31-l             JOHNS HOPKINS HOSPITAL BULLETIN           1 so. :m
                                                                           --
              STUDIES ON BLOOD

THE VITALLY STAINABLE GRANULES AS A SPECIFIC CRITERION FOR ERYTHRO-
  BLASTS AND THE DIFFERENTIATION OF ,THE THREE STRAINS OF THE WHITE
        BLOOD-CELLS AS SEEN IN THE LIVING CHICK'S YOLK-SAC

        By FLORESCE 1~. Sam
(From the Department of Anatomy, The Johns Hopkins Einivcrsity)

In 1920 the writer *' published an account of a study of the
origin of the vascular sytem as it can be made out by watching
the living chick blastoderm of the second day of incubation.
The method of the origin of vessels can be made out in such
specimens, in the area pellucida of the yolk-sac, in stages
which range from the time just before the first somite through
the stage of about twenty somites.

In the area pellucida there are three well-known layers, the
ectoderm, the double layer of the mesoderm lining the extra-
embryonal ccrlom, and the endoderm.  The blastoderms are
mounted in a hanging drop preparation, in Locke-Lewis solu-
tion, with the endoderm against the coverslip. The area
pellucida is so thin that the endoderm, the vascular zone
between the endoderm and mesoderm, and the mesoderm can
all be analyzed with an oil immersion lens.  The technique
was described in 1920.

Blood-vessels begin by the differentiation of a new type of
cell from mesoderm. This cell moves out of the mesoderm,
develops a dense, basophilic, azurophilic cytoplasm and be-
comes physically more refractive than mesoderm.  As soon as
this cell divides, it shows one of its essential characteristics,
namely, the tendency of the cells to stay together to form
syncitial masses. These masses of cells put out sprouts by
which they join similar masses to make a plexus.  Both,
while such solid clumps are still isolated, and after the plexus
is formed, the cells become transformed into vessels by a lique-
faction of the central part of the mass to make blood-plasma,
while the periphery differentiates into endothelium.

There is a progressive differentiation of angioblasts, begin-
ning in the periphery of the area vasculosa at the stage of two
somites; the cells gradually appearing nearer and nearer the
embryo, until, at the stage of five or six somites, angioblasts
differentiate in the axial line of the embryo as forerunners
of the endothelium of the heart and aorta. The heart, aorta
and main vessels of the embryo differentiate in situ from
angioblasts and increase by the addition of newly differen-
tiated cells as well as by the cell-division and the sprouting of
their endothelial walls. The amount of the differentiation
of new cells grows less but at what stage it ceases is not known.
Thus there is established the fundamental m.orphology of the
vascular system.

Blood-vessels arise by the development of a new type of
cell, the vasoformative cell of Ranvier, or the angioblast of
His. This cell produces the first fluid of the blood; thus,
endothelium is primary and blood-plasma secondasy.  Since
there is a tissue fluid before angioblasts arise, endothelium is

from the start a membrane between two different fluids, tissue-
fluid and plasma.  Moreover, the process of liquefaction is
intra-cellular, that is, it can be seen in chains of single angio-
blasts which become vessels and it can also be seen to take
place in the sprouts which are processes of cells, hence the
lumen of vessels is embryologically intracellular and thus not
a tissue-space.

In the living chick of the second day, it can be seen that
both angioblasta and the endothelial cells give rise to red
blood-cells. Erythroblasts begin in the chick in the vessels of
the outer margin of the area opaqua in the stages of from `7
to 11 somit.es. In the area pellucida, where they can be seen
in the living sfiecimen, angioblasts diffcrcntiatc during the
stages of from 5 to 11 somites, while the vessels form and
erythroblasts begin during the stage of from 11 to 14 somites.
The heart begins to beat at the stage of 10 somites and the
circulation starts when the chick has 1G to 17 somites.

During the past year, I have been continuing these studies
on the living chick, and have found that it is possible to mount
the entire blastoderm on a large cover-slip, one measuring
42 by XI mm., throughout the third and fourth days of incuba-
tion. The cover-slips must be entirely free from grease or
the membrane will not flatten out on the glass. From the
fifth day on, the chick is too heavy to mount in the hanging-
drop form, but if the specimen be transferred to a dish of
Locke-Lewis solution, the amnion can be opened, the allantois
pushed aside and the yolk-sac cut off close to the embryo and
then spread out and mounted. The circulation, of course,
stops but the membrane can be mounted and kept alive cer-
tainly for from three to five hours. So far, I have studied
these living membranes only through the first seven days of
incubation. It is an advantage to mount the chick with the
membranes because the preparations are all fixed after the
vital studies have been made and many of them are stained
and mounted in toto. If the embryo has been left attached,
it can be cut off in the alcohol and then the entire ectoderm
can be dissected off from the area vasculosa.  The specimen
is thus made thinner and much easier to analyze.  When the
embryo has been cut away at the start, the ectoderm clings too
closely to the specimen to be taken off and makes one more
layer of stained cells in the final specimen.

