STUDIES OF LIVING HUMAN BLOOD-CELLS  j'

   By FLORENCE R. SAB;N
(From the Department of Anatomy, The Johns Hopkins
            University)

The techuique of staining smears of blood by the various
Iuo~liticatious of Romanovski's method, that is, by mix-
tures of azur, methylene blue and eosin diss&ed in the
alcohol which serves as a fixative, has reached SO high a
degree of perfection and the surveys of blood-cells by
this method are so complete, that further progress must
now depend on the use of some new method. Such a
Wthotl is, I think. furuixhed by the differential staining
of living blood-cells, and by this method a whole new
range of hematological problems has been opened up.

The method of studying blood-cells with aupravital
dyes was applied by the writer 1 in 1021 to the study of
developing blood-cells in the embryo chick, both within
the vessels as seen in the area vasculosa, and in smears
of the living cells.
E. Simpson *    During the same year, Niss Miriam
     made a study of the reactions of living

blood-cells to the large group of dyes which Evans and
Scott 3 hacl studied with reference to an analysis of the
~11s of the connective-tissues.. She also used * the vital

method to separate the group of monocytes, first, in
human blood and second, in rabbits under experimental
conditions.

The, technique of studying living blood-cells is so
simple, that it is readily applicable to the study of clin-
ical cases. All that is needed is clean slides and covers,
a few drops of a stain and a warm box for the micro
scope or warm stage incubator, so that the slides can be
kept at body temperature.  The details of the method are
as follows: the coverslips are cleaned as for fixed smears.
The slides are also cleaned by the usual technique, that
is, they are kept for three or four days in concentrated
sulphuric acid to which a few crystals of potassium
bichromate have been added;..' From the acid they are
placed in running wate+f.for 2 to 3 hours, care being
taken that the water gets between the slides so that all
acid is removed. This is essential, since living cells are
sensitive to an acid. They are then rinsed 2 to 3 times
in distilled water in which they are left over-night. and `-


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[So. 391

finally stored in 80% alcohol. Before using they are
dried on a clean towel and given a high polish with jew-
`eler's rouge applied on a piece of clean silk. For the
suggestion of this method of polishing I am indebted to
Dr. Edward Malone; it is a great advantage because it
ensures an even film of the vital dye. To get an even film
of the dye, flame the slide; be sure that it is free from
dust and then flood it with a dilute solution of any dye
dissolved in absolute alcohol, drain off the stain quickly
and stand the slide upright until it is dry. The stains
can be used over and over but must be filtered frequently
to keep them free from dust. The film should be faint
but spread evenly on the slide. The slides can then be
stored until they are needed, care being taken to keep
them free from dust. Slides with any grease will have
blotches on the stain and should be discarded. In drying
the slides, do not blow on them, for again the stain will
be uneven ; if the stain is uneven, some of the cells will
get too heavy a dose and be killed or damaged.
I/  For stains, I have used neutral red, a combination of
neutral red and Janus green, brilliant cresyl blue and
azur. For the most part I have used neutral red which
is relatively nontoxic. The stock solution of neutral red
is made by adding 100 mg. of the dye to 10 C.C. of abso-
lute alcohol.  I have used only the dye put up by Grtibler
before the war, marked "Zu Injekt. i. vital Oezo. n.
Ehrlich." For making the films on the slides, take 10 C.C.
of absolute alcohol and add 0.4 CC. of the stock solution.
The strength of the. solution can be estimated by the
color after one has had a little experience; it should be
a clear rose color. The proper strength of the dyes can
be readily determined by testing them with normal living
blood-cells; the slightest staining of the nucleus will in-
dicate that the stain is too strong. For the neutral red
Janus green mixture, take 2 CC. of the dilute solution
of neutral red and add 3 drops of a saturated solution
of the Janus green in absolute alcohol. The staining
qualities of the mixture last only a few days.  This mix-
ture is much more toxic than the neutral red.   The
eosinophiles are amceboid in it but the nentrophilic
leucocytes are not.  As Idiss Simpson discovered,
the Janus green has the very great advantage of
being differential for the lymphocytic strain of cells,
since all of the lymphocytes show a characteristic
arrangement of mitochondria.  Azur, which may be
obtained by using about 10 to 12 drops of Wright's
blood stain in 10 CC. of absolute alcohol, is a con-
venient dye but much more toxic than neutral red.
The brilliant cresyl blue originally prepared by ,Griibler
is an excellent dye; but the American dye, which I have
used, has been mixed with a red dye which is far too toxic
for supravital use.

It will be readily recognized that the thickness of
these Alms of dye must vary with the number of cells
to be stained in the preparation. For example, in a film
of bonemarrow where practically all of the cells take

up the dye, the strength of the solution must be greater
than for blood where only a small proportion of the cells
take any stain. The same is true in studying smears of
lymph glands and spleen.  For smears of blood a solu-
tion of the dye, of such strength that it will stain all of
the cells immediately, can be safely and readily used,
while for smears of lymph glands, spleen and bone-mar-
row, better results are- obtained with a dye that takes
10 to 15 minutes to react. In a film of embryonic blood,
where every single red blood-cell also takes the vital dye,
a stronger solution should be used. It should also be
noted that the vital staining of the white blood-cells
takes a much weaker dose of any dye than the staining
of the reticular substance of the red blood-cells, so that
some of the white cells may be damaged if all the red
cells stain. This fact explains the difficulty of staining
the reticular substance of the red cells in marrow, as
contrasted with the method used for films of blood,
since with the marrow a dilute stain may all be taken up
by the white cells.
/ Films of blood are made on the stained slide exactly as
I for any fresh film of blood.  They must be sealed imme
diately with a vaseline of high melting point, for which
we use Salroliue.  Formerly we used melted Salvoline,
but have found it better to smear it, while cold, along
the edge of the coverslip, lest the heat damage the cells.
Vaseline mixed with paraffin can be used; the melting
P oint must be above the temperature of the warm box,
otherwise the vaseline will melt, mix with the immer-
sion oil and make the film opaque. After the film has
been sealed, it should be put into a warm box ; it is not
necessary to hurry in getting it into the box, for the cells
will show active motility even after having been kept at
room temperature for 15 to 20 minutes.. This makes it
possible to carry the slides from the ward to the labor-
atory. III case considerable time'would have to elapse, an
arrangement of carrying the slides on a hot-water bottle
could be devised.  It will be found that blood from which
a considerable amount of fibrin is deposited on the slide
tends to have the red cells clump. It is not necessary
1 to have every single red blood-cell distinct as for stained
smears, but very much clumping of the red cells makes
a film in which the count of the lymphocytes will be too
small. The leucocytes will wander from the clumps of
I red cells but the lymphocytes will not, and may therefore
i be missed in the count.  Of course films with any rou-
lenux are too thick. As with fixed smears, the cells at the
edges m,ust not be counted because an excess of dye col-
lects at the eclges so that some of the cells may be killed
or damaged.

Since the living cells are sensitive both to acid left
on the s1id.e and to the strength of the dye, each new box
of slides and each new bottle of the dilute stain should
be tested with normal blood to avoid any false inferences.
concerning abnormal blood. The cells should all stain
characteristically without any staining of the nuclei.


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JOHNS HOPKINS HOWTAL BULLETIN       .

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,,.e reqrrd any coloring of the nucleus as an evidence of
,I;,l,lLlge to the cell, regardless of the fact that the Cyte
I,lilsnl may continue to move a short time after the
,,,~&us has begun to St&i. The amount of motility and
the duration of motility are certainly reduced.

