3^7
ON THE SPHERICAL FORM OF THE MAMMALIAN
ERYTHROCYTE
BY ERIC PONDER.
(From Washington Square College, New York University.)
(Received 27th March 1929.)
(With One Plate.)
IN 1895 Hamburger described a remarkable phenomenon which can be observed
in suspensions of mammalian red cells in isotonic, hypotonic, or hypertonic saline
and in sugar solutions, but not in serum or plasma. The normally biconcave discoidal cells become perfect spheres, without undergoing any apparent alteration
in volume; the addition of serum to the suspension of spherical cells, however,
converts the spheres back again into the typical discoidal form. Hamburger has no
explanation to offer for the occurrence of this phenomenon, but suggests that it may
be due to changes in surface tension.
In 1920 Brinkman and van Dam published a study of the phenomenon, in
which they record the changes undergone by the red cells of man and of the rabbit
when placed in a haemocytometer chamber. They correct Hamburger's statement
in one important respect, for they observe that red cells in saline are not spherical
but discoidal, and that they become spherical only when placed in the haemocytometer chamber. In becoming spheres they pass through an intermediate form,
"the crab-apple form," which is spherical but finely crenated. Brinkman and van
Dam believe that the cause of the phenomenon is that the cells receive an electrostatic charge from the glass when they come into contact with the floor of the chamber.
They confirm Hamburger's statement that the change from disc to sphere is prevented by the presence of serum, and claim that it is the cholesterol contained in
the serum which inhibits the change of form.
Unaware of these two investigations, Gough in 1924 described the same phenomenon once gain, but fell into Hamburger's error of believing that the red cells
are spherical when suspended in a volume of saline. This error is very easy to make,
and both Millar and I have made it also; indeed, as will be seen below, it is impossible to avoid it if an oil immersion objective is used for examining the cells.
Gough adds two interesting observations to the previous descriptions: that ammonium oxalate and ammonium chloride act like serum in preventing the cells
from becoming spherical in saline, and that the spherical form becomes crenated
after a few hours, passing spontaneously into the discoidal form in about 24 hours.
He does not give any detailed explanation of the phenomenon.
Both Millar and myself (Millar 1925, Ponder 1925) made the same mistake as
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ERIC PONDER
Gough and Hamburger, in believing that the spherical form is due to the immersion
of the cells in saline and that cells are spherical when freely suspended in this
medium. On such an assumption, I carried out many measurements of sedimentation velocity without receiving any indication that the assumption was wrong1.
It appears, however, on a more detailed examination, that the essential condition
under which the spherical form occurs has escaped all the observers who have worked
on the subject, and that the phenomenon is much more complex than has hitherto
been believed.
1. THE OCCURRENCE OF THE SPHERICAL FORM.
The conditions under which the spherical form of the red cell occurs are best
described by enumerating a series of simple experiments which illustrate various
points. The phenomena are most easily observed with a suspension of human red
cells which is prepared either by adding 0*05 c.c. of blood to 5 c.c. of 0*85 per cent.
NaCl or, preferably, by suspending the washed cells from 1 c.c. of blood in 20 c.c.
of 0*85 per cent. NaCl.
(i) If mammalian red cells, immersed in 0-85 per cent. NaCl or in any of the
ordinary physiological salines, are examined in a hanging drop or in a drop enclosed
in a moist chamber, they will be observed to have the typical biconcave discoidal
form. The discoidal form is rarely so perfect as it is in serum or plasma, for there
is often a considerable amount of coarse crenation in isotonic NaCl and a certain
amount of "cupping" in isotonic citrate, but there is nevertheless no difficulty in
recognising that the cells are essentially discoidal and not spherical.
