled taut. In a very few seconds the body became a
purplish-blue and bow motions almost ceased. As soon as

unconsciousness was assured, a longitudinal incision was made

'22-

in the skin about half way between the mid-ventral line and the
left side in onch a way as to avoid severing the internal
memory arteries and veins which course down the ventral body
wall as far as the end of the sternun and on either side of it.
All other main vessels were also carefully avoided. This
incision extended frm the posterior boundary of the abdmen
forward to the clavicle, and was ordinarily supplemented by a
transverse incision in the region of the axillae. men, using
fine scissors, the underlying muscles, peritoneum, and the ribs
were out along the line of the skin incision. The diaphragn
was severed frm the ventral body wall with a sharp pointed
knife. Thus both the abdminal and thoracic cavities were
laid well Open, and with practically no loss of blood. The
animal was then quickly transferred to a small paraffin-lined
dish where it was pinned out (with wooden pins) ventral side up,
in such a way that the viscera were thoroughly exposed. Up to
this point the heart was still beating. The fixative to be
used was squirted into all parts of the body cavities with a
small pipette, and the dish was then filled with the fixative,
so that the animal was entirely submerged. In this way the
fixative was quickly applied to all of the living tissues -

a factor greatly to be desired in histological technique.

Each fixed animal was assigned a number by which it was
designated throughout the entire process which followed. to
"A" following a number indicates an anmic animal in all cases,

the nonnals being designated with numbers without letters.

 

,Illl. Ill]

-33..

Fixation, Dehydration, EnbeddingL etc. McClung's
”Microscopical Technique'la was used quite extensively as a
source for histological technique, formulae, etc. The fixative
particularly recmmended therein for blood work is Holly's Fluid

or Zenker-formol, the formula for which is:

Potassium bichrmate 25 an.
Sodium sulphate 10 gm.
later 100 c.c.
Mercuric chloride 50 an.

Formalin (added when ready for use)50 0.0.

This was the first fixative used. It was found that the
mice should be left in this solution for about 20 hours to
obtain complete fixation. Specimens were left pinned to the
dish with wooden pegs throughout fixation.

They were then washed in running water for 24 hours, after
which they were put in 35% alcohol, to which had been added a
few drops of iodine, for 6 to 12 hours. Then they were moved
into 50% alcohol plus iodine until all signs of crystalized
sublimate produced by the fixative had been completely removed;
this was usually accdplished in 12 hours, during which time
there were two or three changes of the alcohol - iodine solution.
The head was cut off to reduce the size of the animal to be
sectioned. Dehydration was completed in the higher alcohols,
the animals rmaining in each for four to eight hours. Frm
absolute alcohol they were put into cedar oil for clearing
(usually three to four hours) and frm this into paraffin oil
for 12 to 24 hours, after which they were transferred to paraffin

with a melting point of 54°- 58° for 4 to 6 hours, then blocked

in paraffin.

 

 

-24—

Several animals were given this treatment but of these
only'mouse No.12 and mouse NO.13 were used in the final study
made. This method did not prove very satisfactory. In the
first place, the crystalized sublimate formed by Holly's Fluid
is extremely difficult to remove and dangerous to the microtane
knife if not entirely dissolved. In the second place, considerable
distortion of cells was noticed in some parts of the tissues,
especially in the blood cells. In the third place, the tissues
were so brittle that it was almost impossible to obtain thin
sections that were fit to use. It was decided to use another
fixative and a different embedding method.

The second method used was as follows:

1. Killing:- same as above.
2. Fixation in Allen's Fluid, P.F.A.3 - 24 hours.
Formula:

Picric acid (sat.aqueous sol.) 150 c.c.

Formalin 30 c.c.
Glacial acetic acid 20 c.c.
Urea crystals 2 gm.

3. wash in running tap water - 24 hours.
4. Dehydration:
a. 50% alcohol (60 6.0.) plus 20 drops of
lithium carbonate - 4 hours.
b. 70% alcohol - 4 hours.
0. 80% alcohol - 4 hours.
d. 95% alcohol - 4 tea hours.
e. Abs. alcohol - 4'to 8 hours.
5. Infiltration and embedding.

a. Solution of 1.5 gm. celloidin in 100 c.c.

methyl benzoate - until clear, 10 to 12 hours.

 

 

(til
.9 .1 .

-25-

b. Pure benzine - 3 changes of 3 hours each -
total 12 hours.
0. Solution equal parts benzine and 46°- 48°
paraffin at 50°C - 12 hours.
d. Pure paraffin (460-4801 - 4 to 6 hours.
6. Block in paraffin.

This method proved quite satisfactory, so was used for all
of the other animals killed.

Sectionigg: The animals were sectioned in a sagittal plane,
as various trials showed that this was most satisfactory because
so much of the general.morphology can then be viewed in any one
sedtion. Sections were cut Siothick, and three to five were
mounted on each glass slip. Each slide was then numbered with
the number of the mouse from.which sedtions were taken and a
number for the slide series.

Stainigg: Of all the difficulties encountered, perhaps
those concerned with the staining technique were the most
serious and the most baffling. Some preliminary attempts were
made to determine whether wright's stain for blood smears could
be used successfully as a tissue stain. The results were very
unsatisfactory. The tissues took the stain beautifully but in
the process of dehydrating and clearing (a procedure unnecessary
in smears) the delicate stain was so washed out that the resulting
slide was useless. This technique was abandoned. A few slides
were stained by the ordinary hematoxylin-eosin laboratory method.
This is a good process but does not give the delicate differentiation

between different blood cell types necessary for this work.

t
'2‘“ k'

 

 

 

-26-

It was decided that perhaps one of the best stains for
blood work ofithis kind would be the hematoxylin-eosin-azure
stain, as used by Maximow, and by deAberle in some of her work.
The procedure followed was that given by McClung, with certain
variations which I found necessary to suit this material. I
should add here that all of the directions given in the various
sources for blood histological technique are for smears (either
for bone marrow, spleen.material, or fresh blood) or for living
tissue cultures. Nowhere have I found any instructions for the
differential staining of blood in sections, except those given
by deAberle who studied the general.merphology of her animals
in this way, so I have had to adapt these other methods to this
work as best I could.

The method used for this hematoxylin-eosin-azure stain is
as follows:

1. Iylol #1 - 10 minutes
3. Xylol #2 - 10 minutes
3. Alcohol 95% - 10 minutes
4. Alcohol 82% - 10 minutes
5. Alcohol 50% - 10 minutes
6. Water (tap) - 10 minutes

7. water (distilled) - 10 minutes

8. Delafield's Hematoxylin - 24 hours (5 draps to 60 c.c.

distilled water)
9. Water (tap) - 15 minutes
10. Water (distilled) - 24 hours

11. Eosin-azure stain (as follows) - 2.4 hours

 

 

 

 

 

-37-

Stock solution A

Eoein, water soluble yellowish 0.5 gm

Pure distilled water 500 c.c. 5'0 °~°o

Stock solution 3

Pure distilled water 500 c.c. } 4 to 4’5 °'°’

Pure distilled water 50.0 c.c.
12. Alcohol 95% f 5 to 20 minutes
13. Alcohol, absolute - 5 to 10 minutes
14. Xylol - 5 to 10 minutes
15. Mounted in Damar Balsam.