The descriptions of the vitally stained cells which
rollow are limited to human blood; it has been found
i,, the progress of experimental work on animals, which
is ,,ov in progress, that there are greater variations be-
t,,.wu the blood-cells of different species than had been
,stablished by other methods. The observations on Corn-
,,ilri,tive studies will be brought out later. In normal
l,,mlan blood, every type of cell stains so characteris-
tically with a vital dye, that there is rarely a cell that
c.ulll,ot be distinguished with certainty. In abnormal
blood, on the other hand, when a given group of cells has
been stimulated or damaged, its reactions to the vital dyes
n,aY be markedly changed ; or when unusual cells have
been called into the blood-stream, it may be difficult or
even impossible to distinguish them.  The difhcultiea are,
I think, not greater than with tlxed smears, at any rate
they are not ,always identical with the difficulties in
asmars ; in consequence there are great advantages in a
combination of the two methods.

The very marked gain in the use of the supravital
laethod for the study of the white cells is the possibility
of distinguishing cells which have been markedly stimu-
lated to activity from cells which are degenerating. The
second great advantage is the ability to distinguish the
old and dying cells so as to comple& the life cycle of
each of the three types of white cells.'

In making these studies I am very deeply indebted to
the physicians of the Johns Hopkins Hospital for the
opportunity to study abnormal blood-cells. Through the.
COUd@ of Dr.-W. -P. tingcope, Dr. H. L. -Amoss `and`
Dr. J. G. Huck I have been able to follow cases on the
wards `&d Dr. ,W. G. MacCallum and Dr. A: R. Rich
have allowed ,me to study material from autopsies in
which individual cells were still alive. Without these
advantages this work could not have been done.

TB~RMINOL~GY

There has been considerable discussion regarding the
U8e Of the terms, gnunulo and maole in the reactions
of living cells to dyes, so I wish to detlne as exactly as
possible the sense in which I have used these terms.  By
the term vitally 8tainabZf3 gvxmule, I mean particles in
the Q'toplasm of living cells that take a dYe in a uni-
form manner, and do not increase in size during the
functional activity. That is to say, by granules, I mean
particles that do not change their reaction to `vital dyes
nor increase in size during different functional states of
the Cells. As an example of granules, there are the
neutrophilic, eosinophilic and basophilia granules of the
leucocYtes.  It `is true that with the Romanovski methods
there is some variation of the staining of the neutro-

philic granules. Beside these, all of the white cells con-
tain mitochondria in the form of rods that react to Janus
green.  In the use of the term grunzclo, I do not intend
to indicate any opinion concerning the physico-chemical
state of the substance.  On the other hand, by the .term
vacllole I mean certain round droplets of fluid within
the cytoplasm that take the stain and also change .very
greatly in size, according to different functional states
of the cells. They may change also in color, provided that
the dye used is a chemical indicator like neutral red. These
are the digestive vacuoles which Shipley 6 has shown con-
tained phagocytized material. He stained'clasmatocytes
with neutral red after they had stored trypan blue; he
could then see particles of the blue dye within the red
vacuoles. Evans and Scott 9 have called these vacuoles the
"segregation apparatus" of the cell. They are organs of the
cell into which phagocytized material is aggregated. It
may not be possible, at the present time, to discriminate
completely between granules and vacuoles according to
these definitions, indeed, as we come to know more about
the functions of the blood-cells, these definitions may
be shown to be crude; nevertheless, I believe that at the
present time these conventions in the use of terms will
contribute to clearness.  In the specific instances -where
I do not know whether a given substance is to be classed
with the more constant granules or with the function-
ally changing vacuoles, I shall use the term staked pa+
title.` Beside the vacuoles which take a. vital dye, there
are other clear spots in some. cells which do not take a
vital dye; they occur in cells which evince signs of de-
generation and are, I think, to be considered as evidence
of cell-death. Thus, there are two types of structures
called vacuoles, fltit, those that take'the vital dyes, and
vary with the activity of the cell, and second, those.that
I_~i_.- .
49 not tal&                              .- _--.-. -..
    Yp -e-s&& a&l ian ^be~corr&ted with degen-
erativo *processes-
      .
             LElJcocYTEs

1. PoZym.orphunu4~Zew neutrophilic leucmyt~e.
The neutrophilic leucocytes ,are very characteristic in
these preparations.both in their reaction to the stain and
in their striking motilitY. In a successful preparation,
they are never still except for the brief second ;when
they stop in order to change their direction.  Onaccount
of their motilitg the eye picks them .up with great ease
in making differential counts. In neutral red, azur or
brilliant- cresyl blue the neutrophilic granules of human
blood-cells - stain a uniform faint color. The pseudo-
eosinophilic granules of other mammalian forms stain
differently. The granules are of equal size and are ih
constant motion, streaming through the cytoplasm. In
Janus green, the neutrophilic granules stain a faint green
color while the mitochondria stain a brilliant blue. Un-
less they are stained with Janus green the mitochondria
cannot be discriminated in the lencocytes.  Besides .these
two types of granules in the leucocytes, there may also


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JOHNS HOPKINS HOSPITAL BULLETIN

[No. 391

be one or two vacuoles in the normal leucocyte.  These
vacuoles react to the dye more slowly, so that it may be
5 to 15 minutes before they are well stained. They are
always larger than the neutrophilic granules and may
be even as large as the nucleus. These vacuoles vary
greatly in sixe, in number and in color. They also move
in the cytoplasm along with the granules. They can, I
think, be used as an evidence of how much phagocytic
activity is going on in the leucocytes and, if this conclu-
sion is correct, the presence of these vacuoles indicates
that the cells perform a physiological function within
the blood-stream. In normal blood there may be none
of these vacuoles in the leucocytes ; in one case of pneu-
monia, the vacuoles were very large, even as large as
the nuclei, and they had the brilliant scarlet color of the
acid reaction of neutraI red. In this blood there was a
great deal of debris in the plasma. This debris cannot
be seen within the vacuoles, for it is colorless, but occa-
sionaIly a little particle of the jeweler's rouge has
been left on the slide and can be seen within the cell.  In
.one of our experimental rabbits, I saw one of the pseudo-
eosinophilic leuc&ytes in which there were four vacu-
oles, three small ones which had the orange color (neu-
tral reaction of neutral red) while the fourth, a very
large vacuole, had a brilliant scarlet color (acid reaction
to neutral red).

The type of motility of the leucocytes in these prepara-
tions is very striking to hematologists who are familiar
only with leucocytes in fixed smears. The normal, living
leucocyte is never round and never still.  The clear blunt
pseudopods*are constantly being put out from the cell
and the granules stream largely in the direction of the
flow of the cell, with, however, many counter currents
within the cytoplasm. The nucleus is usually in the rear
of the cell and, though it does change in form while the
cell is moving, strangely enough it seems to be not as
amo%oid as the nuclei of the lymphocytes. This, how-
ever, may be due to the fact that the nucleus is often ob-
scured by the streaming of the cytoplasmic granules. The
type of motility which the leucocytes manifest could only
exist in a cell in which the cytoplasm is fluid. In the
chick of the second and third days of incubation,, I have
never seen any cells with true amceboid motion, that is,
with motion of great speed associated with marked
' streaming of granules, except in the case i of certain
wandering endodermal cells loaded with yolk.   The
young granulocytes which develop outside of the blood-
vessels, and can be seen to wander into them, move but
slowly and with very little streaming of granules.  They
require hours to move a distance which a mature' leu-
cocyte could accomplish in minutes or seconds.  This
point seems to me to be very important with reference
to the motility of myelocytes.  Myelocytes, like the early
granulocytes of the living chick, do not have amceboid
motion; they progress only slowly, with slight changes
of the form of the cell and without streaming of the

granules. Hence we may say that the cytoplasm of the
young granulocyte of the embryo or of the myelocyte of
adult bone-marrow has a cytoplasm which it too nluch of
a gel to permit of rapid motion. This is interesting in
connection with the findings of Chambers 6 that a ,,ell
undergoing division has its cytoplasm in the form of a
gel. The contrast in motility of the myelocyte and leu-
cocyte of circulating blood is very striking in smears of
leukemic blood. The point is also evident if one studieR
a bit of living bone-marrow with the supravital method,
because the leucocytes will wander from the central mass
and leave the myelocytes in place. This point is probably
of importance with regard to the regulation of the supply
of leucocytes to the blood-stream, the myelocytes belug
usually retained in the marrow.