(ii) If the same preparation is covered with a coverglass in such a way that
only a thin layer of fluid is left between the coverglass and the slide, all the cells
will be observed to have become perfect spheres of about 5*6/x in diameter. At
first sight they seem to have a perfectly smooth surface, but examination under
critical conditions or by dark-ground illumination shows them to be covered with
very small punctate markings which probably correspond to exceedingly fine
crenations. As the distance between the slide and the coverglass is usually about
50-100/A, these small spherical forms move about freely between the glass surfaces
if currents are set up in the fluid. The assumption of the spherical form is thus
determined by the fact that the cells are enclosed between two surfaces and not,
as Brinkman and van Dam believe, by the contact of the cells with the surface of the
slide.
(iii) That the change of form is largely dependent on the distance between the
coverglass and the slide can be shown in the following way. A No. o coverglass,
24 by 50 mm., is supported on a slide, to one end of which is cemented a small
glass strip about 1 mm. thick. A wedge-shaped chamber, whose depth varies from
1
Sedimentation experiments give no indication that the cells are discoidal, for the reason that
a sphere falls through a fluid with very nearly the same velocity as a flat disc of the same volume. The
figures for the velocity of fall as obtained in such experiments are as applicable to discs as to spheres;
an examination of the cells under a coverglass shows them to be spheres, and so the error remains
undetected.
On the Spherical Form of the Mammalian Erythrocyte
389
about 4 0 ^ to about 1000 /*, is thus formed, and can be filled with cell suspension.
Examination of the end of the chamber at which the slide and coverglass are widely
separated shows the cells to be discoidal, while at the narrow end of the chamber,
where the distance between the glass surfaces is small, they are perfect spheres. At
an intermediate position in the chamber the cells are extremely crenated, and look
like small spheres covered with fine spicules. As the deep end of the chamber is
approached, the spherical forms become less marked and the crenation coarser, and
in a like manner the cells with the finest crenations are found nearest to the narrow
end of the chamber. After some minutes crenated forms may be seen even at the
deep end, by which time the finely crenated forms first observed in the middle
parts of the chamber have become perfectly spherical. This experiment indicates
that the discoidal forms pass into the spherical forms by first becoming coarsely
crenated, then by becoming approximately spherical and covered with very fine
crenations, and finally by losing all crenations and assuming the spherical form.
The rate at which these changes take place, however, depends on the distance
between the two opposing surfaces; when they are close together the discoidal
form passes into the crenated form and then into the spherical form with great
rapidity, but when they are far apart the changes take place so slowly that intermediate stages can be observed. In a haemocytometer chamber (100 fx in depth)
changes are slow, and occur much as described by Brinkman and van Dam.
(iv) The rapidity with which the conversion of the discoidal form into the
spherical form takes place may be observed in the following way. A drop of red
cell suspension in isotonic saline is placed on the edge of a coverglass, in such a way
that the cells are carried under the latter by the flow of the fluid. The cells are
discoidal while in the uncovered drop, but almost immediately after they pass under
the coverglass they assume the spherical form. The change may take place in a
fraction of a second at first, but, as more fluid passes under the coverglass and
raises it from the slide, the time taken for the change becomes greater and the
intermediate crenated forms can be observed. Finally, the coverglass may be raised
as much as 150 [x from the slide (under which conditions it will slip on the surface
of the slide when the latter is tilted vertically) and the discoidal form of the cells
entering the space between the two glass surfaces may now be maintained for quite a
considerable time. Firm pressure on the coverglass or removal of all excess fluid
by means of filter paper, however, causes all the cells in the preparation to assume
the spherical form.
(v) The pressure of the coverglass plays no part in the production of the
phenomenon, as may be shown by turning the stage of the microscope at right
angles to the table, or even by turning the slide upside down so that the coverslip
and the fluid containing the cells are on its under surface.