This staining method proved to be relatively satisfactory.
It gave nicely differentiated cytological structure which was of
utmost importance.

The main difficulty encountered, and one which was never very
successfully surmounted was that of getting the normoblasts, as
well as the erythrocytes to show their customary eosin staining
reaction. Attanpts to renedy this by increasing the amount of
eosin in pr0portion to the azure were not successful. Resilts
were the same as with the weaker solution. A fewcells showed
the acidic reaction but most of them remained definitely basic.
Furthermore, the use of more eosin than indicated above did not
give as satisfactory differentiations between the other types of
cells. The actual staining results obtained will be discussed
more fully later. However, one other type of stain was used in
an attanpt to reveal the relatively high affinity of nomoblast
cytoplasm and of erythrocytes for eosin. It was decided to try

an acid eosin-hematoxylin stain which is used in the pathological

 

 

 

I all ehla

 

~28-

laboratories at the University of Missouri. The method for

treating the sections is as follows.

1. Stqs from xylol down to distilled water same as above.

3.

3.

4.

5.

6.

7.

8.
9.
10.
11.
12.
l3.

l4.

Stain in Harris' Hematoxylin - 5 minutes
(50 c.c. Harris' Hematoxylin plus 2 c.c. glacial
acetic acid)

Wash in tap water.

Decolorize in acid alcohol (50 c.c. of 70% alcohol
plus 0.5 c.c. H01.)

Neutralize in weak ammonia solution (three or four
drops amonia to 50 c.c. water)

Wash in tap water - 3 to 5 minutes.

Stain in a 1% alcoholic solution of eosin (1 gr. alcohol-
soluble eosin to 100 c.c. 70% alcohol),
acidulated in the proportion of 5 drops of
a 2% solution of acetic acid to 45 c.c. of
the eosin solution - 4% minutes.

Wash in tap water - 5 minutes.

82% alcohol ~ 5 to 10 minutes.

95% alcohol - 5 to 1.0 minutes.

Abs. alcohol - 5 minutes.

Xylol #1 - 2 to 3 minutes.

Iylol #2 - 2. to 5 minutes.

Mount in Damar Balsam.

This method served to bring out the acid staining reactions

of the erythrocytes and of the cytoplasm in sane normoblasts, but

it was not very effective for the differentiation of types of

blood cells other than normoblasts. Only mouse No.12, Mouse No.13,

 

...?

to
g

and some of the sections of mouse No.26 were treated with this
stain. All the rest of the slides used were stained with
hematoxylin-sosin-azure except three slides of No.18A. (one of
which entered into this study) which were stained in hematoxylin
and eosin only.

From one-half to two dozen slides were made from.each
animal. If satisfactory results were gained in the first half
dozen slides, no more were made. or these, the most satisfactory
slide was selected for making cell counts.

method of Obtaining_the Data; As has been said the first
step was to make a survey of the general morphology of the
animal at birth. This preliminary examination revealed no very
noticeable differences in the histological morphology between
the enemies and normals except in the liver. It was seen at
once that the liver was still actively functioning as a
hemopoietic organ, for in both normals and enemies its peculiar
adult morphology, i.e., the cords of parenchymatous cells
arranged in radial fashion around central veins, was almost
obscured in nmmerous areas by masses of cells which filled the
interstices. (See Plates I to VII and compare with Plate XII).
These cells were identified as blood-forming cells of the
different types.

The first two animals studied, one an anemic and one a
normal, showed quite an obvious difference in the relative
numbers of the different types of cells in the erythropoietic
series. There seemed to be in the normal animal a great many
more normoblasts than any other cell of the series, whereas in

the anemics, although there were many normoblasts present, they

-30-

appeared to be fewer in number than the erythroblasts, the
predecessors of the normoblasts.

It was decided to make a quantitative analysis of these
pre-blood cells in the liver and see whether this difference
was Just a chance occurrence in these animals or a characteristic
and constant difference between the two types of mice at birth.

Before doing this, however, the bone marrow was studied to
see whether the same picture was presented here. No conclusions
could be drawn, however, due to two factors. The first was a
technical difficulty. The blood cells in the bone marrow were
not clearly stained but appeared undifferentiated dark blue
masses of various sizes. Also, these cells seemed to be
considerably distorted, often presenting rather geometrical
designs. (Plate VIII shows a little of this) The red blood
cells present in the capillaries of the marrow were also, as a
rule, quite distorted. It was evident that the technique used
was not adequate for bone marrow. Probably this distortion was
due to poor fixation. It had not been deemed necessary to
subject these animals to a decalcification process as the bones
seemed very soft and cartilagenous. However, it is quite possible
that the dense cartilage prevented the fixative from.penetrating
thoroughly into the marrow, and poor fixation there resulted.
A few slides were obtained later in.which staining reactions
were more satisfactory (Plate II shows one of these), but at
best the cells were not nearly as well differentiated as in the
liver. It was obvious that if any study of the marrow were to

be made, a special technique should be used. The secand difficulty

 

 

 

-31-

encountered in the search for hemopoietic cells in the marrow
is the fact that at this stage extensive bone histogenesis is
in progress and groups of osteoblasts are present which so
complicate the picture that it is difficult to differentiate
all of the various bone and blood elements present. For these
reasons, it was decided not to attempt a quantitative study of
the blood cells in the marrow.

An examination was:made of the spleen also to see what
possibilities it presented for this study. .Although primarily
a lymphoid organ later on, it is capable of some red blood cell
fomation at this early age. Plate I shows why the spleen was
not used in this study. It was so densely packed with lymphocytes
among which were scattered a few cells of the red cell line, that
a quantitative study was next to impossible.

.Attention was then centered on the liver. The method of
making a quantitative study of the hemopoietic cells of this
organ was quite simple. A.suitable area was located with the
_low power of the microsc0pe. This area was sketched roughly
on paper and its position noted by means of the scale on the
mechanical stage of the microscope. I then counted the
hemopoietic cdlls in sixteen, non-overlapping, high power fields
from the liver of each of four normal mice. The same thing was
done for each of four enemies. The number of cells belonging to
each type in the erythrocytic genetic lineage was observed and
recorded for every field. The totals for the sixteen fields
constituted the cell count for the animal. Even more accurate

information as to the cell counts was desired. So 48 high power

 

35...}!

 

-5 3-

fields were counted in an additional normal mouse. These

4.8 fields counted were not all on the same slide. Sixteen
high power fields were counted on one slide. men sixteen
fields were counted on another slide which had been made from
tissue some distance away from that on the first slide. The
count of the last sixteen fields of the 48 was made from a
third slide made from a still different region of the animal.
Thus these 48 counts represented three widely separated sleas
within the same liver. This same procedure was followed in
making 48 counts of an additional normal mouse. Altogether
then, counts were made of 5 normal and 5 anemic animals.