In watching the living leucocytes, it is of course esaen-
tial to know the normal rate of motility or rather the
normal limits of rate before we can make any judgment
concerning variations from the normal. So far I have
made only qualitative estimates between active and slug
gish leucocytes, calling the cells hyperactive when they
move in and out of the field while the count is being
made so that it is necessary to skip fields in order to
avoid counting cells twice.  Dr M. McCutcheon,* of the
University of Pennsylvania, has an ingenious method for
measuring the rate in p per minute which must be usetl
in studying deviations from the normal.

The leucocytes are in such rapid motion that no draw-
ing could record their form. Often, in their movement.
they put out a pseudopod which becomes a very sleudel
filament, stretching across the entire field and ending ii1
a little ball of cytoplasm at the tip.  Such a cell is showll
in Fig. 1, though many of these filaments are much finer.
Such excessively fine strands of cytoplasm show how the
leucocytes can get out between endothelial cells 01
through the tiniest crevices. Often the ball of cytoplasni
at the tip of such a process is lost without seeming to
damage the cell at all. These fragmented bits of cyt()-
plasm are very characteristic and are never to be Con.
fused with platelets because they always show Browuiill~
movement of the discrete particles. As will be descrihcrl
later, the granules of platelets are in clumps which (lo
not show Brownian movement. Within the cytoplaani of
the normal leucocyte, there is, I think, no Browubul
movement. The granules do not vibrate but either floN
with a steady movement or occasionally dart past others
in the cytoplasm. As a cell is beginning to die in these
films, especially as the power of locomotion begills to
diminish and the cell begins to round up, Browllin"
`motion develops which may mean that the cell has beg""
to take in too much fluid from the surrounding nl~lil"n~

* McCutcheon:- Studies on the locomotion of leucovtes. "
The normal rate of locomotion of human 1euCoCYtes in Gtrl"
Studies on the locomotion of leucocytes. 2. The effect of tyLz;.
ature on the rate of locomotion of human 1eUcocYtes.
Jour. Phyeiol., ,1923, vol. 66.


&YI'BJIBEB, 19231

JOHNS HOPKINS HOSPITAL BULLETIN

. .--
  -
,ll ,,lle clinical case (Med. No. 49329) which I studied,
thrnl ,vas Brownian motion in the leucocytes while the
(,(,ll ,,.as actually progressing.  It was a case of late gen-
,,r;ll carclnoma in which the bone-marrow was markedly
i,l,.ol,.etl in the carcinoma as demonstrated by the X-ray
,,l,otographs.  There' was another indication that the
l,l,,o,l.cells were damaged in this case, namely, they moved
,.,B,.f slolvly, showing many Ane filaments which were
,,r+~ll put out and withdrawn without leading to loco-
Ilwtion.  Numerous fine tllaments of cytoplasm I had
,,ftCll peen in studying living cells in the chicks in arti-
tic:,1 media. In 1920, Miss M. A. Herwerden ' published
iI n&hod for demonstrating amo?boid movement of leU-
cocytes by keeping them in a warm box for half an hour
111 Deetjen's solution, and then fixing and staining them.
la this treatment the leucoeytes become completely cov-
rr~l with similar fine illaments as shown in her Fig. 2
o1l plate 1. This is, I think,, a very interesting reaction,
lult iu my experience it is a sign, not of locomotion but
rather of a paralysis of a leucocyte due to an abnormal
surrounding fluid. In a moving cell the pseudopods are
few in number and much more blunt. VAt the last meet-
ing of the American Association of Auatomists, held in
(%icngo in 1923, E. R. and E. L. Clark * described the
reaction of living amphibian leucocytes after injections
of croton oil into the tadpole's tail. They said that the
leucocytes wandered from the vessels, and then became
stationary, putting out processes so that they looked
like "prickle cells." Subsequently they withdrew the pro-
cesses and became motile again. I have seen such "prickle
cells" in studying mammalian leucocytm from marrow
in Locke solution ; in the one instance in which I have
seen human blood after transfusion, the leucocytes were
in the form of prickle cells for about an hour. During
this time they did not show any locomotion, then they
gradually pulled in the processes and began to move
ilgdn. It may be, then, that the prickle leucocyte is a
c*ell temporarily paralyzed and the observation may be
of great value in studying the effect of transfusion on the
activity of the leucocytp.

The vital preparations remain in good condition al-
ways for an hour, and usually for four or tlve hours.
When the preparation is tlrst made, the leucocytes may
round UP for a minute or so, but usually they have start-
ed to move almost as soon as the oil immersion lens
has been adjusted, provided that the box is warm. It
is not necessary to warm the slides before making the
film- The length of time the slides last makes it per-
fectly feasible to make differential counts of the cells,
though they can perhaps not be made quite as quickly
as with fixed smears. In making the differential counts
One should record whether the leucocytes move actively
Or sluggishly, and whether they show the vacuoles
that indicate phagocytic activity. Also a tendency to
fragment or any other peculiarity of the leucocytes
should be `noted.  In this way we shall gradually accu-

mulate a knowledge of a new set of criteria of the activity
or one might even say of the physiology of the leucocytez

2. Non-motile Zeuwcytes.
All of the white cells of the blood take a vital dye

except one small group, and these I have called the non-
motile leucocytes. In studying smears of normal blood;
it soon appeared that there were peculiar, granular
forms which neither took up the dye nor showed any
motility. IvIoreover, in these cells the proportion of the
nucleus to cytoplasm is not like that of any other granu-
lar form, for the nucleus almost completely fills the cell,
the cytoplasm being represented by a rim of irregular, re-
fractile granules around the nucleus. Such a cell is
shown in Fig.- 2. This particular cell has perhaps a
more irregular outline than many non-motile leucocytes,
because they tend to be round, but the t$pe of outline will
bring out the fact that one may not infer anneboid acti-
vity from the mere fact of irregularity of contour of a
cell. This cell was described by &hilling e in 1908, who
discovered it in studying living blood-cells with dark-
field illumination.  He noted that the granules till the
ectoplasm whch is usually free from granules. He re-
garded them as dead leucocytes and described similar
cells, from sputum, which he thought came from very
young leucocytes, the nucleus being smaller and directly
in the center of the cell. In this paper Schilling also
gave some very interesting observations on following the
centrosphere in the ameboid leucocytes. He found that
in the moving cell the centrosphere maintained its cen-
tral position regardless of the position of the nucleus.
In these non-motile forms he found no centrosphere
whatever and thought it was the loss of the centrosome
that allowed the nucleus to take the central position.

By studying living smears, I have seen every transition
between the non-motile leucocytes and the actively amo+
boid neutrophilic leucocytes, indicating that, as a leu-
cocyte ages, it stops moving and becomes round ; its mem-
brane then becomes impervious to the vital dyes, the
nucleus becomes oedematous and the neutrophilic gran-
ules change into large, more refractive particles. Schil-
ling calls this process a coagulation of the granules.  As
will be seen in Fig. 2, these particles vary in size; the
nucleus loses all appearance of structure and appears
like ground glass. Streaks across the nucleus indicate
where the lobes were before the nucleus became oedema-
tous. They may come from cells which had nuclei with,
three, four or five lobes, rarely from those with nuclei of
two lobes.