(vi) If the coverglass is removed from a preparation in which all the red cells
are spherical, the spherical form persists for some time in the uncovered drop,
which should be examined in a moist chamber for the purpose of preventing
evaporation. In a few minutes, however, the spheres begin to show fine crenations,
and after four or five minutes may all have assumed the crenated form which is
390
ERIC
PONDER
intermediate between the disc and the sphere. The process may go further and some
of these crenated forms may suddenly flatten out to form more or less typical discs,
which usually present, however, a certain irregularity of contour. This complete
recovery of the discoidal form usually occurs at the edges of the uncovered film,
where drying is more likely to occur than elsewhere, and I am doubtful whether it
should be regarded as a true reversal of the disc-sphere transformation.
(vii) If a covered preparation containing spherical forms is left standing for
from 15 to 20 minutes in the air of the room, it will be found to have undergone
certain changes. Instead of all the cells being spherical and free from recognisable
crenations, there are two additional forms present: (a) crenated cells, which are
probably similar to those which occur when the coverslip is removed from a preparation of spherical cells, and which may represent a form intermediate between
the sphere and the disc, and (b) large and apparently spheroidal cells, which can be
seen to be stuck to the slide. These large cells are peculiarly "plastic," and may be
deformed into curious shapes without becoming detached from the slide. If a little
isotonic saline is run under the coverglass, these cells first crenate, then become
detached from the glass, and finally assume the spherical form; in this form they
are carried along by the currents in the fluid. It is important to note, however, that
the detachment of the spheroidal forms and their conversion into spherical forms
is often accompanied by a considerable amount of haemolysis, haemoglobin-free
ghosts appearing together with the newly formed spheres. It is probable that the
cells which haemolyse are those which have been dislodged from the glass since a
corresponding haemolysis does not seem to take place if no sticking to the slide
occurs (see viii below).
(viii) The fact that the spheroidal cells referred to above occur most frequently
in preparations which have stood for a sufficiently long time under conditions in
which a certain amount of drying occurs at the edges of the coverslip suggests that
the changes observed might be different if the covered preparation of spherical
cells were kept in a moist chamber. This is the case, for under these circumstances
the sticking of cells to the slide does not occur appreciably. A certain small degree
of crenation may make its appearance in the moist chamber, but a preparation can
be kept for 12 hours without the cells losing their spherical shape. The introduction
of a small quantity of fresh saline beneath the coverglass has little effect and is not
followed by haemolysis.
The above descriptions (vi, vii, viii) are based on many hundreds of experiments,
but nevertheless I cannot feel certain that no significant detail has been omitted,
for it is not always possible to reproduce some of the results described in a single
experiment, although they can be observed in the majority of a number. The probable explanation of this variability is one which must be strongly emphasised, for
I believe that a considerable number of erroneous observations can be put down
to the same cause. For two experiments to give comparable results, it is necessary
that the distance between the slide and the coverglass should be the same in each
case, and also that observations should be made at the same interval of time after
the application of the coverslip to the drop. It is safe to assume that these conditions
On the Spherical Form of the Mammalian Erythrocyte
391
were left unfulfilled in earlier work on this subject, for the importance of the
presence of the coverglass does not seem to have been appreciated; I have myself
fallen frequently into this error, and realise how difficult it is to avoid it. The best
method is to measure out with a capillary pipette the quantities of suspension
which are to be placed on the slides; if this is done, it will be found that o-oi c.c.
of suspension, enclosed between a slide and a coverglass measuring 18 mm. by
18 mm., shows excellent spherical forms and may be used as a kind of "standard
preparation."
Even when the strictest precautions are taken, however, it is difficult to decide
to what extent the disc-sphere transformation is reversible. I have arrived at the
conclusion that the spherical form may pass into the slightly crenated spherical
form which is an intermediate stage in the transition from disc to sphere, but that
under most circumstances the possible further transition from crenated sphere to
disc does not occur spontaneously. Gough states that cells which have been left
standing for a few hours in isotonic saline appear as crenated forms rather than as
spheres, and that many of the cells are discoidal after standing 24 hours. I have not
been able to verify this statement; one is apt to be misled by the appearance of
spheroidal cells which are stuck to the glass slide, and it is quite possible that the
changes described might be accounted for by variations in the volume of the fluid
between the slide and coverglass.