Two or three different low power areas (indicated in the tables
by Ranannumerals I, II, III) were necessary to give these
sixteen non-overlapping fields. For my own convenience in
keeping the data, each field was numbered and is so designated
in the tables of the individual counts. (Tables III to III
inclusive - left hand column).

The cells were classified according to their resemblance
to Maximow's four types of cells in the erythrOpoietic series:
hemocytoblasts, proerythroblasts, erythroblasts, and normoblasts.
Megakaryocytes also were counted. Identification was based upon
a comparison with Maximow's color plates? and descriptive text.

I was unable to find in the literature any measurements of
the hemopoietic cells in mice, so it was decided to determine
the average diameter of each type. This was done with a Zeiss
eye-piece micraneter which had been calibrated with a stage

micrometer. These measurements are tabiisted in Table II.

~35-
A certain amount of overlapping in the size ranges of the
different types is to be noticed, a factor which contributed
to the difficulty in differentiating one type from.another.
Frequently, however, these measurements were an aid in
identification.

Difficulties in 0611 Identification: Before presenting
and discussing the data obtained, there is much to be said
regarding the principle difficulties encountered in identifying
these cells. Reference has already been made in the section on
staining to the fact that the cytoplasm of the erythrocytes
and normoblasts had a tendency to take a basic rather than an
acid stain, or not to stain at all. This unusual condition
could be seen very well in sections of blood vessels. As a
rule cut vessels showed a mottled appearance. Some erythrocytes
were deeply pink, as they should be. Some were very faintly
pink and many were but clear, transparent cells only the outlines
of which could be seen. These cells resembled blood cells that
had been laked. Plate II shows this condition very well. The ‘
unstained cells show in the picture as thin gray circles,
whereas the stained cells are dark. No reason could be found
for this reaction, but it must have been due to faulty technique
“nowhere in the process, for some sections, especially those
which received the acid eosin treatment, showed much less of
this condition than the rest.

As to the normoblasts, very few of them, except those
stained in acid eosin, showed any sign of pink in the cytoplasm.
.A great number of the cells classed as normoblasts were only

extruded normoblast nuclei, but these were counted on the

~34-

grounds that such nuclei showed where a normoblast had but
recently been formed and transformed into a circulating
erythrocyte. When cytoplasm was present it usually showed
only as a clear, sometimes faintly bluish halo around the
nucleus. This halo was usually hard to see unless the light
was greatly reduced and then it was frequently obscured by
other cells being packed in close to the normoblsst, or partly
superimposed on it. Color plate XIII gives fairly accurate
representations ogyyarious groups of normoblasts. In the
microphotcgraphs (Plates I to VII) the cytoplasm of the
normoblasts, if present and in focus, appears either as a
white or slightly gray halo. Nermoblasts designated on these
plates as *nbl‘) are examples of this.

.A few normoblasts showed a very faint pinkish tint in the
cytoplasm, but identification had to be based largely on the
size, shape, and staining reaction of the nuclei. However, none
of these factors were constant. The size was subject to
variation, due to the pyknotic condition of the normoblast
nuclei that are about to be extruded, or have been extruded.

A "young" normoblast, that is one but recently formed from an
erythroblast, would be expected to have an almost spherical,
relatively large and.somewhat:mottled nucleus, whereas in an
older normoblast this nucleus has undergone pyknosis which

both shrinks and distorts it, and no mottling is visible due

to its compactness. Therefore, a certain range had to be allowed
in the size of the nucleus. These differences also may be

observed in color plate XIII. The normoblasts measured were

~55-

those in which the limits of the cytoplasm, whether stained
or unstained, could be definitely determined. These measurements
showed the cell diameter to be quite constant. (See Table II)
No difference was observed in the staining reaction of any of
these cell types as between.normal and anemic animals, so I
do not believe there is any correlation between affinity for
stains and the anemia. '
Perhaps the most difficult cell of all to identify was
the erythroblast. As has already been mentioned in this
general description of cells, a relatively large amount of
variation is to be observed in the cells of this group even
when the staining technique is excellent. To this is to be
added the technical difficulties already described, so it is
evident that the identification of this cell type constituted
a real problem. According to Maximow's color chart, the
earliest erythroblasts should be easily differentiated from
the proerythroblasts by their noticeably smaller size and
lighter cytoplasm. Reference to Table II shows that so far
as could be determined there is no sharp line of demarkation
between them as to size. Cells which I identified (on the basis
of cytological structure plus staining) as proerythroblasts, were
frequently as small as 8.§/¢in diameter, whereas erythroblasts
were frequently as large as 8f~in diameter. The color of the
cytoplasm was no sure criterion because the cells were sometimes
in closely packed clusters and superimposed on each other.
Erythroblasts at the other end of the series should,
theoretically, be easily differentiated from.normoblasts by

the greatly increased amount of eosin in the cytOplams of the

-3 6—

latter. Since this increased affinity for eosin was usually
not evident, and since here too, the size limits are not
sharply marked off, there was only the nucleus to rely upon.
Ordinarily the nucleus of the erythroblast showed a marked,

if dense, reticulation with blotches of massed chromatin
scattered through this reticulation. I have already referred
to it as being of a mottled appearance. Such cells were

easily identified. Occasionally cells were feund in which the
nucleus had begun to condense and therefore to stain more
deeply and.more homogeneously. In such a borderline case it
was a choice between calling the cell an erythroblast or a
normoblast and some guesswork was inevitable. Color plate XIV
shows the different types of erythroblasts observed. Errors

in identification were probably about as frequent in the counts
of the normal.mice as in those for the anenics, so that the
distribution of cell types in one kind of mouse can be compared
with the distribution for the other.

The smaller proerythroblasts were semetrmes hard to tell
from.erythroblasts, due to similarity in size and in nuclear
structure, but ordinarily this type was not hard to identify.
(See Plate XV)

Hemocytoblasts were the least numerous of all the cell
types. In fact they were rare. According to Maximow they
should be unlike any of these other cells. IMany hemocytoblasts
were observed, however, having an irregular cytoplasmic outline

similar to that of the megakaryocytes. See Plate XVII.

Hemocytoblasts can be confused with small:mononucleated.megakaryocytes,
but on the basis of the fact that most of the megakaryocytes have
multilobed nuclei and are of a.muoh larger size ordinarily, all
cells of this type having but a single nucleus and being under
about 14 in diameter were classed as hemocytoblasts.

In summary then, it may be said thatzas to diameter there is
a continuous series of types which blend into each other, from the
normoblasts up to the megakaryocytes; as to staining reactions,
all of these cells did not react in a typical fashion and do
not clearly demonstrate the progressive changes from the
intensely basic hemocytoblasts to the intensely acidic
erythrocytes; and as to structure,:msny deviations from the

type descriptions were observed.