I tested these cells by letting a drop of Turk's solution
run under the coverslip and found that they are retained
and so are probably included in total counts of the white
cells. It is a possibility that they may have been included
in the differential counts with the Ehrlich technique,:
their cytoplasm having been ilxed by heat. Schilling
thinks that they show also in Giemsa stain, appearing
as cells with a centrally placed, faintly stained round


282

JOHNS HOPKINS HOSPITAL BULLETIN

[No.391

nucleus, which had been described as modified lymph-
ocytes by Giitt and Weidenreich. I think, however, that
they are not retained in the technique which involves
a &ration in alcohol. I tested them in 33% alcohol, and
the granules are dissolved in 13 minutes, while the neu-
trophilic granules are unchanged. It is difficult to let
stronger alcohol run under a coverslip far enough to
reach them, since too much coagulation is produced. If
they are retained by the Ehrlich technique, this might
account for the fact that the Ehrlich data gave `a higher
percentage of leucocytes, namely 70 to 72 per cent, as
contrasted with the more modern data. In 1922 Bunting lo
pointed out this discrepancy from the Ehrlich data and

gave the figure 54.6 as the normal percentage of leuco-
cytes from a large group of students from the University
of Wisconsin. Corresponding data from the clinical lab-
oratory of the Johns Hopkins Medical School give 65 as
the normal percentage.

In the course of some experimental work on the for-
mation of blood-cells in rabbits which Dr. R. 8. Cun-
ningbam and I are carrying out, it became evident that,
though there may be some rhythm in the occurrence of
these non-motile cells, there must be exceedingly careful
controls concerning their production. The question must
certainly be raised as to whether they actually occur in
the circulating blood or not, and wherever direct obser-
vation of the circulation can be made, an effort should
be made to search for them. In the meantime, lacking
such complete proof, it has been only possible to elimi-
nate from the technique all factors which can be shown
to increase the number of degenerating forms. Among
these factors, are excessive heat, pressure from the cover-
slip when the preparation is too thin, and accidental
pressure in focusing the microscope. Notwithstanding
these points, which indicate that errors in technique
produce and increase the number of these forms, the non-
motile leucocyte, even when the coverslip presses too
heavily, as in the very thin areas, or when the specimen
has been overheated, will appear side by side with per-
fectly active leucocytes, indicating that there are marked
differences in fragility in the different leucocytes in mur
specimens.                                   .~
.Taking every precaution which we have up to the
present time analyzed as affecting the production, of
these cells, namely, securing free flow of blood, counting
only 6lms in which the red cells are thickly spread or
even overlapping a little, avoiding undue heat, using a
regulator in the warm box and maintaining a uniform
temperature of 37.5"C., we'still find the non-motile cells
in our fllm& We have been making complete counts of
the-w% cells, with differential counts of fixed smears
and ef the, living cells as often as the vital counts can
be made, which proves to be about every 40 minutes
throughout the day.  We make on an average of 12
counts a day. This experiment we have been making
with rabbit's blood. and have five such series of counts

-

with normal human blood. We find that there are a
few non-motile cells scattered through the different
counts, or perhaps none, but at one time, usually about
the middle of the day, there appears to be a shower of
the non-motile forms, which may reach fairly high per-
centages, as for example 16 per cent or more.  When the
Axed smears from blood, taken at the same time as the
supravital preparations, were counted, it was found that
there were six different structures usually regarde&as
smudges that had to be correlated with the vital counts.
First, there are definite, naked nuclei which are either
square or rectangular; they have shadows of granules
around them and I think correspond to the non-motile
leucocytes. Second, there are very long, naked nuclei
also with shadows of granules which correspond with
degenerating or damaged transitional cells. Third, iu
making so many counts from the same individual we
find small, round nuclei from the small lymphocytes.
Fourth, there are compact masses that are pink in
Wright's blood-stain that correspond to masses of debris
in the living preparations. In the vital counts, these
masses of cellular debris, that are so far disintegrated
that they cannot be related to the type of cell from which
they came, vary on different days from the same indi-
vidual.  These masses are not included in the total
counts. In smears they look more like nuclear material
than cytoplasmic; in the vital prepa&tions they take
none of the vital dyes, so that there is never any question
of confusing them with platelets. These masses are being
studied by Dr. David T. Smith in connection with lymph-
atic leukemias of children, in which they occur in lnrgc
numbers. Fifth, there are the well known masses of
fibrin. These are clearly fibrin in the fresh preparations.
and in smears they are large pink masses, looking viIC'II-
olated and with delicate fibers on their edges. Sixth
there are the so-called fragile leucocytes, in which the
cell membrane ruptures while the covers are being dr:l\vII
apart. These fragile leucocytes in which the scattevetl
and stained granules make it perfectly clear from wl~t
cell they came, whether- neutrophilic or eosinophilic, arc
usually disregarded in making ditferential counts. There
is no doubt but that they are mechanically increased IbY
faulty technique, by dust on the coverslip, or by an 111~
even sliding apart of the two coverslips.  They never
occur in the living preparations, where there is no drag
ging of the cells across the illm, nevertheless, in correlnt-
ing the counts of fixed smears with vital counts the!
must be `included. They can be shown to have bee'* rel'-
resented in a given double count by actively motile form'
in the living state. On the other hand, in one of Our
series the fragile leucocytes had to be added to the naketl
nuclei of the fixed smear to give any correlation with thr
count of the non-motile leucocytes in the fresh sPe~i~xxex'~
Such preparations show that there is the same difiCnby-
in analyzing the fragile leucocytes in smears as the x'o'1-
motile forms in the living, to discriminate how nluch i'


SEPTEMBER, 19231

JOHNS HOPKINS HOSPITAL BULLETIN

due to technique and how much to the condition of the
original cell. It may be stated that fragile leucocytes
may be produced at any time by rough handling, but that
when the leucocytes are about to go into the non-motile
phase, they may appear as the fragile forms in the f&d
smears.

In the series from human blood, there is also some indi-
cation of a period during the day when there is a con.
siderable increase in the proportion of the small lymph-
ocytes which we regard as the old form. I am well
aware that this report of the non-motile forms and of the
aging of lymphocytes is incomplete, indeed quite frag-
mentary, and I hope subsequently to give an analysis of
the daily cycle of the white cells with a more standard-
ized technique and with more adequate data. It would
then be possible to give a curve of the rhythm of the types
of white cells. I mention these experiments at this time,
however, because in the study of blood in clinical cases,
there are often very marked discrepancies which are
usually disregarded. For example, there are often smears
of blood with great numbers of smudges which cannot
be repeated a few hours later. In our experience the
actual showers of the non-motile form last only a half
hour or so, which would easily account for such cases.

The significance of the non-motile cells, if we can prove
that they do occur in the circulating blood, seems to me
to be that they give us a chance for the first time to
estimate the normal death rate of the leucocytes, indeed
to study the normal rhythm of the blood-cells. In the
studies made by Arneth *l on the number of lobes of the
nuclei of the leucocytes, there was given an indication of
the relative age of the leucocytes to be found in the circu-
lating blood, but we have never had any way. of esti-
mating the number that were dying. These non-motile
cells seem to me to show that there is a real chemical
transformation of the cytoplasm as the cell dies, so that
they should be studied from the standpoint of whether
they give a substance to the plasma which has to do with
maintaining the normal rhythm of the leucocytes. If we
can secure such cells, the point might be subjected to
experiment. In this connection, the cells of pus must be
studied with the vital method. It is obvious that we
need to know the mechanism of maintaining the normal
rhythm of tbe different forms of the white cells as a
preliminary for studying the causes of variation from
the normal and it is certainly a rational idea that the
normal death rate might be correlated with this rhythm,
that is, that the dying cells might produce the substances
that would attract similar cells into the blood-stream.
Here it is obvious that there are two factors to be. anal-
yzed, the actual production of new leucocytes in the mar-
row and the calling of them into the blood-stream.  The
maintenance of the normal rhythm is certainly complex;
when large numbers of cells, leucocytes or lymphocytes,
have been called from the blood into the tissues, the fac-
tors are even more complex, and to analyze this mech-

. .

anism is now one of the major problems connected with
the blood.
L/ 3. BamphiZic Zeucocytes.