It should be noted that the transition from sphere to crenated sphere, which
represents the maximum reversal of the disc-sphere transformation which ordinarily
occurs, takes place even when the cells are covered with a coverslip, i.e. under
apparently the very conditions in which the spherical form is first produced.
(ix) In view of Brinkman's statement that the cause of the phenomenon is the
contact of the cell with the glass, it is important to show that the spherical form
does not occur unless the cells are enclosed between two surfaces. If a drop of
suspension is placed on a slide in a moist chamber, the cells quickly sediment and
come to rest on the glass surface; their form, however, is discoidal, and only a few
show crenations. These crenations are not fine but coarse, and the spherical form
is never observed. As soon as the drop is covered with a coverglass, however, the
discoidal cells first crenate and then rapidly become spherical. This experiment can
be carried out on a haemocytometer slide such as used by Brinkman and van Dam
as well as on ordinary glass slides.
(x) If the suspension of red cells in isotonic NaCl is drawn into a capillary tube
of more than about 150/A in diameter, the cells will be observed to retain their
discoidal form, very little crenation and no spherical forms being observed. If the
suspension is drawn into a capillary tube of about 50^ in diameter, on the other
hand, all of the changes associated with the disc-sphere transformation can be
observed to occur in due order. At first the cells in the tube are typically discoidal;
they then undergo fine crenation, and ultimately become perfect spheres. Experiments with a number of capillaries show that the diameter of the tube is the essential
factor in the production of the spherical form, just as the distance between slide
and coverslip appears to determine whether or not the phenomenon occurs between
B
JEB'Vliv
26
392
ERIC PONDER
two plane surfaces; it is obvious, however, that the formation of spheres is very
much slower in a capillary of 50 ju, in diameter than it is in a chamber bounded by
slide and cover slip 50 JU, apart. In the latter case, the transformation takes place
almost instantly, but in the former it may take as long as fifteen minutes.
(xi) The same phenomena as described above can be observed with suspensions
of the red cells of any mammal which possesses discoidal erythrocytes; nothing
analogous to the disc-sphere transformation, however, occurs with the ellipsoidal
red cells of the camels (Ponder, Yeager, and Charipper, 1928). I have already
described a change in the cells of birds which may be related to the subject under
discussion (Ponder, 1925), but I know of no analogous occurrence in the case of the
red cells of reptiles, amphibians or fishes.
(xii) The change from the discoidal to the spherical form occurs when the cells
are suspended in isotonic NaCl, in isotonic sodium citrate, and in Brinkman's fluid,
Fleischl's fluid, Tyrode's fluid or Clarke's fluid. As will be seen below, even considerable variations of the tonicity of the suspension medium does not change the
essential character of the phenomena.
2. THE EFFECT OF VARYING THE NATURE OF THE
OPPOSING SURFACES.
Brinkman and van Dam, the only authors who attempt to give any explanation
of the formation of the spherical form, conclude that the phenomenon is due to
discoidal cells coming into contact with the glass floor of the haemocytometer
chamber, to the latter possessing an electrostatic charge as a result of previous
rubbing, and to the transference of a part of the charge to the surface of the cell.
They state that the changes in form occur more readily on a glass haemocytometer
chamber which has been rubbed with linen or silk than on one which has not, and
that they do not occur at all on chambers which have been flamed so as to remove
electrostatic charge. This statement is confirmed by McGlone (1926) and by Kasten
and Zucker (1928), both of whom believe that the phenomenon is electrostatic in
origin, but neither of whom have observed that the presence of the coverglass plays
an important part in its production.
The hypothesis of Brinkman and van Dam may be tested in three ways at least,
for, if it is true, (a) the formation of spherical forms should not proceed between
slides and coverglasses which have been flamed, (b) spheres should be readily formed
on surfaces, other than glass, to which an electrostatic charge can be imparted by
rubbing, and (c) the discoidal form should be maintained when cells are enclosed
between an earthed plate of metal and a flamed coverslip.