I have found it a great advantage to study the embryos wit11
a vital dye and add 1 to 3 drops of a 1 per cent aqueous solu-
tion of neutral red to 10 c. c. of the Locke-Lewis solution.
making a dilution of possibly 1 to 10,000 of the dye, or less.
This may be termed the physiological dilution of the dye.


()CTOBER, 19211

JOHNS HOPKINS HOSPITAL BULLETIN

After the specimens have been studied, they are fixed by
lloating the cover-slip, embryo down, on Bouin's picro-form01
and then keeping them in 70 per cent alcohol until all the
picric acid is removed. They are then stained in haematoxylin
;lml counterstained in eosin with a little orange G.  This fixa-
tion is excellent for the granulocgtes, but quite worthless for
the erythrocytes after the primitive stage. After fixation in
12ouin's solution the young erythrocytes show only a wide-
meshed. reticulation having no relation whatever to `any of
the substances that can be made out in the living cell. NO
fixation of the blastoderms is adequate to follow the changes
in the red cells, but they can be identified best after fixation in
the vapor of formalin if not applied tod long. I use it for
from ten minutes to half an hour. In this the hemoglobin is
well preserved but not the basophilic cytoplasm and indeed
the earliest traces of hemoglobin which can be seen in the
liring cell by a distinct yellow color cannot be detected in
the fixed specimens. Helly's fluid, which preserves the baso-
philic substance better, cannot be used, because the blastoderms
float off from the cover-slip almost immediately and wrinkle
so that they can never be studied as a section with an oil
immersion lens.

In connection with the study of these blastoderms, a drop
of blood is drawn from the vessels when the egg is first opened
and used for a film.  These films of blood are studied vitally
1)~ Pappenheim's method. They are made as follows : A clean
glass rod is dipped into a dye and drawn across a perfectly
clean glass slide. The two dyes which I have used are neutral
red and brilliant cresyl blue, made up either in a 1 per cent
aqueous solution or in a saturated alcoholic solution.  The
even film of the stain dries quickly and its strength is esti-
mated by the color. The film must not be dense enough to
stain any nuclei. A drop of blood is then drawn out with a
fine glass cannula, placed on a cover-slip which is then in-
verted on the film of stain, ringed at once with salvoline and
placed in a.warm box.  The vital stains develop slowly, qn an
average, in ten minutes, and if permanent preparations are
wanted, the specimen is watched under an oil immersion lens
until the staining is best, then the cover is drawn off from the
slide and the film of blood counterstained with one of the blood
stains. I have used Wright's eosin-methylene blue.  In blood
from chicks on the second and third days of incubation the
amount of the specific substance of the red cells, that is, the
megaloblasts, stainable in the vital dye is so massive, that it
is necessary to differentiate the specimens after staining by
Wright's method. This can be done in absolute alcohol and
the decolorizing stopped with xylol. The white cells are so
very unevenly distributed in the vessels of the early blasto-
derms that no film of blood represents adequately the amount
of differentiation for the stage. Hence, each stage must be
studied by both methods, by a survey of the total area pellu-
cida and by drops of blood with specific stains.

In these studies, it can be seen that there are three different
strains of blood-cells, first, those that arise from endothelium,
which include both the red cells and the monocyte strain of
the white cells; secondly, the granulocytes, and thirdly, the

lymphocytes. The red cells begin to differentiate on the
second day of incubation. On the third day the endothelium
gives rise to the monocytes, that is, to the large mononuclears
and the transitional forms of Ehrlich.  In two different
specimens I have seen an occasional monocyte on the second
day, but the process becomes active only on the third day.
The group of the monocytes of the blood is especially well
illustrated by Pappenheim on his Plate 1," as the third row of
cells, and as the fourth and fifth rows on Ferrata's Plate 12."

At the same time that the endothelium gives rise to the
monocytes, namely, beginning on the third day, it gives rise
to a much more numerous extravascular group of cells, iden-
tical with the monocytes, which are the clasmatocytes of the
connective tissues.  On the third day also the granulocytes
begin to differentiate as a new type of cell from the mesod-.
These cells develop a specific type of granulation and wander
into the blood-vessels. The third strain is the lymphocytes.
These I have never seen differentiating in the wall of the
yolk-sac ; they begin to appear in the blood stream on the
fourth day but do not become marked until the fifth and
sixth days. It may be that they arise only within the embryo
itself.