-38-
PRESENTATION OF THE DATA

The counts for the five normal animals studied are
shown in Tables III, IV, Va, Vb, Vc, VI, and VII. Each
horizontal column in these tables represents the count in
one high power field, and shows the numbers of the five types of
cells classified. Fer example; reference to Table III will show
that field No.1 within low power area I contained 66 normoblasts,
46 erythroblasts, 5 proerythroblasts, no hemocytoblasts, and l
megakaryocyte - a total of 116 hanopoietic cells. Reading the
totals at the bottom.of each vertical column, we see that the
sixteen fields counted in mouse No.18 yielded 802 normoblasts,
575 erythroblasts, dl proerythroblasts, 5 hemocytoblasts, and
27 megakaryocytes, or a total of 1450 cells. The last horizontal
column shows that of these 1450 cells, 55.31% were normoblasts,
59.65% were erythroblasts, 2.82% were proerythroblasts, 0.34%
were hanocytoblasts, and 1.86% were megakaryocytes. The counts
of normal animals are summarized in Table XIII. It will be
recalled that sixteen fields were used for four of these
animals, but that fortybeight fields were counted for mouse No.20.
To make the cell numbers for No.20 comparable with those of the
other mice, the totals for the forty-eight fields were divided
by three and incorporated in Table XIII. The percentage which
eadh class of cells comprised of the total normal count was
obtained and its probable error computed. (See last vertical
column.)

Tables VIII, IXa, IXb, IXc, X, XI and XII present in

exactly the same way the counts made of anemic mice. Table XIV

summarizes these counts for the enemies and is comparable to Table XIII.

 

 

-39-

-Table XV shows the differences in the percentages of each
type of cell for normals and enemies. The probable error of
these differences was computed, and its statistical significance
is indicated. (See the last vertical column)

Tables I and II have already been explained.

-40-
DISCUSSION or THE DATA

Table XV shows the final results of this study. The
first thing which attracts one's attention is the difference
in the relative number of normoblasts present in the enemies and
the normals. The normoblasts of the normals comprised 52.51 .t
.425% of the total hemopoietic cell content, whereas in the
enemies they comprised only 42.371.4l8%. The difference is
10.141 .6%. This difference is seventeen times the size of
its probable error, which shows that so far as these data go
it is not due to chance and is certainly significant.

It is interesting to note further that there is also a
significant difference observable in the relative proportions
of erythroblasts. This type comprises only 40.89 1.418% of the
total in the normal and 50.31I.425% in the enemies. The
difference is 9.421,6%, which is'sixteen times its probable
error. This difference is also unquestionably significant so
far as these data go.

The difference between the relative proportions of
proerythroblasts is not so great. There are 1.15% more
proerythroblasts present in anenics than in normals. The
difference is 4.2 times its probable error in this case, which
is probably significant, yet the difference here is not nearly
as marked as it is between the first two cell types.

As to the proportions of hemocytoblasts and megakaryocytes,
there was no significant difference as between normals and
enemies. The differences between them are probably due to

chance. The hemocytoblasts comprised less than 1% and the

-4l-

megakaryocytes only slightly more than 1% of either the normal
or anemic totals.

It would seem then that the deficiency of normoblasts and
the excess of erythroblasts and proerythroblasts in these
anemic animals at birth are the most significant facts that
these data present.

Two possible explanations of the lack of normoblasts in
the enemies at once present themselves. first: it might be
due to some factor which was destroying the normoblasts in the
anemic animals with the result that fewer erythrocytes were
formed; or, second: it might be that the development of the
blood was retarded in the enemies so that they were born in
a more immature condition than the normals, and hence have a
lower blood count.

How do the facts support or discredit these theories?
were the normoblasts undergoing destruction? I am inclined to
think they were not. In the first place there was no visible
evidence of any destructive processes in the liver,- no
degenerative changes, pyknosis, or dissolution of tissue.

If these cells, manufactured in the liver, were destroyed,

it would probably be within the same organ, as very few
normoblasts were found in the circulating blood. Most of

them become mature erythrocytes before even entering the blood
stream. They are incapable of locomotion by themselves, so they
remain in the liver sinusoids until carried away as erythrocytes.
Furthermore, it is quite possible that any destructive agent
would have some effect on other cells in the vicinity, but

nothing of this kind was observable histologically, nor was

there any statistical evidence of it, for the total of all cells
was about the same for enemies as for normals (6296 to 6240).

It might be suggested that the destruction occurred Just
after the erythrocytes enter the blood stream, which would then
account for the anemia found by Mixter in his work. In that
case the hemOpoietic centers would contain a full quota of
normoblasts and a study of liver tissue only would not show any
difference between normals and enemies - only erythrocytic
counts would detect the anemia. Such is not the case. The
_blood showed a deficiency of erythrocytes, and the liver a lack
of normoblasts.

Let us consider the second theory. Were these two types
of animals equally developed at birth? The greater body size
of the new-born normals would indicate a difference in prenatal
growth-rate. This theory is supported by the contemporary work
being done by A. A. Andrews. If we may postulate a differential
growth-rate, then, it would be quite logical to interpret“"if;(Emiliamon
the result of retarded blood development. Blood is a tissue.
Growth in blood tissue would be expressed in terms of the
numerical increase in its cells, and would have to be initiated
by factors operating on the hemopoietic organs. Let us see
what the evidence is in support of this theory. In the first
place we know that ordinarily the hemopoietic function of the
liver is lost shortly after birth and this role is assumed by
the bone marrow. The [cunts of the normal animals would seem
to indicate that hemopoiesis is in process of being concluded,

because in these animals the cells of the generations preceding

the normoblasts are in a minority. Although a.great many

-43-

normmblasts are still present, their source (the erythroblasts)
is becoming exhausted and no new supply of the earlier stem
cells is being proliferated to take their place.

If the normal animals contained just as many erythroblasts
and proerythroblasts as did the enemies per unit area, then we
might be justified in supposing the deficiency of normoblasts
in the anemics to be due to destruction. Since, however, the
normals have fewer erythroblasts and proerythroblasts as well
as more normoblasts, it seems logical to conclude that a.majority
of the erythroblasts in these normals has but recently transformed
into normoblasts, and the number of erythroblasts is depleted
due to their production having practically ceased. If it is
proved that development is retarded in these flexed-tailed
animals, then the deficiency of normoblasts could very well
be interpreted as meaning that the proliferation and
differentiation of homepoietic cells in the liver has not yet
reached its postnatal climax in these retarded flexed-tailed
animals but is still in the ascendancy. In normals of the same
age, on the other hand, the process (due to more rapid growth)
has reached its climax and is on a descendency leading
eventually to complete cessation. If this is the case then
the so-called anemia with which we are dealing is not a
pathological anemia, i.e., it is not a condition due either to
loss of red blood cells or to an inability of the animal to
‘manufacture them. These animals would then be anemic in the
sense that a sixteen day embryo would be "anemic" - i.e., have

less blood cells and less hemoglobin - if compared with a twenty

-44-

day embryo in which blood volumn and hemoglobin perdentage
has greatly increased.