(a) I have been unable to confirm the statement that the formation of spherical
forms takes place either more rapidly or more extensively when the glass surfaces
have been vigorously rubbed, or that the discoidal form is maintained when the
glass surfaces are discharged by flaming. It is not difficult, however, to obtain
misleading results if the experiments are done on ordinary slides, and if the volume
of fluid between the slide and coverglass is not the same on successive occasions.
On the Spherical Form of the Mammalian Erythrocyte
393
Further, as has been observed above, spherical forms can be seen in narrow glass
capillaries; these, from their very manner of preparation, must be almost completely uncharged.
(b) With a view to ascertaining whether the spherical form is produced with
equal rapidity and uniformity on all surfaces to which an electrostatic charge can be
imparted by rubbing, I have examined suspensions of cells, both covered with a
coverglass and uncovered, lying on slides made of the following materials: glass,
ebonite, Bakelite, vulcanite, sealing wax, celluloid, and amber. In cases where the
substance composing the slide is not transparent, the examination of the cells must
be made with a vertical illuminator.
The results of these experiments may be summarised by saying that spherical
forms are never observed when an uncovered drop of a suspension is placed on a
slide composed of one of these substances, and that rubbing the substance until it
carries a considerable electrostatic charge makes no difference to this result. Crenated forms are often observed when the cells have settled down to make contact
with the slide, especially at the edges of the drop, but the crenations are coarse,
and the extent to which they occur is not influenced by imparting a charge to the
slide by rubbing. If the drop of suspension is covered with a glass coverslip,
whether rubbed or flamed, the spherical forms are produced irrespective of the
nature of the insulating substance of which the slide is composed. The completeness
and rapidity of the disc-sphere transformation, however, varies to some extent with
the nature of the opposing surfaces; it is remarkably rapid and complete, for example,
when the cells are enclosed between two glass surfaces, but is slow and sometimes
stops short at the production of crenated spheres when the slide is composed of
celluloid. The same incompleteness in the transformation is observed if celluloid
coverglasses are substituted for glass ones, while if both slide and coverslip are
composed of celluloid, the discoidal form may be maintained for a considerable
time. In carrying out these experiments, however, one cannot escape the conclusion
that the difference in result can be largely accounted for by the fact that plates of
some substances, e.g. celluloid, do not cohere strongly when separated by a thin
layer of fluid, and that plates of such substances are accordingly more widely
separated than are plates of substances which tend to cohere (e.g. glass). Two plates
of celluloid, for example, will not form spontaneously a chamber of 50-100/* deep,
for they tend to repel each other when separated by a layer of saline; only by
pressing them together can the necessary thin film of fluid be formed between them,
and even under these circumstances the formation of the spherical form is slow
and sometimes incomplete.
(c) If drops of suspension are placed on metal surfaces, the discoidal form
of the red cells is maintained exactly as in the case of the glass surface, but if such
drops are covered with a coverslip, the discoidal cells crenate and pass into the
spherical form. This occurs even if the metal slide is connected to earth during the
experiment and for a long time previously, and occurs also if the coverglass is
flamed. These facts show that the phenomenon is not the result of electrostatic
effects, and do not support the hypothesis of Brinkman and van Dam.
26-3
394
ERIC PONDER
I have used slides made of each of the following metals: gold, silver, copper,
iron, brass, lead, zinc, tin, phosphor-bronze, aluminium, and Monell metal; the
cells being examined by means of the vertical illuminator. Provided that the
volume of fluid between the slide and the coverglass is the same in each case, the
formation of spherical forms is equally complete. If celluloid coverglasses are used,
the discoidal form may be preserved on these metal surfaces, but only until the
coverslip is pressed into close contact with the slide.