ERYTHROBLASTS

In these studies it has been possible to establish a criterion
for a primitive red cell. As was discovered by Pappenheim,"
the primitive red cell has a basophilic, azurophilic cytoplasm
so finely granular as to appear like ground glass. Fixed to
show this basophilic cytoplasm, the cell looks like a single
angioblast. In the living cell a droplet of yolk is occasionally
to be seen. If, however, one stains the cell supravitally with
either neutral red or with brilliant cresyl blue, there appears
a very massive granulation which at first completely fills the
cell. This granulation is completely soluble in alcohol and
in all of the usual fixatives; it disappears also in the Gapor of
formalin. If, however, it be stained with neutral red or with
brilliant cresyl blue, it becomes insoluble in methyl alcohol
and hence can be seen in films stained with Wright's eosin-
methylene blue. For these double stains brilliant cresyl blue
is slightly better. This special granulation is then very easy
to bring out in films of blood. It is not so easy to stain in the
total blastoderm, because the dilution which stains the neutral
red granules of endothelium, the yolk and all of the stainable
substances of the clasmatocytes, that is, the diIution of 1 to
10,000, is too weak to stain the granulation of the reds, but it
does, nevertheless, make it just visible. The .primitive red
cell in the living blastoderm can be stained, however, by in-
jecting the dye directly into the blood-stream ; or if a drop of   '

1 per cent solution of neutral red is put on the blastoderm for
a few seconds and then washed off with clear Locke-Lewis
solution, the red cells show the stain well.

The question must come up as to whether the stainable sub-
stance actually exists in the living cell in some state from
which it is precipitated by the dye, just as Mott has shown that
the Nissl substance, in its stainable form, develops only as the
nerve cell dies. Indeed, when Israel and Pappenheim I' first
described the vital staining of substances in erythrocytes, they


JOHNS HOPKINS HOSPITAL BULLETIN         ,

[Ko. 3G8

saw that the staining commenced only as the cell began to
die. This may be true.  The criterion I have used for the
actual death of the cell is whether the nucleus stains or not,
and this specific substance does stain in dilutions which do
not stain the chromatin of the nucleus at all. The`exact dilu-
tion necessary to stain it must be worked out.

For the permanent. films of blood on the second and third
days, the granulation is so dense that, after counterstaining
with Wright's stain, the cells must be differentiated in absolute
alcohol, the decoloration being stopped with xylol.

On the second and third days, the red cells being megalo-
blasts, both the granulation and the basophilic cytoplasm com-
pletely fill the cell. On the fourth day a narrow rim of clear
cytoplasm appears in' many of the cells making erythroblasts,
sho%ing hemoglobin around the edge free from granules, while
the granules make a very dense rosette or wreath around the
nucleus.  These rosettes are very characteristic and in stained
preparations they greatly obscure the nucleus. By the fourth
day the red cells have no longer the somewhat uniformly round
shape of the earlier stage and there is no longer a comparative
uniformity in size, but rather there are many much larger
forms together with many small irregular or oval cells.  This
period of great variation in size is a stage of active division of
the cells as well as of growth of individual cells.  In the small
oval cells, the rosettes are oval and in division the rosettes
divide, so that each daughter nucleus is surrounded by a
wreath of granules before the cells separate.

On the fifth day a few of the red cells begin to show a
diminution of the granulation, some of the cells grow much
larger and the granules and rods begin to spread out into the
cytoplasm, the cells showing the polychromasia and the retic-
ulation which is known to be characteristic of the so-called
reticular forms occuring in anaemias in human blood. By the
seventh day there are large numbers of the cells in the retic-
ular stage, while there are still many of the primitive cells
and the wreath forms. Gradually the amount of the basophilic
cytoplasm and of the vitally stainable granulation decreases
while the hemoglobin increases. Finally, the basophilic cyto-
plasm disappears, but a small amount of the specific grrtnula-
tion remains in each erythrocyte. At the time of hatching
and for three days afterward, all of the red cells in the
circulating blood show from one to eight or ten vitally stain-
able granules. I have not carried the studies farther nor
studied the stages between the seventh day of incubation and
the time of hatching. It is clear, however, that one can work
out the types of cells that are characteristic for each stage of
the developing chick. Of course, at any stage there are some
cells characteristic of preceding stages. On the second day
only the primitive stage, the megaloblast, is present,, and then,
with a given increment of time, the different stages'in the
development of this specific granulation are added. When
such/a study has been made for a mammalian form and
especially for the human embryos, as is now feasible, we
shall be in a position to estimate just how primitive are the
cells that appear in the circulation in anemias.