One more fact of interest should be brought out. Mr. Mixter
found (see Table I) that the erythrocytic count in enemies at
birth (5,520,000) is approximately 70% of the erythrocytic
count in normals at the same age (4,880,000). In other words,
there is a deficiency of about 50%k I have shown that
according to these data the normoblast count in the anemic's
liver at birth (2668) is approximately 80% of the normoblast
count in normals (5277) - or is about 20% deficient. This
indicates that some relationship undoubtedly exists between
the production of normoblasts and the anemia. Perhaps the
50% deficiency he noted would be entirely accounted for were
my work based upon counts cf'more individuals.

The data seem to support our second theory. However, the
theory can?:: accepted as definitely proved on the basis of
these findings alone. Studies should be made of both the
normals and enemies at different age levels preceding and
succeeding the one I have used. This investigation could be
carried out in the manner described in this paper by making
quantitative studies of hemopoietic cells in the liver,
beginning with the embryonic period at which this process is
initiated and continuing into postnatal life as long as any
signs of hemopoiesis are visible in the liver.

The only step in this direction which I have taken is
the examination of a seventeeneday-old anemic animal. As may

be seen from Plate XII, hemopoiesis in the liver has stopped.

-45-

Mixter's data also (Table I) show that the anemia has disappeared
by that time. This fragmentary evidence, inadequate as it is,
supports our theory that hemopoiesis in enemies is merely retarded,
but that it runs its course eventually as in normal animals.

However, it must be admitted that the weakest part of this
work is the insufficiency in the number of animals studied. A
study of Tables Va, Vb, and Vc (normals) and of IXa, IXb, 110
(enemies) shows how'much variation.may be found in the widely
separated areas of the same liver. Also, much variation was
encountered among different mice of the same type. Some of
this variation is admittedly due to inaccurate classification
of some of the cells, but much of it is undoubtedly attributed
to intrinsic difference in the animals. Greater accuracy might
have been attained had.more cells been counted in each animal,
as was done in two of the cases. This should have been done to
several.more animals of each type. Although these data offer
clear-cut points of difference between flexed and normal
animals, the possibility still exists, due to the small number
of animals used, that these differences are due to a chance
selection of animals.

Furthermore, fixed sections of tissues, although valuable
in any histological study, do not always give a true picture
of the cytology of the tissues being studied. Wbrk of this
type should undoubtedly be supplemented by observations on the
living cells, using the methods of supravital staining and
tissue cultures. Such methods might enable one to identify

cells of the hemoPoietic series with greater certainty.

 

 

 

LI?! .

a1. 3.1.1.,

.t

 

-45-
CON CLUS IONS

l. Hemopoiesis occurs in the liver of’mics less than a day old.

8. Although fixed and stained sections of tissue may be used
for such a study, other methods (such as supravital and intravital
staining, and tissue cultures) should be used to supplement this
one.

3. The livers of anemic animals contain only 80% as‘many
normoblasts as do the livers of normals.

4. The livers of normal animals contain only'80% as many
erythroblasts and proerythroblasts as do the livers of the anemics.

5. This difference in the percentages of normoblasts,
erythroblasts and proerythroblasts in these two types at birth
is interpreted as indicating that hemopoiesis is being concluded
in the normals but is delayed in the anemics.

6. These data are significant as far as they go, but they
are insufficient. Mere animals should be studied and a more
complete analysis of the condition.made of the animals at

birth, and at preceding and succeeding ages.

-47-

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~48-

Table II

Distribution of Sizes of the Different Cells
of the Erythrocytic Genetic Lineage Based Upon Absolute Measurements.

Diameter of
cell
(in.micra)

4.5 -
5.0 -
5.5 -
6.0 -

6.5 "

8.5

9.5 '-

4.9
5.4
5.9
6.4
7.4
8.4
9.4

10.4

10.5 - 11.4

11.5 - 1204

1305 "

13.4

13.5 - 14.4

14.5 - 15.4

15.5 - 2004

20.5 -

25.5

30.5

5595 "

25.4
30.4
35.4

40.4

No.

Nbl

3
10
50

6

l

of No. of No. of No. of No. of

Ebl Pbl Hbl iMQg

35

so

15* 25*
18
21

18 5

30
52

10

 

Total No. of Cells
Measured.

50

5*“ “/5

80 80 25 80

*All'but one of these 88 proerythroblasts measured 8.4u'which was
practically the:minimum.size of this type. NOne of the erythroblasts
measured over 8;».
be in the table.

This overlapping is not as large as it seems to

-49-

Table III

Count of Blood Cells in Mouse No.12 (Normal)

 

Field Nb1 Ebl Pbl Bbl Meg Total

No!

I 1 66 46 3 O 1 116
2 57 38 3 0 0 98
3 6'7 52 2 0 1 122
4 35 28 2 0 3 68
5 64 48 1 0 0 113
6 32 20 0 0 2 54
7 34 33 3 1 2 '73
8 51 45 4 2 1 103

II 9 6'7 39 4 2 2 114
10 45 45 2 0 1 93
11 42 43 3 0 0 88
12 68 36 1 0 3 108
13 50 38 4 0 1 93
14 46 27 4 O 4 81
15 40 20 3 0 4 67
16 38 17 2 0 2 59

m
16 802 575 41 5 27 1450

fof total W m

I'Key to abbreviations used in Tables III to IV inclusive:
Nbl - normoblasts; Ebl - erythroblasts; Pbl - proerythroblasts;
Hbl - hemcytoblasts; Meg - megakaryocytes.

Table IV

Count of Blood Cells in Mouse No.13 (Normal)

Field Nbl Ebl Pbl Hbl Meg Total

 

2 59 4o 5 o 5 as
5 57 57 7 o 1 102
4 35 52 s o 5_ 75
5 14 32 1 1 1 49
6 4o 26 2 o 1 59
7 so 55 5 o o 68
e 67 45 2 o o 114
II 9 48 53 5 o 2 as
10 54 20 ‘ o o 1 55
11 50 25 5 o 1 so
12 28 44 7 o o 79
15 51 59 5 o o 75
14 36 34 2 o 1 75
15 40 55 5 o 0 so
15 27 22 5 o o 54

W
16 598 532 56 2 17 1205

ifbr total *”
count 49 o 62% 4.4. 14% 4o 64% 0. 16% 1.41%

-51-

Table Va

Count of Blood Cells in Mouse No.20 (Normal)

 

11 old Nbl Eb]. Pbl Hbl Meg Total
Hm

I 1 51 17 4 O O 72

8 48 15 5 l 1 70

3 27 26 11 1 0 65

4 50 55 7 O 2 114

5 46 25 5 2 3 81

6 28 51 3 O 0 82

7 58 36 8 0 5 107

II 8 51 30 4 1 1 .7

9 34 23 4 0 0 61

10 63 53 5 0 1 122

11 35 4O 7 0 6 88

13 44 2.6 4 O l 75

13 30 25 0 1 1 57

14 36 40 2 0 3 81

III 15 50 13 0 1 0 64

16 38 11 2 0 O 45
m

16 683 486 71 7 84 1271

fl 2 of total

0011115 53. 73% as.“ 5.50% 0.55% 1.88%

-52-

Table Vb

Count of Blood Cells in Mouse No.20 (Con't.)