As a result of a large number of experiments with various types of surfaces, I
have arrived at the conclusion that the condition essential for the formation of the
spherical form is that the saline in which the cells are suspended shall wet the
surface of the slide or coverslip, or of both. The most striking way of demonstrating
this is to cover the surfaces of the slide and coverslip with paraffin, a process which
may be carried out in the following manner. Two slides are warmed and a small
piece of paraffin wax placed between them; the wax melts and forms a thin layer
between the two surfaces, the slides are drawn apart much in the same manner as
in making a blood film, and with a little practice an exceedingly thin but perfectly
continuous layer of paraffin can be left covering the surface of both slides. Coverslips
are paraffined in a similar way, and it is convenient to use slips which measure
about 50 mm. by 24 mm., to paraffin only one half of the surface, and then to cut
off the unwaxed part. If properly prepared, the paraffin film is so thin that objects
such as red cells may be seen through it with a remarkable degree of clearness.
If the suspension of red cells in saline is placed between two such paraffined
surfaces, the cells retain their discoidal form for an indefinite period. The same
experiment may be carried out using a number of different kinds of wax, and it is
probable that the slowness and incompleteness of the disc-sphere transformation
which is observed when both slide and coverslip are made of celluloid or of
similar substances is principally due to the fact that celluloid is not readily wetted
by saline.
In order that the discoidal form shall be retained it is necessary that both the
opposing surfaces shall be unwetted by the suspension medium. Between a
paraffined slide and a glass coverslip, or between a paraffined coverslip and a glass
slide, the formation of spheres proceeds much as between two glass surfaces,
although not with the same rapidity. It is important also that the opposing surfaces
shall be completely paraffined if the discoidal form is to be maintained, for if cracks
or fissures appear in the wax film, crenation of the cells slowly occurs and spherical
forms may ultimately appear.
3. THE EFFECT OF ADDED SUBSTANCES.
So far as is known, only two classes of substance prevent the disc-sphere transformation, and, when added to a preparation of spherical cells, cause the rapid
reassumption of the discoidal form: all observers agree as to the effect of serum or
plasma, and Gough states that ammonium oxalate and ammonium chloride act
similarly.
On the Spherical Form of the Mammalian Erythrocyte
395
(1) Brinkman and van Dam claim that it is the cholesterol contained in serum
which is responsible for the prevention of the change from disc to sphere, and state
that red cells suspended in saline containing o-i per cent, cholesterol and 0-5 per
cent, serum albumin retain their discoidal form when placed in a rubbed haemocytometer chamber, whereas cells in saline containing 0-5 per cent, serum albumin become spherical. I have been quite unable to confirm this statement, and have also
been unable to observe that the alcohol soluble constituents of serum, when
extracted and redissolved in saline, have any effect on the phenomenon.
The serum (or plasma) constituent which is responsible for the maintenance of
the discoidal form is not contained in the protein-free filtrate of serum, and is not
dialysable. As mentioned above, it cannot be extracted, either with hot acetone,
alcohol, or ether, nor is it associated with the fraction of the serum globulin which
can be precipitated by CO 2 . The protein fraction which remains unprecipitated by
the CO2 method, however, is apparently as effective in preventing the assumption
of the spherical form as is serum itself; the substance which is responsible for the
maintenance of the normal form is accordingly either an albumin or a substance
which is carried down with the albumin fraction.
As all previous investigators have observed, serum or plasma is effective in
preventing the formation of spheres even when in comparatively high dilution, but
since the disc-sphere transformation is dependent upon several variables which are
very difficult to control, it is by no means easy to obtain any quantitative comparison
of the effect of one serum with that of another. In the case of human serum the
discoidal form is usually maintained in serum diluted about 1 in 25 with saline, but
higher dilutions are ineffective in preventing formation of spheres. Similar figures
are found for the serum of the rabbit, ox, sheep and guinea-pig, and lead to the
conclusion that different sera vary very little in this respect.