The method of studying blood-cells in the living form
disproves conclusively the idea of Weidenreich,12JaJ4
that the human basophilic leucocytes of the blood-stream
are degenerating cells. They are unquestionably living
cells, as. has been maintained by Naegeli l6 and by Bunt-
ing.`O  C)f course it will now be possible to find the
changes in the basophilic leucocytes of the blood-stream
that indicate cell death, but so far I have seen only
living basophilic cells. Some of them have nuclei that
are slightly polymorphic but even the ones with the
peculiar rosette-shaped nuclei, such aa are shown in Weid-
enreich's 12~1a figure, are living cells, as proved by the fact
that the nuclei do not stain in vital dyes and that the
cells themselves are amaeboid. In human blood the baso-
philic granules are intermediate in size between the neu-
trophilic and the eosinophilic granules and they have the
special characteristic of taking the dye unevenly, some be-
ing much darker than others. The granules are all. round
but not all of the same size as are those of the neutro-
philie and eosinophilic types. They give a deeper red re-
action in neutral red than either of the other forms.

In the motility of the basophilic cells of human blood
there is but little streaming of the granules and their
rate of motion is much less than that of either of the other
two groups of the leucocytes. I have not seen any changes
in these cells to be related to function, that is to say, I
have not analyzed any of the stained particles as vacuoles.
The observations on them have been, of course, limited
on account of their small numbers. With the supravital
methods the relations of the basophilic cells of the blood
to those of the marrow and of the diffuse connective tis-
sues can now be attacked again.
pi 4. Eosinophilic leucoc3(te8.

The eosinophilic leueocytes have. the large granules
which take the dye uniformly and more intensely than
the granules of the neutrophilic leucocytes.. Like- the neu-
trophilic cells, they show an active amcaboid motion with
a streaming of the granules.  I have never seen them re-
main motile as long as the neutrophilic cells, but I have
seen them move as fast.  Usually they move at a slower
rate and do not remain in motion in the preparations more
than half an hour. I have never made out any vacuoles in
the eosinophilic cells. So far I have not studied any cases
in which there was a marked eosinophilia. The nuclei
are usually two-lobed. When the cell moves, the two
lobes may be dragged far apart, a slender thread keeping
them from separating. The two lobes change in form
as the cell moves, but not as much as the nu&%the
lymphocytes.
cr 5. Mmoeytes.

In studying the living blastoderms of chicks on the
third dayof incubation, I l6 found that there is one group
of the white cells that comes from endothelium. `On the


384

JOHXS HOPKINS HOSPITAL BULLETIN

second day of. incubation, the origin of the red cells from
the endothelium of the vessels can be seen with great
clearness. On the second day, all of the single cells or
clumps of cells clinging to the inner lining.of the vessels
of the area vasculosa can be seen to contain hemoglobin
by the color of the living cell.  The amount of the hemo-
globin may be too .small to show in the deeply basophilic
cytoplasm of the 5xed cell, but the yellow tinge is evi-
dent in the living state. On the third day, an occasional
cell becomes free from the inner wall of the vessel which
not only does not shou- hemoglobin, but does show the
stained particles and the stained vacuoles that charac-
terize the monoc.yte stem. These cells I think are analo-
gous to the monocytes of the adult blood. 9t the same
time, for the nest few days of incubation, very large
masses of similar cells wander from the outer surface
of the endothelium of the vessels of the area vasculosa.
These are the clasmatocytes. Aschoff and Kiyono Ii be.
lieve that the clasmatocytes of the connective tissues and
the mono@es of the blood should be grouped together
under the common name of histiocytes.
..- -

In studying normal, human blood, it becomes evident
that there are two strikingly different types of cells in
the blood belonging to the monoc.ytic strain. Indeed
they are so different in appearance as to require some
proof that they are only tz-o phases in the life of a single
cell. The first form corresponds to the large mononu-
clear form of Ehrlich, a cell entirely different from the
large lymphocytet the second is the transitional cell of
the Ehrlich classification.
I/ The large mononuclear cell is shown in Fig, 3.  It is
a large cell, almost always round, varying in size from
a cell slightly larger than a leucocyte to one the size of
a myelocyte. The nucleus is usually round, may be
slightly indented. and is always eccentric.  The most
striking characteristic of the cell is that its cytoplasm is
completely 511ed with fine uniform particles that stain
slightly darker than the neutrophilic granules in neutral
red. The particles of the cells are usually still, or they
may be in slight motion. In the very young forms, a
spot free from granules in the center of the cell opposite ,
-the nucleus indicates the centrosphere. The cell shows
no active locomotion. but it may move very slowly so
that by watching it 10 to 15 minutes one mav perceive a
change.!' After one is familiar with this cell with the
vital dye, it can be distinguished without any stain at all,
that is, the same gmnnles are very plain in the living
cell. It could never be mistaken for the large lymph-
ocyte, as can ,be Sef?D by comparing Figs. 3 and 7, The
cell is to be distinguthed from a neutrophilic myelocyte
by the fact that the particles are smaller than the neu-
trophilic granules and have a slightly different shade
of color in neutral red. The two cells may be found side
by side in bone-marrow and compared. There are, how.
ever, but few large mononuclear cells in bone-marrow.
It will be noticed that I have used the term "particle,"

in connection with this cell, advisedly, because so far it
has not been possible to determine the nature of the sub-
stance in the terms of my definitions.

The transitional cell, on the other hand, varies much
more in size than the large mononuclear just described,
though the typical ones are very large. They are shown
in Figs. 4 and 5. In the living preparations, they are
seldom round, in fact they are usually very long, but may
be quite irregular, as in Fig. 4. They show considerably
more motility than the large mononuclear form and the
particles in the cytoplasm are often in quite active
motion. The locomotion of the cell is slow and not as
correlated u-ith a flowing of granules as is the case of the
leucocytes. The cell is especially conspicuous for the
amount and the variety pf the substance stainable with
vital dyes. Its cytoplasm is 5lled with particles stain-
able with neutral red, which vary in size all the way from
a fine dust to huge vacuoles. At present I do not know
the relationship between the particles and the vacuoles,
but when the cell is highly stimulated to phagocytic
activity so that its cytoplasm is filled with large vacu-
oles, as in Fig. 5, the fine particles have disappeared.
The fine particles also stain in Janus green, giving a
greenish tinge to the cell in which can be seen small,
scattered mitoehondria. Perhaps it is the same fine par-
ticles that give the cytoplasm its color in 5xed smears.
Of all the cells of the circulating blood the transitionals
change the most with function, the large mononuclears
the least. The only change I have seen in the latter is
a slight increase in the motility of its cytoplasmic par-
ticles while in the case of the former, if my interpretation
of the stainable vacuoles is correct, the transitional is
the most phagocytic of all of the cells in the blood
stream ; that is to say, a single cell can phagocytize a
greater amount of substance than any other cell. They
vary all the way from a cell filled with fine particles and
no vacuoles to a cell whose cytoplasm is so loaded with
vacuoles that nothing else is visible.