 

 

Field Nbl Ebl Pbl Hbl Meg Total
I 1 50 38 7 0 0 95
2 43 34 4 0 0 81
3 60 45 5 0 0 110
4 65 48 3 0 1 117
5 3O 45 3 0 1 79
6 40 47 7 O 0 94
7 14 26 6 0 1 47
8 65 46 13 0 0 114
9 34 36 3 2 0 75
10 25 34 3 0 1 63
II 11 36 36 5 0 1 78
12 37 32 3 0 1 73
13 58 41 1 0 2 102
14 68 35 8 0 2 113
15 72 4O 3 1 1 117
16 50 5O 4 0 1 105
W
16 747 633 78 3 12 1473
f of total A I ‘5

count 50. 71% 42.97% 5. 29% 00% 0.81%

Table Vb

Count of Blood Cells in Mouse No.20 (Con't.)

 

 

r1614 Nbl 5:51 Pbl Hbl Meg Total
I 1 50 38 7 o o 95
2 43 34 4 o o 81
3 6o 45 5 o o 110
4 65 4s 3 o 1 117
5 so 45 3 o 1 79
6 4o 47 7 o o 94
7 14 26 6 o 1 47
s 65 46 13 o o 114
9 34 36 3 2 o 75
10 25 34 3 o 1 63
II 11 36 36 5 o 1 7s
12 37 32 3 o 1 73
13 58 41 1 o 2 102
14 68 35 s o 2 113
15 72 4o 3 1 1 117
16 50 50 4 o 1 105
W
16 747 633 78 3 12 1473
i 0:: total I

count 50.71% 42.97% 5.29% 0.20% 0.81%

-53..

Table Vc

Count of Blood Cells in Mouse No.20 (Con'd.)

 

 

 

 

 

Field Nbl Ebl Pbl Hbl Meg Total
=h;-°.-.’==: 1 L ha: ~~~ ‘
I 1 60 41 5 O l 107
2 62 34 l l l 99
3 12 24 3 O 0 39
4 47 41 7 O 2 97
5 42 48 7 0 l 98
6 43 48 3 O l 89
7 48 6O 10 2 2 122
8 73 72 9 0 3 157
II 9 24 40 9 O 2 75
10 56 38 4 1 1 100
11 26 4O 8 l O 75
12 21 45 5 O 0 71
13 49 53 6 0 O 108
III 14 38 14 1 0 0 53
13 27 13 O O 0 40
16 11 10 1 O O 22
W
16 639 615 79 5 14 1352
f of total 1

count 47.26% 45.48% 5.84% 0.36% 1.03%

Table VI

Count of Blood Cells in Mouse No.23 (Nomal)

 

 

F1011 Nb1 Ebl Pbl ‘Hbl II!‘ Total
I 1 64 45 5 0 3 117
2 57 52 4 0 1 114
3 35 26 3 0 O 64
4 56 43 7 1 1 108
5 50 23 4 0 2 79
6 57 36 3 0 3 101
7 57 44 7 0 1 109
II 8 56 42 2 0 O 100
9 29 32 2 O 3 66
10 11 16 2 0 O 29
11 41 35 3 0 0 79
12 3O 37 3 2 O 72
13 32 4O 4 0 3 79
III 14 3O 31 5 0 0 66
15 37 11 2 0 1 51
16 52 34 4 0 1 91
[mull .i , .
16 694 5" 62 3 19 1325
T{fo‘ftotel I W I

001313 53.37% 41. 28% ‘0 68% 0e 22% 1.43%

_l 1‘

-55-

Table VII

Count of Blood Cells 1n.Mbuse No.26 (NOrmal)

 

 

 

 

rield Nbl Eb]. Pbl Hbl Meg Total
NO.
I 1 36 22 6 2 O 66
2 31 29 3 O 2 65
3 35 24 2 1 0 62
4 28 15 8 1 O 52
5 4O 11 4 O O 55
6 32 3O 9 1 1 73
7 15 14 3 0 2 34
II 8 37 19 4 O 0 6O
9 25 20 6 1 0 52
10 27 17 8 O O 52
11 28 32 3 O O 63
12 39 27 4 0 O 70
13 33 15 5 0 O 53
14 26 18 2 0 1 47
III 15 42 15 1 0 O 58
16 19 12 1 0 0 32
W
16 493 320 69 6 6 894
$50: total

~56-

Table VIII

Counts of Blood Cells in Mouse No.17A (Ananio)

 

 

 

 

 

 

Held Nb1 Ebl Pbl Hbl 1163 Total
332:» 1 I
I 1 21 35 5 O O 61
2 35 45 9 O 2 89
3 7O 42 7 0 O 119
4 20 42 7 O 0 69
5 49 47 8 0 0 104
6 52 47 5 1 O 105
7 38 45 8 0 0 91
8 39 46 9 O 1 95
9 27 36 7 0 2 72
10 32 43 5 0 0 80
11 51 36 4 0 3 94
II 12 28 59 8 0 0 95
13 32 37 3 0 1 73
14 29 5O 3 O 0 82
15 63 6O 8 0 0 131
16 37 46 4 O 0 87
W
16 623 714 100 1 9 1447
% or total
count 43.05% 49.34% 6.91% 0.07% 0.62%

wu.~—- .1”.- . ~
I . ‘ ." -.. .

-57-

Table IKE

Count of Blood Cells of’Mouse No.18A. (Anemic)

 

 

 

 

 

Yield Nbl Ebl Pbl Hbl Meg Total
No.

I 1 14 29 3 0 0 46
2 44 26 6 0 l 77

3 27 40 4 0 O 71

4 11 8 3 0 0 22

5 8 12 3 0 2 25

6 6 20 0 0 0 26

7 47 16 0 0 0 63

II 8 27 30 3 O 2 62
9 11 37 2 1 2 53

10 28 32 3 0 1 64

11 24 37 1 0 2 64

18 36 14 5 0 0 55

13 21 39 3 0 l 64

14 21 33 3 0 1 58
III 15 35 43 l 0 4 83
16 30 34 6 1 l 72

16 390 450 46 2 17 905

%'of total

count 43.09% 49.72% 5.08% 0.22% 1.87%

Table IXb

Count of Blood Cells of Mouse No.18A. (Con't.)

Field

 

Nbl

Ebl

Pbl

 

Hbl

Meg

 

 

Tbtal

 

2 28 27 7 0 2 64
3 19 37 8 0 0 64
4 15 42 6 0 2 65
5 35 56 4 o 1 96
6 23 40 8 0 1 72
7 31 38 9 1 0 79
II 8 21 23 5 0 o 49
9 25 26 3 0 1 55
10 40 37 5 1 1 84
11 17 55 9 0 1 82
12 30 67 7 0 0 104
13 15 40 6 0 1 62
14 16 38 3 0 2 59
15 29 25 4 0 0 58
III 16 33 35 5 0 o 73
WY * :=====:=====
16 421 630 94 3 12 1160
%“62 total 5‘ 5
count 36.29% 54.31% 8.1% 0. 25% 1.03%

“‘7’: F "in

~58-

Table IXb

Count of Blood Cells of Mouse No.18A (Con't.)