Although the addition of serum or plasma to a preparation containing spherical
cells causes the spheres to reassume the discoidal form, the resulting discs differ
from normal erythrocytes in at least three respects: their edges are usually scalloped
or slightly crenated, their surface appears mottled if examined under dark-ground
illumination, and they show an unusual degree of stickiness, adhering sometimes to
each other and sometimes to the slide. These changes have been observed by
Millar (1925).
(ii) Gough's statement that 1 per cent, ammonium oxalate or ammonium
chloride in saline prevents the formation of the spherical form is not altogether
correct. If red cells suspended in such solutions are enclosed between slide and
coverslip the discoidal form may be maintained for some minutes; later, however,
crenation begins, and all the changes which are intermediate between the discoidal
and the spherical form are observed. The perfectly spherical form which occurs in
isotonic NaCl, however, is rarely seen, and the disc-sphere transformation may be
incomplete even after six hours, all the cells tending to be spherical but being
covered with fine crenations.
A comparison of the effect of different ammonium salts shows that the discoidal
form of the cell is retained longest in 1 per cent, ammonium chloride dissolved in
396
ERIC PONDER
isotonic saline, that ammonium oxalate is not quite so effective, that ammonium
sulphate, nitrate and thiocyanate are much less effective than the oxalate, and that
ammonium tartrate is altogether ineffective. The property of delaying or partially
preventing the disc-sphere transformation is accordingly not common to all
ammonium salts. Other oxalates, e.g. those of sodium, potassium, and lithium,
have no effect on the formation of spheres.
(iii) I have examined the effect of a number of substances on the disc-sphere
transformation with a view to finding whether any substance other than serum
albumin is effective in preventing it. Among the substances examined were saponin,
bile salts, soaps and other surface active substances, egg albumin, haemoglobin and
a number of other proteins, a number of substances, such as urea, which pass freely
through the red cell membrane, and a large number of organic and inorganic salts.
It is sufficient to say that none of these were effective in preventing the formation
of spheres.
It has already been shown that the diameter of the spherical form is from 5-6 to
5'7/x. This corresponds to a volume of about 100 ^ 3 , which is approximately the
same as that possessed by the cell in its discoidal form. In hypertonic saline, the
spheres which result from the enclosing of the cells between slide and coverslip
have a diameter somewhat less than 5*6 JX and a volume considerably less than that
of the sphere in isotonic saline; in hypotonic saline the diameter of the spheres is
greater, while their volume may be increased by as much as 50 per cent. Except
for this difference in the volume of the spherical forms, the phenomenon of the
sphere formation is the same in salines of widely differing tonicity (from 2 per cent,
to o*6 per cent.), as Hamburger observes.
4. CONCLUSIONS.
The foregoing experiments show that mammalian red cells suspended in saline
differ from similar cells suspended in serum or plasma in that the latter retain
their biconcave discoidal shape when enclosed between two surfaces which are
wetted by the suspension medium, while the former are converted into spheres.
As has been seen, this alteration of form is not accompanied by any alteration in
volume; the essential change, accordingly, is one in which the normally extended
surface of the erythrocyte takes up the smallest possible value compatible with
its enclosing a volume of about 100 /J,S.
The principal difficulty in dealing with this change of form lies in the fact that
the shape which the cell possesses when freely suspended in fluid itself requires
explanation, whereas the spherical form which is assumed between two surfaces
corresponds to a condition of minimal surface energy and is accordingly to be
expected; the problem of the disc-sphere transformation is thus intimately bound
up with the problem of why the normal erythrocyte is biconcave and discoidal
instead of spherical, and can be approached in as many ways as there are explanations of the typical discoidal shape. Essentially, however, only two types of explanation are possible.