These tu-o cells are so strikingly different in the living
state, that it would be impossible from normal blood to
conclude that they both belong to the same group, namely.
the monocytes. But I had the opportunity to study one
case of abnormal blood in which it became clear that
they are merely two phases in the life cycle of the same
cell. This very interesting case was one of Malta fever.
The patient was admitted to the Johns Hopkins Hospital
October 18, 1922. (l&xl. lSo. 48S13) and showed a fever
of a moderate grade correlated with a blood-count in
which the total number of the white cells was normal
but there were ten per cent of monocytes. The diagnosis
of Malta fever was made by Dr. Amoss by finding the
organism in the circulating blood. He made an auto-
vaccine and gave it in four doses at intervals of four
and five days. The first vital differential count which 1
made on November 25th was as follows:


SEPTBIYBER, 19231       JOHNS HOPKINS HOSPITAL

   Polymorphonuclear ueutrophiles . . . . 32%
    Non-motile leucocytes  . . . . . . . . . . . . . .   6.2
    Fksfnophilee  . . . . . . . . . . . . . . . . . . . . . . . .  1
    Basophiles  . . . . . . . . . . . . . . . . . . . . . . . . . .  1
    LYmPl-w tes   . . . . . . . . . . . . . . . . . . . . . . . 39.6
    Large mononuclears  . . . . . . . . . . . . . . . .   .7
    Trausitionals  . , . . . . . . . . . . . . . . . . . . . . . . 16.6
In actual percentage the count did not diEer much

from this throughout the course of the disease, the mon-
ocytes remaining about twenty per cent. After the set-
ond dose all of the monocytes were of the transitional
form ; after the third dose there was a very marked in-
crease in the stainable substance of the transitional cells,
the cytoplasm being completely filled with large stained
vacuoles. As a sign that they were actively phagocytic,
red blood-cells were found engulfed in them. After the
fourth dose there was a brilliant demonstration of the
fact that the transitionals are merely older, more active
large mononuclear forms.  There were a few large mono-
nuclear forms of a young stage, that is, the centrosphere
was plainer in them than I have ever found it in these
cells. From these young forms there was every transi-
tion from the mononuclear to the transitional form, of
the inactive and active state, on to cells that were de.
generating. No sharp line could be drawn between the
large mononuclear or young forms and the transitional.
Dying cells have very little reaction to the vital dye,
show no movement of particles, no locomotion and have
clear unstained vacuoles in the cytoplasm,  The entire
life cycle of the monocytic strain from the youngest to
the dying cells could be seen in one preparation.

The subsequent history of the case was also interest-
ing. I had a chance to follow the case long after the
acute illness was over. The leucocytes remaining very
low, the increase in monocytes being at the expense of
the leucocytes, Dr. Amoss gave the patient a series of
three doses of Gphoid vaccine, in the hope that there
might be an increase in the percentage of leucocytes.
Instead, there followed a stimulation of the lymphocytic
strain .of cells. Lymphocytes showed a marked increase
in the number and size of the vacuoles in the neutral
red and there was a marked variation in their staining
reaction in Wright's stain. This gradually subsided but
the increased vacuolization of the monocytes continued
over a period of some months.  Gradually this subsided.
The patient was discharged from the hospital with a
negative blood culture on December 10, 1922, but it was
not until April that the staining reaction of the mon-
ocytes became normal. On April 24, 1923, the monocytes
were still ten per cent of the white cells, but they were
normal in staining reaction. Blood cultures were still
negative.

From the study of this case I became entirely con-
vinced that the large mononuclear cell is the younger
stage of the transitional form. The transitional form is
more active as shown both by increased motility and by

LETIN                  285

increased phig%ytic power as indicated by the large
numbers of stainable vacuoles.

In connection with these studies of blood, I have been
making smears of living cells from lymph glands and
from the spleen in different animals. I press out, a little
fluid from the gland, put it on a slide prepared as for
the blood films and then add a small amount of a scraping
from a freshly cut surface. In such preparations, as is
well known, tihere are always many clasmatocytes, that
is, cells. of the transitional type from lymph glands,
spleen and bone-marrow, but the cells identical in type
with the large mononuclear of the blood stream I have
found in any considerable numbers only in scrapings
from the spleen. The animals used have been cats and
rabbits.

6. Lymphocyte8. .

5 The lymphocytes have proved to be the most dif5cult
of the white cells to analyze. As a matter of fact we
have had no convincing proof as to which is the young
form, one group of hematologists thinking that it is the
large lymphocyte, the other that it is the small. I am
confident that these vital studies offer very considerable
evidence toward the view that $he large lymphocyte is
the young stage. As was shown by Miss Simpson, Iymph-
ocytes are characterized by definite clumps of large mito-
chondria. All of the lymphocytes, with one occasional
exception, have a, cytoplasm that is strikingly clear in -
the living form. This is in ma.rked contrast to the 5ne
granulaton of the monocytes. In the cytopiasm of all
lymphocytes there are two substances that take vital
dyes ; first,, they all have clumps of mitochondria, and
second, two or more small vacuoles reacting to neutral
red. `t In Fig. 7, is the large lymphocyte, showing three
vaeubles and the characteristic mitochondria, placed as
usual opposite the nucleus. Both of the substances are
often in motion and an occasional rod or vacuole is to
be found along the rim of the nucleus. The large lympho-
cyte is almost always rounded up, as shown in this figure;
its nucleus is round or oval, seldom irregular and the
cell very seldom shows any locomotion.  The stri!ing
characteristic of the intermediate form is that it is fre
quently in motion.  Also it shows much more variation in
the reaction to neutral red. Such a cell is shown in Fig.
6. The intermediate Cell is distinctly smaller than the
large lymphocyte; it has the same cluster of mitochon-    .
dria opposite an eccentric nucleus. Among the rods are
a few vacuoles of which several are shown in Fig. 6.  The
cell moves much more slowly than the leueocyte; when
*he cell is moving, the nucleus is usually in the front
end and its form is constantly changing.  Both the mito-
chondria and the vacuoles are in active motion, not in
the least correlated with the locomotion of the cell;
indeed the particles may be in very active motion when
the cell itself is quite still. The particles stainable with
neutral red in the lymphocytes vary from one or two up
to about ten or even more. The small lymphocytes al-


286

JOHNS -KINS HOSPITAL BULLETIN

[No. 391

ways show the same clump of mitochondria, and at least
one or two vacuoles. The vacuoles may increase in size
and number but less markedly than in the intermediate
forms. The small cell seldom moves at, all.

In all of the lymphocytes tshe cytoplasm is much more
clear than that of the monocytes; that is to say, the
lymphocyte lacks the fine granulation of the mono&e.
Indeed it is on this account that the mitochondria are
very readily seen in the living cell without being stained
at all.  This is not true of any of the other cells of the
blood.  There seems to me to be little doubt but that the
substances that stain in neutral red in the lymphocytes
belong to the category of vacuoles in my definition ; that
is to say, they are structures that vary markedly in differ-
ent functional states of the cell. `This can often be made
out very clearly in the case of the small lymphocyte
where the two or three vacuoles can become markedly
larger than usual without causing the least di!Ilculty in
discriminating the cell from all other types in the blood.
The size of the cell is so distinctive and the massive
blotches of chromatin in the nucleus are so plainly seen
in the living state that the cell cannot be confused with
any other. With the intermediate and the large lymph-
ocyte the case is different. When they are highly stimu-
lated, the neutral red vacuoles may approach in size and
number the condition of the monocytes. The mitoehon-
dria remain the same and it is then necessary to use the
double stain vitally and to correlate the differential
counts with studies of fixed smears.

There is one form of the lymphocyte which occurs
occasionally in normal blood in which the nucleus is ex-
actly in the center of the cell instead of having th;! usual
eccentric position, and in these cells, which belong to the
large and intermediate group, the cytoplasm shows a
very fine granulation like ground glass. In the fixed
smears these are the cells with markedly basophilic cyto-
plasm. I think that they will be understood only when
we have a complete account of the life cycle of the lymph-
ocytes.