The first account of the staining of the specific granulation
of the red cells, which I have been able to find is in a paper
by Israel and Pappenheim " in 1896, in which they say that
if a few dr? grains of neutral red are placed on a slide and
used for a film of fresh blood, there will appear a granulation
in some of the red cells just as the cell begins to die. In
1901, Bettmann * described the use of vital neutral red in
the staining of red cells in pathological conditions, but did
not discriminate between a staining of the nucleus and the
specific grantilation around it. Three years later, Rosin and
Bigergeil Km described the methods of vital staining
and the dyes to be used, but it was not until 1907 that we
have a clear account of the vitally stainable granulation of
the red cells. In 1907 there appeared three papers in the
Folia Hsmatologica, by Caesaris-Demel,' by Pappenheim JI
and by Ferrata," ' m which the vitally stained granules are
described and illustrated. Caesaris-Demel shows the stage of
the wreath around the nucleus, not, however, in as primitive a
stage as on the fourth day of incubation in the chick. He
shows also the reticular stages and the final stage of a few
granules.  He distinguished between the deeply staining
granules and the more faintly staining filaments.

The primitive basophilic cell, which is the first red blood-
cell, was first. differentiated by Pappenheim and called the
megaloblast. It has a basophilic cytoplasm and a large
nucleus, poor in chromatin, and a conspicuous nucleolus.
It becomes an erythioblast and all the stages of the develop-
ment of this cell, as far as concerns the decreasing of its
basophilic cytoplasm, the increasing of its content of hemo-
globin and the changes of its nucleus, have been worked out
with the eosin-azur technique in final perfection by Pappen-
heim,iLY Ferrata,12 Danchakoff,J'* Maximow II-M and Weiden-
reich.`-q  The development of this cell with reference to its
specific granulation is now necessary to complete its life
history.

In the chick, it has been shown that all of the primitive
blood-cells on the second day of incubation are megaloblasts
which become' erythroblasts as soon a.s a trace of hemoglobin
can be made out.  These cells are derived from angioblasts
and from endothelium. As far as the specific granulation is
concerned, the first stage, on the second and third days of
incubation, has the granulation throughout the cell. From
the fourth to the sixth day, there are the rosette or wreath
forms in which the granulation is around the nucleus. On
the seventh day most of the cells show the reticular form of
the granulation. All of these stages show diffuse basophilia.
With further studies it will be possible to tell just when
basophilia and the reticular forms disappeas in the majority of
the red cells. At the time of hatching all of the cells in the
circulation have acidophilic, hemoglobin-bearing cytoplaanl
with a few vitally stainable granules. It is, of course, clear
that at, any stage in development, while the majority of the
cells are in a specific phase, a few of the earlier types may bc
found. In normal human blood about one per cent or less
of the erythrocytes show a few vitally stainable granules.


OCTOBER, 19211

JOHNS HOPKINS HOSPITAL BULLETIN

The question which must come up first in connection with
this granulation is its relation to the so-called basophilic
punctation, and both Pappenheim and Ferrata agree that
these two substances are entirely different. The basophilic
punctation stains in azur after fixation and Ferrata" thinks
that it is an abnormal clumping " conglobation " of the azur-
ophilic cytoplasm, and he shows the gradual production of
the punctate forms in red cells after experimental lead poison-
ing on hie Plate VIII. It is thus easy to see why basophilic
punctation does not occur in embryonic blood, whereas the
vitally stainable granulation is specifically characteristic of
development.

Another point in which this specific granulation may prove
of interest is that it offers a chance to study the development
of hemoglobin in the cell by testing the granulation for the
presence or the absence of iron. Both the azurophilic cyto-
plasm and the granulation disappear as hemoglobin develops,
but the granulation alone is characteristic of the red cell as
distinct from all other blood cells.

In the developing blood there are always a few cells contain-
ing the Howell-Jolly bodies.  These are fragments of nuclei
staining just like chromatin, which were discovered by
Howell,`" in 1890, in a study of the blood of the cat after
hemorrhage. Of course the corpuscles of the chick are all
nucleated, so that the question of the extrusion of nuclei
does not come up, but an occasional cell, from the very begin-
ning of the formation of blood on the second day, shows a
fragmented nucleus. I interpret such cells as dead. They
are to be found in the early blood islands before the cells
become free and are very interesting as showing that cell
death occurs in the early stages of marked cell-division and
growth.