On the Spherical Form of the Mammalian Erythrocyte
397
Since the surface energy at the surface of the discoidal cell is some i-6 times
greater than at the surface of a sphere of the same volume, we require to postulate
two forces: (1) a force which maintains the surface of the discoidal form some 60^ 2
in excess of the minimum value, and which may be regarded either as a force which
causes the surface membrane to increase in area until an equilibrium position (the
normal form) is reached, or as a resistance offered by some components in the cell
or its membrane to the force of surface tension, and (2) the force of surface tension
which tends to make the surface minimal, i.e. to produce the spherical form. To
account for the disc-sphere transformation we require to add a third force, which
either {a) lessens or abolishes the first force or (b) which intensifies or augments
the second; this force acts on the cell only when the latter is enclosed between two
wetted surfaces, and then only in the absence of serum.
In this simplest statement of the problem we have three forces involved, regarding none of which anything definite is known. No adequate explanation has
yet been advanced for the maintenance of the normal discoidal shape of the erythrocyte, there is no means of measuring the surface tension at the red cell surface, and
we can do little more than speculate as to the nature of the force which is developed
between the opposed surfaces of slide and coverslip. It is certainly not electrostatic
in nature, as Brinkman and van Dam believe, but is more likely to be accounted for
by considering the "molecular attraction fields" which have been shown to exist
between two closely opposed surfaces (Hardy and Nottage, 1926) and which may
extend outwards from a surface for a distance as great as 10 //,.. If the normal biconcave shape of the red cell were due to repulsive forces arising from some particular
molecular arrangement in the plane of the membrane and tending to increase
surface area, as has been frequently suggested, it is conceivable that the form of the
cell might be profoundly altered by molecular fields existing between two wetted
surfaces.
Although we have at present no way of accounting for the facts recorded in this
paper, we may be certain that the explanation, when finally found, will indicate
that the cell is not a spongy mass containing haemoglobin in its meshes, but a sac
surrounded by a membrane to the special properties of which the special shape is
due. As Gough remarks, it is virtually impossible to imagine the changes from disc
to sphere taking place in a structure composed of a close spongework, the meshes
of which would require to suffer an elongation to more than twice their entire length
in one direction and a compression to nearly half of their initial length in another
when the disc-sphere transformation occurs; these meshes, moreover, would require
to be completely elastic in order that the disc might be reformed. It is quite possible,
however, to imagine the changes occurring in a balloon-like body surrounded by a
membrane, especially if the normal shape of this body is regarded as due to the
action of forces situated in its own membrane, which normally keep its surface
extended in excess of the minimum required to enclose its volume.
398
E R I C PONDER
REFERENCES.
BRINKMAN and VAN DAM (1920). Biochem. Zeitschr. 108, 52.
GOUGH, A. (1924). Biochem. Journ. 18, 202.
HAMBURGER (1895). Arch.f. d. ges. Physiol. 141, 230.
HARDY and NOTTAGE (1926). Proc. Roy. Soc. A, 112, 52.
KASTEN and ZUCKER (1928). Amer. Journ. Physiol. 89, 263.
M C G L O N E (1926). Amer. Journ. Med. Science, 172, 155.
MILLAR (1925). Quart. Journ. Exper. Physiol. 15, 253.
PONDER (1925). Quart. Journ. Exper. Physiol. 15, 235.
PONDER, YEAGER and CHARIPPER (1928). Quart. Journ. Exper.
Physiol. 19, 181.
DESCRIPTION OF PLATE VII.
FIG. A. Human erythrocytes suspended in saline, uncovered. Magnification, 560.
FIG. B. Cells of same preparation, immediately after being covered with coverglass. The cells are
markedly crenated and tending to the spherical form—the "crab-apple form."
FIG. C. Cells of same preparation, after about 30 seconds. The cells are spherical.
FIG. D. Human erythrocytes on a surface of brass, and covered with a coverglass. (Vertical illumination.) Magnification, 390. The cells will be observed to be spherical; the slightly hazy outline is
due to the slight movement of the cells during the long exposure necessary (2 minutes).
JOURNAL OF EXPERIMENTAL BIOLOGY
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VI, PLATE VII
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D
PONDER—ON THE SPHERICAL FORM OF THE MAMMALIAN ERYTHROCYTE