In studying lymphocytes in the living forms, it is strik-
ing how seldom the nuclei are as round as in the fixed
smears. This is true even of the small f.orms.  In cases of
lymphoid leukaemia, I have found marked changes in the
nuclei, especially of the small forms. In one case (Med.
No. 49241) a large proportion of the small lymphocytes
had nuclei which were split in half or fragmented into
three parts. Wehile this was very clear in the living
specimens, fixed smears did not show these degenerations
of the nuclei at all. In this case I saw a nucleus of a
large lymphocyte divide by direct division, division
of the cell did not follow but there was a division of the
mitochondria into two parts opposite the cleft in the
nucleus.  Subsequently I found two more large lymph-
ocytes with two nuclei and with the same arrangement
of mitochondria.  All of these observations indicated
unusual or pathological conditions of the lymphocytes

and it was very striking in this case that there was no
increase in phagocytic power, that is to say, there were
almost no vacuoles in neutral red in the entire strain of
the lymphocytes in this case. Thus there were marked
changes in the nuclei of the cells correlated with low
functional activity.

It has been striking that the lymphocytes taken directly
from the lymph glands have not shown as many or as
large vacuoles as t,hose of the blood. I do not know
whether there is any correlation to be made between
the azurophilic granules and the vacuoles that stain in
neutral red, but I think that there is no such change in
the size of the azurophilic granules as there is with the
vacuoles.

There was a patient in the Johns Hopkins Hospital
(Med. No. 489984, Path. No. 7383)) whose case was very
interesting in connection with the lymphocytes.  The
condition was exceedingly complex; `no definite diag-
nosis could be made even from the autopsy, but it was
probably related to the lymphoid leukemias.  The patient
was in the hospital during the last month of life. The
striking point in the case was the enormous enlarge
ment of spleen and lymph glands. Until the last few
days the total count of the white cells was normal and
the differential count showed only signs of the so-called
pathological lymphocytes.  Finally, there was a marked
leucocytosis reaching as high as 110,000 cells, during
which strangely enough the proportions of the cells were
still approximately normal.  Myelocytes appeared in the
blood-stream during the last four days of life and at
autopsy the bone-marrow was found to be active.

I had an opportunity to study smears from the lymph
glands, spleen and bone-marrow from this case while the
cells were still living. Similar smears from the lymph
glands and spleen of cats and rabbits show groups of
cells, stuck together in clumps, which are in no sense
syncytial masses, since the cells can be separated by
pressure.  These cells have the following characteristics:
the cytoplasm is as basophilic as the nucleus ; the nuclei
are the least visible of any living nuclei I have yet worked
with. In smears with Wright's blood stain, the cyto-
plasm of these cells is almost as basophilic as the nuclei.
In the living state there are no granules of any sort to be
made out in these cells. This type of cell may well be
the stem cell of the blood-cells that do not come from
endothelium, namely, of the leucocytes and lymphocytes.
In this case in which the striking changes were in the
lymph glands and spleen, the spleen had similar masses
except that every cell had a clump of mitochondria like
those that characterize lymphocytes. This may represent
a differentiation of the primitive cell into a forerunner
of the lymphocytic series.

From these studies, it is my opinion that the large
lymphocyte is the young form of which only a few occur
in the normal blood ; that they are comparable to the
myelocytes of the granulocytic strain except for the fact


SEPTPIMBER, 19231

JOHNS HOPKINS HOSPITAL ~LLETIN

287

that a few do occur in the blood. The intermediate
forms are, I think, more active functionally, first, because
they show the greatest amount of motility, and second,
the greatest variations in phagocytic power. The small
forms are the oldest, but most of them are active cells
with power of phagocytosis. They show the greatest
number of degenerating forms.  The only suggestion
which I have concerning the cells with marked increase
of the baaophilic reaction of the cytoplasm is that they
may be near the phase of cell division, in which there is
a general increase in basophilic reaction of cytoplasm.

It haa already been indicated that there are some dif-
ficulties in analyzing the blood-cells in the vital method.
The most frequent difficulty is the one just mentioned,
that of discriminating lymphocytes, when they are in
a state of unusual phagocytic activity, from moderately
active monocytes.  In such cases comparison must be
made with fixed. smears and the case must be followed
over a period of time. In other instances, as for example
in certain cases of infectious mononucleosis, I think
that it is possible to say definitely that it is the lymph-
ocytes that cause the increase in mononuclear forms and
not the monocytes. Another difliculty, I have found, is
the distinguishing of certain very sluggish leucocytes,
in which there has been a marked depletion of the neu-
trophilic granules, from inactive monocytes. As with all
other methods, there are from time to time cells which
must be regarded as unclassified.

7. Platelets and hfegalokaryocytes.

Platelets are very characteristic in the vital dyes.
They have two types of granules, particles that occur in
clumps and stain with neutral red and discrete particles
that stain blue in Janus green. These particles are
always still, never in Brownian motion. These charac-
teristics distinguish them from all other forms of debris
in vital preparations. In these vital films platelets are
more often found in large clumps than they are in fixed
smears. '                   .

In a case of chronic myeloid leukemia (Med. No.
48856) admitted on November 25, 1922, we were sur-
prised to find very large numbers of megalokaryocytes
in the circulating blood. In this case there were 80,240
white cells on November 26th. On November 28th I
counted 306 white blood-cells and 19 megalokaryocytes,
making 325 cells counted. Besides these there were 18
large masses of platelets without nuclei.  Thus the nucle-
ated giant cells made approximately five per cent of the
total white cells in the blood. The next total white
count was 89,280, made on March 5th. Estimating the
number of white cells on November 28th as 80,000, there
were then 4000 megalokaryocytes per cu. mm., in the
circulating blood. In studying these giant cells on the
slide, the granules stainable in neutral red gradually
formed in little clumps and the cytoplasm then frag-
mented into typical platelets on the slide. In Fig. 8 is
a typical giant cell as it appeared on November 28th. In

Fig. 9 is one with very little cytoplasm but with a nucleus
very typical of the megalokaryocyte to be found in the
bonemarrow and spleen. On March 4th, the patient Was
given a dose of X-rays, and two days later the total num-
ber of the white cells dropped to 55,350 and all the giant
cells had disappeared from the blood.

In Fig. 10 is a cell taken on March 4th, in which I
followed the disintegration of the outer mass of CytO-
plasm into platelets. As will be seen, the nucleus with
a very small amount of unfragmented cytoplasm is in
the upper left hand corner of the mass, most of the
cytoplasm being in the form of platelets. The contents
of some of the platelets were not drawn, since the cell
became too disintegrated to show their character.  These
observations seem to me to he a confirmation of Wright's
theory that it is the giant cell which gives rise to plate.
lets. Certainly platelets can be detlnitely separated from
the debris of the blood-cells in the vital preparations.
Since, according to Minot,lg the occurrence of giant cells
in the blood stream is not as uncommon as had been
heretofore supposed, the number of cases identified will
I am sure increase where the technique of studying blood
supravitally is introduced, since the cells are so much
more easily identilled in these preparations than in
stained smears where the giant cells are almost certainly
damaged. Moreover, the occurrence of any very large
clumps of platelets in the circulating blood will suggest
that. giant cells be searched for.

In conclusion, in these studies of white blood cells, it
has been shown that there is much evidence to favor the
view that the large mononuclear cell is the young form of
the monocytic strain and that the large lymphocyte is
the young stage of the lymphocytes. The method offers
a chance to distinguish cells which have been stimulated
from those which are degenerating. I am convinced that
the method opens a new phase of the physiology of the
white blood-cells.  The next step will be to complete the
life cycle of each type of white cell.  With these data in
hand we shall be in position to complete the study of
the normal rhythm of! the leucocytes and thus have a
foundation for analyzing the method for maintaing this
rhythm.