ORIGIN OF M~XOCYTES ASD CLASYATOCYTES FRON
           ENM)THELIUN

The separation of the clasmatocyte as a distinct type of
cell of the connective tissues is due to Naximow.I"  He showed
that by introducing two sterile cover-slips under the skin in
rabbits, one could separate three types of cells by the speed
with which they passed between the covers, leucocytes appear-
ing first; a special cell, the clasmatocyte, wandered in during
the first nineteen hours and the fibroblast in from two to four
days. Then he showed that the clasmatocyte was specifically
sensitive to neutral red,= while Bouffard,' Goldman `*p"
Evans,`e" Schulemann `--I and a large group of workers have
demonstrated that it is the cell of the connective tissues most
specifically differentiated to phagocytize and store particulate
matter. The specific reaction of this cell to vital, neutral red
is that the dye stains certain granules of the cell and certain
large fluid spheres which are called vacuoles, the so-called
"neutral red granules and vacuoles " of Lewis and Lewis."
These vacuoles are organs into which the cell passes phagocy-
tized particulate matter.  In the vacuoles the fine particles
ahich the cell has taken up become clumped and, as Evans
and Scot,t'" have shown, may even be re-crystallized.

Aschoff and Kiyono ' then showed that an identical reaction
to a vital dye could be obtained by certain cells of the blood,
namely the group Naegeli B" has called the monocytes, which
are the large mononuclear and transitional forms of the
Ehrlich school. Thus they distinguished and related histio-
cytes of the blood and histiocytes of the connective tissues.
Moreover, they regarded the histiocytes of the blood as of
endothelial origin.

Pappenheim and Ferrata have illustrated the separation
of the monocytes, the leucocytes, and the lymphocytes on
purely morphological grounds, believing that there is a com-
mon stem-cell, a hypothetical haematoblast for them all.
Aschoff and Hiyono separate the monocytes, calling them his-
tiocytes of the blood, on a physiological basis, and I think that
I can demonstrate on a fundamental embryological basis that
the monocyte and the clasmatocyte are identical cells, derived
from endothelium, thereby making one of the' three great
groups of connective-tissue cells that contribute to the blood.

,~ If a blastoderm of the third day of incubation be stained
in vital neutral red, the endothelium stands out with num-
erous granules staining in the dye which are both around the
nuclei and scattered in the thin periphery of the cytoplasm.
The endothelium of the capillaries and the veins often becomes
reduplicated. Endothelium is more refractive than meso-
derm, and this property, as well as the granules stainable with
neutral red, characterizes both of these layers of endothelium.
One of the cells of the inner row can then be seen to enlarge,
protrude into the lumen and develop the vacuoles which are
characteristic of clasmatocytes.  The periphery of the cell
then puts out a film of cytoplasm in which there is a central
process more refractive than the rest, simulating a flagellum,
and these films are in constant motion.  In fact the eye is
attracted to these cells both by the stained vacuoles and by the
motion of the peripheral films of cytoplasm.  Such a cell then
gradually becomes free.  The characteristic motion of the
peripheral films continues, keeping the surrounding fluid
moving, though the cell itself shows very little locomotion.
In the study of the origin of the red blood-cells on the second
day of incubation (Sabin)" it was noted that the erythroblasts
formed great clumps of cells attached to the inner surface of
a complete endothelium.  The monocytes, on the other hand,
differentiate and drop off as single cells, leaving the original
endothelial cell from which they came as the wall of the
vessel.

An endothelial cell may become phagocytic while it is yet
in place, for I have seen them with red cells engulfed just as
Maximow p shows for a mammal in his Fig. 4 on Plate
XVIII. This means that an endothelial cell which is actually
a part of the wall of a vessel, not one of the reduplicated forms
already on the road toward becoming free, may be phagocytic.
That is to say, endothelium is'itself phagocytic as well as
having the power to give off free cells which are phagocytic.
In the same figure quoted above, Maximow shows three free
monocytes, very characteristic, labeled Edph. He recognized
them as desquamated endothelium but did not identify them
as monocytes. In fact all of the early stages of the develop-


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JOHNS HOPKINS HOSPITAL BULLETIN

[X0. 365

ment of blood are beautifully illustrated by the two plates of
Maximow in this same article.

While these few cells are getting free in the lumen of the
vessel to make the monocytes of the blood, the outer row of
the reduplicated enclothelium divides rapidly in irregular
patches, making the outlines of the vessels, both capillaries
and veins, have an exceedingly irregular contour, very differ-
ent from the smooth contour of the earlier capillaries and
from the wall of the omphalomesenteric arteries which now
have a single layer of smooth muscle. The clumps of cells
along the outer wall of the vessels develop the vacuoles char-
acteristic of clasmatocytes and become fr'ee as clasmatocytes.
Many hundreds of the extra-vascular cells are formed from the
endothelium to one intra-vascular.  The extra-vascular forms
tend to be larger and have larger vacuoles, but I have seen one
of the large cells wander into a vessel. The original endothe-
lium has granules that stain in neutral red ; it may also have
vacuoles. The free cells all have both vacuoles and granules
and a differentiation of the periphery of the cell into motilet
films. Studied with vital neutral red, the monocytes and the
clasmatocytes are conspicuous because they are stained.