Field

 

Ebl

Pbl

Hbl

Meg

 

 

Tbtal

 

 

2 28 27 7 0 2 64
3 19 37 8 0 0 64
4 15 42 6 O 2 65
5 35 56 4 0 1 96
6 23 40 8 0 1 72
7 31 38 9 1 0 79
II 8 21 23 5 0 0 49
9 25 26 3 0 1 55
10 4O 37 5 1 1 84
11 17 55 9 0 1 82
12 30 67 7 0 0 104
13 15 4O 6 0 1 62
14 16 38 3 0 2 59
15 29 25 4 0 0 58
III 16 33 35 5 0 0 73
:======================================51 —~ :=============
16 421 630 94 3 12 1160
% of total
count 36.29% 54.31% 8.1% 0.25% 1.03%

3L.“ ‘1: H‘

Table 1X0

Count of Blood 06118 of Mouse no.181 (Con'd.)

Field Hbl Ebl Pbl Hbl Meg Total

 

 

 

2 16 23 6 0 1 46

3 23 33 4 1 0 61

4 50 40 4 0 0 94

5 30 35 3 0 1 69

6 55 35 l 0 2 93

7 52 47 5 0 0 104

8 60 55 4 0 4 123

II 9 43 46 6 0 0 95

10 46 58 8 0 0 112

11 42 47 4 0 l 94

12 31 44 5 0 2 82

13 49 33 1 1 1 85

14 32 27 6 0 0 65

15 23 25 3 0 3 54

16 34 44 5 0 0 83

16 626 625 69 3 15 1338
{of Yam "‘ ‘ I

count 46.78% 46.71% 5.15% 0.22% 1.12%

”w. o‘ “a“. sane

~60-

Tablo I

Count of Blood Cells of Mouse No.214 (Anemic)

 

Field Nbl Ebl Pbl .Hbl LMeg Total
No: . -
I 1 50 45 O O 1 96
2 33 38 1 O 3 75
3 82 46 O 0 1 129
4 43 27 2 0 1 73
5 33 41 3 1 1 79
6 42 34 2 0 0 78
II 7 18 41 3 0 0 62
8 19 47 2 O 1 69
9 28 43 4 O 0 75
10 34 4O 3 O 1 78
11 39 34 3 O 1 77
12 28 33 4 0 0 65
13 35 22 2 1 4 64
14 38 37 3 O 3 81
15 25 46 1 O O 72
16 19 32 3 O 0 54
W
.16 566 606 36 2 17 1227
76 of total _1 V

count 460 12% 49.38% 809336 0.16% 1.38%

 

-61.

Table II

Count of Blood Cells of Mouse 22A (Ananic)

 

 

 

11614 Nbl n1 Pbl Hbl Meg Total
I 1 26 43 6 0 1 76
2 36 32 8 1 0 77
3 35 48 6 0 1 90
4 29 37 5 0 1 72
5 30 40 2 0 o 72
6 35 37 7 0 3 82
7 51 30 4 0 1 86
II 8 12 42 7 0 2 63
9 27 40 8 0 1 76
10 26 48 6 0 4 84
11 36 35 5 o 1 77
12 45 49 7 0 2 103
13 28 40 6 0 1 75
14 22 37 5 I 0 65
15 38 42 6 0 0 86
16 57 49 5 0 0 111
mfifi? WfiU—fi 1- 1 J “L
16 533 649 93 2 18 1295
%TBI total ' " '55“ “5 "*
count 41.15% 50.11% 7.18% 0.15% 1.38%

-52-

Table.III

Count of Blood 0811. of Mouse No.24A (In-010)

 

 

 

Field Nbl 351 Pbl Hbl Meg Total
No!
I 1 25 29 3 0 1 58
2 56 32 3 0 0 71
3 22 48 6 0 0 76
4 31 34 5 0 0 70
5 28 43 8 0 2 81
6 23 46 5 0 1 75
7 31 29 9 0 0 69
8 33 42 3 0 0 78
9 44 50 4 o 0 98
II 10 35 52 5 0 0 92
11 31 36 6 0 0 73
12 9 29 5 0 0 43
13 19 36 3 1 0 59
14 54 42 7 0 0 103
15 26 35 5 1 3 70
16 20 47 3 1 5 76
16 467 630 80 3 12 1192
% 6:261:11“ ‘ 5'5 A 5'

count 39.17% 52.85% 60 71% O. 25% 1.01%

 

 

 

 

 

 

 

 

 

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Table 1!

Difference Between Total Percentages of the Different
Cell Types for Nomsls and Anemics
(Sumnary of Tables XIII and XIV)

Cell 76 of Total % of Total Difference No. of times
Type Normal Count Ananic Count between difference
. Percents-es exceeds P.E

     

 

 

 

 

 

Nbl 52.51% 1: .43% 42.37% 45.42% 10.14% 1.60% 17.0
E’bl 40.89% 1.42% 50.31% :.43% 9.42% 11.60% 16.0
Pbl 4.87% 1.18% 6.02% :.20% 1.15% 1.27% 4.3
Hbl 0.33% I .05% 0.17% 15.03% 0.16% 1.06% 2.6

 

Meg 1.37% t .09% 1.12% 32.09% 0.25% 1.13% 2.0

 

-66-

BIBLIOGRAPHY

l. Minter, Russell, ”The Blood of the Flexed Tailed Mouse with
Some Observations on the Effects of Inbreeding on
the Flexed Tailed Strain.” Thesis for the Degree
of“Master of Science. Michigan State College, 1930.

2. Kindred and Corey, ”Studies On the Blood of the Fetal
Albino Rat.” Anatomdcal Record, 761.47, Nov.,1930,
pp. 21.3 & ff.

3. Karsner, Howard T., ”Human Pathology”. Chapter XIV,
pp.485~524. "The Hematopoietit System."
J.B.Lippencott &.Co., 1929.

4. Evans, Frank A., "Pernicious Anemia". Williams and Williams
Company, 1926.

5. Strong, 0.8. and Elwyn, A., "Bailey's Text Book of Histology."
Seventh Edition. Chapter IV, pp.125-l43.
William.Wbod & Company, 1928.

6. Maximow, Alexander 3., "Relation of Blood Cells to Connective
Tissues and Endothelium." Physiological Review.
V01. IV, October , 1924 , Pp0535-5630

7. mathow, A. and Bloom, William, "A Text-Boot of Histology."
Chapter III, pp.48-71, Chapter V, PP.99-l50,
W.B.Saunders 00., 1930.

8. now, 00A... Cunningham, R.S., and 8813111, FOR.’
"Experimental Studies on the Origin and'Maturation of
Avian and.Mammalian Red Blood Cells." Contributions
to Embryology No.83, pp.l65-226. Carnegie Institute
of washington, January, 1925.