LEmmDs

Fig. 1-Polgmorphonncleai neutrophilic leucocyte from nor-
mal, human blood, drawn from the living cell in which the neu-
trophilia granules have been supravitally stained `with neutral
red.' This is a free-hand drawing; the motion of the cell was
too great for the uee of a camera lucidal It shows the general
form of an advancing pseudopod becoming a long filament. la
is a red blood-cell for the magnigcation.
Fig. 2.-Non-motile leucocyte from normal human blood. The
preparation from which this cell was taken wae supravitally
stained with neutral red, but this cell did not take any of the
dye. The large msss in the center is the nucleus, which had
four lobes before it became oedematous. The irregular granules
which fill both the endoplasm and the ectoplasm were highly
refractive. 2a is a red blood-cell for the magnification.


JOHNS HOPKINS HOSPITAL BULLETIN

Fig. 3.-Monocyte of the large mononuclear type, from nor-
mal human blood, stained supravltally with neutral red.  The
magnification is not given, but it is the largest type in the cir-
culating blood.

Fig. 4.-Monocyte of the transitional type, from normal human
blood. stained supravitally with neutral red. The cell was in
motion; in the cytoplasm are two types of substance that re-
acted to the dye, fine particles and larger, round vacuoles. 4a
is a red blood-cell for the magnification.

Fig. S.-Monocyte of the transitional type, taken from the
blood of a patient after an attack of Malta fever (Med. No.
48513). but while the monocytes still showed a marked stimulation
to the formation of vacuoles. The vacuoles, which were stained in
neutral red, are shown in gray. This is the same type of cell as
that shown in Fig. 4, except that it was more active functionally.
Drawn with a camera lucida, Leits Obj. l/12, Zeiss Comp. Ck.
pi, and then enlarged 3x.

Fig. B.-Lymphocyte from normal human blood, supravitally
stained with neutral red. The cell was moving in the direction
of the arrow.  In the cytoplasm are 21 vacuoles which were
stained, and and a clump of mitochondria in the form of rods
which were not stained. The nucleus is markedly irregular in
consequence of the movement of the cell.  6a is a red blood-cell
for the magnification.

Fig. 7.-Lymphocyte of the large type, from normal human
blood, supravitally stained with neutral red and Janus green.
It shows the characteristic clear cytoplasm of the lymphocytic
strain.  The nucleus is eccentric; in the main mass of the
cytoplasm are four small vacuoles which stained with neutral
red and a large clump of mitochondria which stained in Janus
gr-.

Fig. I.-Megalokaryocyte from human blood, taken from a
case of myeloid leukemia (Med. No. 488561. The nucleus is in
the upper part of the cell and the cytoplasm shows a marked
granulation which was supravitally stained in neutral red., The
character of the cytoplasm was accurately copied with the
camera lucida. (Leitz Obj. l/12, Zeiss Comp. Gc. vi.) '

Fig. 9.-Megalokaryocyte drawn from the same preparation as
the cell of Fig. 8. It shows a cell which has lost most of its
.oytepl&n;- the-nucleus is mulberry-shaped.  9s is a red blood-cell
which corresponds both to this figure and to Figure 8.

Fig. lO.-Megalokaryocyte from the same case as Figs. 8 and 9.
This preparation was taken from the patient on the morning
of December 4th, immediately before he received the first dose
of X-rays.  The slide was given the same exposure to the X-ray8
as the patient.  The giant cells were watched throughout the
day for the disintegration of the cytoplasm into platelets. The
preparation was kept in the incubator over night and the ciranv-
ing was begun on the next day, the leucocytes being still alive
and moving. On the next day, Dec. 6, an attempt was made
to complete the drawing and the dead cell marked 1Oc was put
in. The character of the platelets, however, had changed so
they were left with the outlines originally drawn with the

camera lucida. On this day all of these cells disappeared from
the blood of the patient. $Oa is a red bloodcell; lob is the
nucleus of the giant cell.  (Leits Obj. l/12, Zeiss Comp. Gc. vi.)

              BIBLIOGRAPHY
1. Sabin: Studies on blood.  The vitally stainable granules
as a specific criterion for erythroblasts and the differentiation
of the three strains of the white blood-cells as seen in the living
chick's yolk-sac.  Johns Hopkins Hospital Bull., 1921, xXx11,
314-321.

2. Simpson: Vital staining of human blood with especial
reference to the separation of the monocytes. Univ. OS Calif.
Pub. in Anat., 1921. I, l-9:

3. Evans and Scott: On the differential reaction to vital dyes
exhibited by the two great groups of connective-tissue cells.
Contributions to Embryology, 1921, X, l-55. Carnegie Inst.,
Wash. Pub. No. 273.

4. Simpson: The experimental production of circulating en-
dothelial macrophages and the relation of these cells to the
monocytes. Univ. of Calif. Pub. in Anat., 1921, I. 11-19.
5. Shipley: The physiological significance of the reaction of
tissue cells to vital benzidene dyes. Amer. Journ. of Physiol.,
1919, XLIX, 284.301.

6. Chambers: Microdissection studies. II. The cell aster: a
reversible gelatlon phenomenon.  Journ. of Exp. ZoSl., 1917.
XXIII, 483-504.

7. Herwerden: A method for fixing films of human blood
cells during the amceboid movement of leucocytes and thrombo-
cytes. Journ. Exp. Med., 1920, XXXH, 135-138.
8. Clark and Clark: Changes in the nuclei of leucocytes in
aseptic inflammation in the tadpole's tail. Proc. Amer. Ass.
Anat., Anat. Record, 1923, XXV, 123.
9. Bchilling: Lebende weisse Blutktirperchen im Dunkelfeld.
Folia Haematol., 1908, VI, 429-443.
10. Bunting: The leukocytes.  Physiol. Reviews, 1922, II, SOS-
520.

11. Ameth: Die Qualitative Blutlehre. Leipzig, 1921.
12. Weidenreich: BeitrSge zur Kenntnis der granulierten Leu-
cocyten. Arch. t. mikr. Anat., 1908. IXXII, 209325.
13. Weidenreich: Die Morphologic der Blutxellen und ihre
Bexiehungen zu einander. Anat. Record, 1910, IV. 317940.
14. Weidenreich: Zur Kenntnis der Zellen mit basophilen
Granulationen im Blut und Bindegewebe.   Folia Haematol.,
1908, V, 135-155.
15. Naegeli: Blutkrankheiten und Blutdiagnostik. Dritte Auf-
lage. Berlin und Leipzig, 1919.
16. Sabin: Studies on the origin of blood-vessels and of red   '

blood corpuscles as seen in the living blastoderm of chicks
during the second day of incubation. Contributions to ,Embry-
ology, 1920, IX, 215-262. Carnegie Inst. Wash., Pub. No. 272.
17. Aschaff and Kiyono: Zur Frage der grossen MOnOUU-
kle&ren. Folia Haematol., 1913, XV, 383-390.
18. Wright:  Histogenesis of the blood platelets.   Journ.
Morphol., 1910, XXI, 263-278.
19. Mhot:  Megacaryocytes in the peripheral circulation.
Journ. Exp. Med., 1922, XXXVI, l-7.

THE JOHNS HOPKINS HOSPITAL BULLETIN

The Hospital Bulletin contains details of hospital and dispensarg practice, abstracts of pa,pers read before the
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THE JOHNS HOPKINS HOSPITAL BULLETIN. SEPTEMBER. 1923

PLATE XXXV