Thus, in the early chick, endothelium gives rise to two
groups of cells, .the megaloblasts which develop hemoglobin
and become erythroblasts and a strain of cells termed histio-
cytes by Aschoff.  The extra-vascular histiocytes have been
termed clasmatocytes, the intra-vascular ones monocytes.
They are identical and are specifically differentiated along
the line of phagocytosis. They take up particulate matter and
debris in solid form which they segregate and store in certain
preformed vacuoles filled with fluid. They do not store this
insoluble material permanently because it is gradually re-
turned to the circulation and excreted by the kidney. So they
represent a mechanism for taking care of foreign matter in
excess of the amount that the body can excrete at the time.
In the blastoderms from the third to the seventh day there is
comparatively little differentiation of new angioblasts in the
area pellucida. In fact, in about tity specimens, I have found
only three masses of solid angioblasts. The hollow isolated
vesicles made from these solid masses are, however, more
numerous, indicating that this stage lasts longer than the
solid stage. In one specimen of the third day of incubation
there was a long mass of solid angioblasts which started to
liquefy to form a vessel, and while the center of the mass was
liquefying to form a vessel, two cells wandered off from the
periphery as clasmatocytes. Thus angioblasts can also give
rise to clasmatocytes.

If one takes the group of monocytes as they are shown in
the third row of Pappenheim's Plate I,= or the fourth and
fifth rows of Ferrata's Plate XII,- it will be seen that they
include all of the large mononuclear forms and the transi-
tionals of the circulating blood. Both of these types can be
seen early in the chick coming from endothelium; an endo-
thelial derivative which is larger and less vacuolated is the
mononuclear cell, a smaller and more vacuolated type, the
transitional.  The transitional is thus shown to be a finished
type of cell like the cell of the adult form, for which the term

transitional is therefore a misnomer. The large mononuclear
type always has an excentric nucleus; it is distinguished most
easily in the.films of blood, stained with brilliant cresyl blue
and counterstained with eosin-azur.  It lacks the specific
granulation of the erythroblast and has a very clear distinctive
blue cytoplasm in Wright's blood-stain. With the group of
the clasmatocyte in the connective tissues, Maximow divided
the cells into resting and active cells. With the group of en-
dothelial derivatives in the blood, it is not wholly clear
whether the larger or mononuclear forms are resting or are old
forms. In the embryo, the large mononuclear forms appear
less specifically differentiated. Both forms can be seen in
the living chick to come from endothelium, the transitional
cell being developed specifically along the line of phagocytosis
and storing of particulate, solid matter and possessing a
certain type of motion of the cell in situ and very slow loco-
motion like the clasmatocyte of the connective tissue.

THE GRANULOCYTES OR LEUCOCYTES

On the third day, also, granulocytes begin, represented by
the cells which are analogous to the neutrophilic myelocyte of
mammals. In the chick the granulocyte with fine granules is
pseudo-eosiniphilic.  The first sign of the beginning of the
granulocytes is that a cell appears close to a vessel which
cannot be distinguished from a single angioblast.  I have not
found in these cells any substance stainable in neutral red
except the specific granulation which stains paler than the
granules of the endothelium, but am not yet entirely sure that
this will be a sufficient distinguishing mark between this cell
and a single angioblast. When, however, such a single cell
divides, there is no longer any difficulty, because two angio-
blasts stay together while two granuloblasts separate. This
criterion is not adequate when one has only sections, but in
watching the living membranes or in studying them after
fixation, where every cell of an entire area can be seen in its
relations to other cells, it is sufficient. Such material has
obvious advantages over sections.  Thus, from one cell comes
a clump of four or more cells with a dense azurophilic cyto-
plasm, the stem cell of the monophyletic school, lying near a
vessel.  These cells then show the following changes. The
nuclei become excentric, while the center of the cell is occupied
by the centrosphere made very obvious by the development of
fine granules, staining pink in neutral red, always arranged
in a crescent around the centrosome. Thus, there is a nucleus
on one side, a clear spot in the center of the cell and on the
other side this crescent of fine granules. The granules are
entirely motionless at the start, there is none of the active
streami,ng of the granules which is always associated with am@
boid movement and which must be associated with a fluid state
of the cytoplasm. The cell itself, however, does move, but
very slowly, directly toward the vessel.  One of the cells
reaches the wall, half-way between two endothelial nuclei, and
then one can see the wall bend inward, until finally the ~~11
enters the lumen. The rest of the clump line up behind the
first and also pass in. Thus, these granulocytes show a
specific chemotactic reaction at once. Throughout these earl?