9. IMedlar, "An Interpretation of the Nature of Hodgkin's
Disease.” American Journal of Pathology, 701.7II,
pp.499 & ff., September, 1931.

10. Jordan, 3.3., "varieties and the Significance of Giant Cells."
Anatomical Record. Vol.30-3l, September, 1925,
PP . 51-65 0

ll. Foa, P., 1899, Ziegler's Beitrage zur Path. Anat.,
Bd. 25, S. '76.

12. Pugliese, A. 1897. 'Uber die physiologieche Rollo der
Riesenzellen.” Fortschr. der Med., Bd.15, 8.729.

13. Denys, J. 1866, "La cytodierese des cellules geantes et des
petite cellules incolores de la moelle des as."
La Cellule. T.2, p.245.

BIBIIOGRAPHY (Continued)

14. Sabin, Florence R., "On The Origin of the Cells of the Blood.”
Physiological Review, Vbl.II, No.1, Jaiuary 1922,
pp. 38-69 0

15. Arey, leslie 3., "Developmental Anatomy." Chapter II,
ppo253-259, Second Edition, W.B.Saunders Co., 1931.

16. Bunting, C.H., "The Leucocytes" Physiological Revhews,
VOleII’ No.4, OCtOber, 1922’ pp.505-520.

l7. deAberle, S. B., ”A Study of the Hereditary Anemia of.Mice."
American lburnal of Anatomy, vo1.4o, Nevember 1927,
pp.219-247.

18. McClung, C. I.. "Handbook of Microscopical Technique."
Pan]. Be Hoeber’ 1110., 1929.

EXPLANATION OF THE FOLLOWING PLATES

The first twelve plates are microphotographs of various
tissues and organs of the normal and anemic animals at birth
(except Plate III which is the liver of a l7-day old anemic
mouse). These photographs were taken with a Leitz
microphotcgraph camera. JMsgnification in all cases was
x450. Plate 711 is an enlargement of the picture used for
Plate V, and is intended to bring out the details of cell
structure.

Plates 1111 to XVI inclusive are free-hand illustrations
done with colored drawing pencil by the author. They are
drawn approximately to scale (11.9ptto the inch).
IMagnification was in all cases 1600. These plates are to
show the various staining reactions observed in the five

groups of hemopoietic cells.

KEY '10 ABBREVIATIONS USED IN PLATE I TO XII INC.

0 - cartilage

c.v. - a central vein

ebl - erythroblasts

ebl' - erythroblasts in mitosos

ecy - erythrofiytes

ecy” - unstained or faintly stained erythrocytes

hbl - hemocytoblasts

L - cord of liver cells

lcy - lymphocytes

lcy' - lymphocytes in mitosis

L.n. - nucleus of a liver cell

meg - megakaryocyte

nbl - normoblast

nbl" - normoblast with unstained or faintly
stained cytoplasm

obl - osteoblasts

pbl - proerythroblasts

P. 1. - polymorphous leucocytes

v-vein

ebl
nbl

nbl

C e 'e

pbl
obl

 

Liver of send me (No.18) at birth.
Note the predeninenee of amounts. (Aeid eosin-h-etomu stein)

 

Plate 11

Same as above but ensuing e different eree.
Note the difference in eiee of ”noblest nuclei.

-11-

hbl

 

Plate III
Liver of e send me (3.”) at birth.
late the elutere of eel-oblate. (anemia-mime etein)

ebl obl

/ nbl.“
ebl

pbl

nbl

 

Plat. IV

fine as above but a different eree.

Note the We of erythrobleete. mere with Pietee 1.11.111"

 

Lieu: of en we we (loan) at birth.
”marked predninenee of Wee ever W.
ampere eith Phtee I end 11. (antenna-eosin etein)

 

P13“ 71

S-euebovebntedifferentfield.

ebl

 

Plate '11

Liver of en ell-1e we (1mm) at birth.

(is Wt of Plate 7)
late the mrphelea of the liver at this ego.

 

 

mete VIII
hemmtheMefeeenelleue (lea?)
late the dieterted mhreeytee inter edudllneteeyiin—uin-eme).

 

M11

haemfrcribefemel-eeee (Item).
lete the intneeiy eteined nor-oblate end errthretleete.
(Hamlin-“meme stein)

 

Plate!
nineteen-ulna (hum) et birth.
“themattheeelh.theirdueeetehiu.ud
hemlitetie rim. (_Wil lid!)

 

Plate 11

x-eeetieeefebieedveeeeiefeeeneineuee(le.w)etbirth.
letetheeteinedeedeeeteined erythrocytee. (kWh-ewe).

L.n.

.e'e

 

mum

Liver of en ell-it nee (1mm) 17 due old.
Note the ehereeterietie wee of the liver eerde. end the
ebeeeeeefh-epeietieeene. Oqeretheeppeereeeeefthie
liver with theee at birth. (Pietee I - m ine.)
(Acid eosin-humus stein)

- viii -

 

 

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Plate XIII
I‘IOR‘IIOBLASTS

Groups A, B, C, and D: Cells stained with hematoxylin-eosin-azure.
Group A: Cells of average size showing the faintly basic-staining
cytoplasm, and dense, homogeneous nuclei. No.1 - in mitosis.
No.2 - shows a faint trace of pink in the cytoplasm.

Group B: Large sized younger normoblasts; somewhat mottled nuclei.
Group C: Extruded nuclei.
Group D: Cells with unstained cytOplasm.

Group E: Cells stained with acid eosin-hematoxylin. No.1 - shows
the nucleus being extruded.

 

 

 

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1' FA. 4.

 

- ix -

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Plate XIV
ERYTHROBLASTS
Nos. 1,2, and 3 -

Group.A: Stained with hematoxylin-eosin-azure.
No. 3 - shows a pinkish tint in

average sized erythroblasts.
No.4 - small sized erythroblast; compare this

the cytoplasm.
with normoblasts (Plate XIII). No.5 - large erythroblast.
Compare No.1 and Yo.

Group B: Stained with acid eosin-hematoxylin.

2 with normoblasts.

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PlateiXV
PROERYTHROBLASTS

No. 1 -.Average sized proerythroblast.
No.2 and No. 3 - Large proerythroblasts.
No.3 - In.mitosis.

Hematoxylin-eosin-azure stain.

“ _... _.—

 

 

Plate m

HIEMOCY‘I'OBIASTS

A pair of average sized hemocytoblasts.
Note the irregular extensions of the cytoplasm.

Hematozylin-eosin—azure stain.

 

 

Plate m

HDJOC'YTOBIASTS

A pair of average sized hemocytoblasts.
Note the irregular extensions of the cytoplamm.

Hematoxylin-eosin-azure stain.

Os.

 

V—f— - ._..-..-, an- . v

 

 

 

-xii -

 

 

 

Plate XVII

A MEMKARYOCYTE
An average sized cell ( about 25/uin diameter ). Note
the multi-lobed nucleus.

Hanatoxylin-eosin-azure stain.

 

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