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319

stages the granules are arranged characteristically around the
centrosome. Thus, the specific granulation of the red cell is
arranged around the nucleus, of the granulocyte around the
centrosome, while the granules of the endothelial cell are
scattered throughout the cell. Even in these early stages the
nuclei of many of these cells become indented, the concave
side always being toward the centrosome ; thus, the primitive
cell may soon be regarded as a leucocyte.

In the case of the monocytes and the clasmatocytes, both of
these cells can be readily found differentiating and dropping
off from the endothelimn, but no relation to endothelium can
be made out in the case of the granulocytes.  They are near
vessels but never form a part of their wall. It was shown by
Danchakoff * in 1908 that the granulocytes are extravascular
in origin.

There are no eosinophilic cells on the third day. The eosino-
philic granule of the chick's blood is in long rods. During the
first seven days I have seen only a few in the circulation and
have not found them differentiating in the area pellucida.
Probably further study will bring them out, since they are
known through the work of Danchakoff' to develop in the
area opaqua of the yolk-sac. The mast cells I have not seen
at all in the first seven days, and Maximow n found that they
develop late in mammals.

From these observations one may offer the theory that the
two stem cells, the angioblast with its power to give rise both
to red cells and to histiocytes in the larger sense, and the
granulocyte are cells whose common ancestor is a mesenchyme
cell instead of a differentiated stem cell or " hsmatoblast."
In other words, the cells of the blood are not so sharply
marked off from the cells of the connective tissues as to have
a specific, common stem cell. At least one would have to
prove that the differentiated cells which normally made the
syncitial masses of angioblasts could be made to develop
granulocytes.  The argument for the mesenchyme cell as the
stem cell, for the three distinct strains of cells which contribute
to the blood, is that three such groups can be isolated embryo-
logically and that they correspond with a functional classifica-
tion. At least one may say that no common differentiated stem
cell has been adequately demonstrated.

LYMPHOCYTES

In these studies of the development of blood in a living form
the account of the origin of the lymphocytes is very incom-
plete. The lymphocytes make a group of cells ranging in the
mammal from the size of a red cell up to cells twice the size.
Likewise in the chick the lymphocytes are the smallest cell.
When the cell first appears, all of them are of the small size.
The cell has a characteristic nucleus and its cytoplasm con-
tains a few azurophilic granules, discovered by Uichaelis and
Wolff .*  The living cell has a nuclear membrane which is
more distinct than in any other cell, but that this criterion is
a difficult one to go by can be realized readily in connection
with the fact that all nuclei become distinct as a cell dies.
The cytoplasm of the lymphocytes contains but few granules
and they do not stain readily in neutral red, but can be made

to by increasing the amount of the dye or the time of staining.
From these facts it is less readily discriminated than the
other types. The reactions of lymphocytes in tissue cultures
have been described by Lewis and Webster." In Wright's
stain, the early lymphocytes are exactly as distinctive as in
adult blood.  The first forms are of the small variety. I
have seen a few on the fourth day, more on the fifth and sixth.
In the blood smears, they occur in small clumps.  The chro-
matin of the nuclei is very dense and has a peculiar violet reac-
tion with azur-eosin. I have never found any indication of
their differentiation in the area pellucida, thus it may be that
they form only within the embryo itself rather than in the
yolk-sac. However, a more extensive study of the yolk-sac
may bring them out. All of the evidence from the study of
this cell in the adult is that it differentiates extra-vascularly
from reticulum. The only evidence, then, of significance in
these studies in regard to this cell is that it occurs later than
the other two groups and hence should not be regarded as a
stem cell. Thus, from these studies, I would stress the use of
the three names of white cells as specific for the three distinct
groups, the leucocytes, the monocytes and the lymphocytes.

In these studies it is very plain that each white blood-cell,
as it first appears, is differentiated. The red cells pass through
a long stage of maturation. The first megaloblasts can be told
as early stages of the red cells by a specific granulation, but the
cell itself passes through a long series of stages, the erythro-
blasts, before it is the erythrocyte of the adult blood. The
monocytes are a phagocytic type, like the cells of the adult,
before they leave the wall of the vessel, the endothelium itself .e
being phagocytic; the granulocytes develop their specific type
of granulation early and soon begin to be leucocytes, and
thirdly, the first lymphocytes are distinctive. When, however,
all of these cells begin to divide; discrimination between all of
the young cells is by no means easy, as the entire history of
hematology attests.  From this it can be seen readily that
one must continue these studies of the development of blood in
these living forms, watching especially the young cells just
after division in all the stages of incubation, before one can
adequately master all of the types of cells that are to be seen
in bone marrow. In a drop of blood taken on the third day of
incubation it is possible to tell all the cells apart, later it be-
comes most difficult. It is the study of the maturation stages
of each group of cells by means of the eosin-azur technique
that has been the great contribution of the monophyletic
school.  To this study must now be added certain specific
criteria that come out through the method of applying dyes to
living cells and we must now follow the stages of the cells
with these vital dyes through the different embryonic periods.

The postulation of three strains of blood-cells on the basis
of embryology fits in with the functional groups as we now
know them. The endothelial or angioblastic group represents
first the hemoglobin-bearing cells, and secondly, that group
of the blood-cells which exhibit a special property of endothe-
hum, namely, phagocytosis. The monocytes have this power
of phagocytosis, they possess a peculiar type of motion in situ
with very slow locomotion.  The granulocytes possess a high


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.%              JOHNS HOPKINS HOSPITAL BULLETIN           [So. 368

degree of anmboid motion, with speed and a flowing of the
granules. They respond to chemotactic influences, are also
phagocytic and have functions probably related to their
specific granulations. The lymphocyte strain, as Murphy rs-D
has shown, are separated off physiologically by their being
more sensitive to X-rays and to the emanation of radium than
other normal cells. Moreover, he has shown that they are re-
lated to immunity toward certain forms of tumors as well as
to certain types of infection.

The study of the blood-cells is a part of the study of the cells
of the connective tissues. The erythrocytes are the only type
that function only within the vessels. Of the other group
from endothelium, the histiocyte in the larger sense, the vast
majority make the clasmatocytes of the connective tissues,
which are the mononuclear forms and the actively phagocytic
forms of sub-acute infection, the resting and active wandering
cells of Maximow. A few of this group make the monocytes,
that is, the large mononuclear and transitional forms of the
blood.

Of the granulocytes, which all differentiate extra-vascularly,
the neutrophilic leucocytes pass into the vessels in large&
numbers. Of the eosinophiles very many remain in the tissues
while the mast cells never enter the vessels in most animal
forms. By mast cell is meant a cell of the connective tissues
occurring along vessels, along nerves and between muscle
fibers, having a special metachromatic, basophilic granule.
The so-called mast cell of human blood has been shown by
Weidenreich,"-* to be a degenerating cell without any centro-
some. The lymphocytes are for the most part extra-vascular,
arising in the lymph glands and in the follicles of the spleen
and in very numerous follicles in the various organs either
associated with lymphatic capillaries or not. Thus, the differ-
entiation of three strains of biood-cells, the endothelial strain,
the granulocyte strain and the lymphocyte strain, that can be
made out in the early stages of the chick embryo, can be shown
to correspond with a functional grouping as far as we yet know
the functions of the types of blood-cells. Moreover, the origin
of the cells of the blood can be shown to be but a part of the
study of the great groups of wandering cells of the connective
tissues, the one type which functions only intra-vascularly
being the erythrotiyte.

The method ,of studying blood with vital dyes, beginning
with the stages of the embryo when the cells first appear gives
a very great advantage in following the maturation of specific
cells and gives a chance of analyzing the complicated young
forms which it is necessary to recognize in order to understand
bone marrow.

The group of the red cells is characterized by a specific
granulation stainable in certain vital dyes, possibly one should
say precipitated by these dyes. This substance is at first
throughout the cytoplasm, then in a wreath around the
nucleus, then a reticular form and finally in scattered grau-
ules or droplets. The arrangement of this granulation around
the nucleus should be stressed, although the substance is of
cytoplasmic not of nuclear origin. Red cells with nuclear frag-
ments, Howell-Jolly bodies, can be shown to be dying cells.

-

The strains of white cells, clasmatocytes and monocptes, that
come from endothelium, are characterized by certain granules
and vacuoles stainable in very dilute neutral red. They are
scattered diffusely throughout the cells. The granulocytes are
characterized by the arrangement of their specific granulation
with reference to the centrosome. The lymphocytes are less
sharply characterized morphologically, but have somewhat
distinctive nuclei and granules stainable in azur.
This work is a part of the new subject of experimental
cytology which seeks to analyze cells by- means of speci&
criteria and to use these criteria to study the reactions of cells
to normel and abnormal conditions.

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