Vol. 49, No. 1
Printed in U.S.A.
T H E AMERICAN JOURNAL OF CLINICAL PATHOLOGY
Copyright © 1968 by The Williams & Wilkins Co.
AN UNSUSPECTED ULTRASTRUCTURAL FAULT IN HUMAN ELLIPTOCYTES
JOHN W. REBUCK, M.D., P H . D . , AND ELLIS J. VAN SLYCK, M.D.
Departments of Pathology and Medicine (Hematology), Henry Ford Hospital, Detroit, Michigan 48208
In 1943 Penfold and Lipscomb13 cited an
incidence of an increased rate of erythrocyte
destruction in hereditary elliptocytosis of
only 12 %, and stated further that only a few
instances of frank hemolytic anemia were
associated with this abnormality of the
erythrocyte. In the 25 intervening years
it has become increasingly apparent that this
concept of the benignancy of the condition is
somewhat distorted. More recent reports 7 ' 10 ' 19 have stressed the hemolytic character of elliptocytosis, not only in the
homozygote, but also in the heterozygote.
Disclosure of instances of compensated and
mildly decompensated hemolysis by more
refined technics accounts for this enlightenment.
To date no satisfactory correlation has
been established between the degree of
aberration and the rate of hemolysis seen in
elliptocytosis. 3 ' 7 ' 11 This may be partly the
result of other factors, such as the inconsistent presence of splenomegaly with variable degrees of hypersplenism, or perhaps to
hidden associated red cell anomalies, as
suggested recently by Ozer and Mills,12 and
Davidson and Strauss.8
In an attempt to gain further insight into
the physical phenomena which render the
elliptocyte susceptible to premature disintegration, a morphologic study was carried
out on the normally elliptical erythrocytes
of the llama (species Camelidae) and on
elliptocytes from patients with hereditary
elliptocytosis. Electron microscopy and a
shadow-casting technic were used, and form
the basis for the present report.
were allowed to stand with the fixative at
room temperature for 4S hr. The formalinized cells were then removed from the tubes
and washed three times in isotonic saline
and three times in distilled water. They were
then spread thinly over Formvar-covered
glass slides and dried.
Direct mounting was achieved by placement of specimen screens over areas suitable
for erythrocytes selected by light microscopy. The plastic coating was cut from the
end of the glass slide and cellophane tape
was pressed over both of the screens and the
coated and uncoated areas of the slide.
Tape removal carried away with it the
erythrocyte-bearing film from the glass surface. Specimen screens, located between the
erythrocyte-bearing film and the tape, were
cut from the tape and mounted directly in
the electron microscope. (Figs. 2a, 5 to 7,
Sa, 9, and 10 are micrographs of erythrocytes so prepared.)
Additional preparations were shadow-cast
with vaporized metallic chromium at an
angle of 25 degrees and then mounted as
above. (Figs. 1, 26, c, 36, and 46 are micrographs of the shadow-cast erythrocytes.)
Elliptocytic erythrocytes were also hemolyzed in distilled water, mounted directly,
and compared with similarly hemolyzed
sickle cells obtained by a modification of the
Beck-Hertz technic (previously described by
llebuck and associates16 for electron microscopy). Figure S, 6 and c, was so prepared.
The erythrocytes depicted in Figure? were
obtained from a 7-year-old Negro boy with
frank hemolytic anemia (Hb. 6.0 Gm. per
100 ml.). They are compared with the
erythrocytes presented in Figures 5 and 6,
which were obtained from his parents, each
of whom displayed the elliptocytic trait.
Blood from five additional unrelated heterozygotes was the source of the other micrographs.
The elliptical erythrocytes of the llama
(Figs. 3 and 4) were generously supplied by
Dr. W. K. Appelhof of the Detroit Zoological
M A T E R I A L S AND METHODS
Blood from seven patients with hereditary
elliptocytosis and one patient with hereditary elliptocytic anemia was studied. A
saline-formalin fixative was added directly to
small glass tubes containing 0.02 ml. of
blood from each subject. The erythrocytes
Received February 27, 1907.
19
20
REBUCK AND VAN SLYCK
Vol. 49
FIG. 1. Three degrees of human elliptocytosis. A, oval; B, elliptical; C, bacilliform. X 8500 (reduced
4250).
to 4250)
2a
FIG. 2. Human elliptocytes. A, direct microscopy; B and C, canoe-shaped shadows. X 8500 (reduced to 4250).
Park. These elliptocytes were studied after
fixation with both direct mounting (Figs. 3a
and 4a) and metallic chromium shadow-casting at an angle of 25 degrees (Figs. 36 and
46). They were also exposed to distilled
water.
RESULTS
The erythrocytes of the llama (species
Camelidae) normally are elliptocytic, and
on ordinary blood smears appear to be
similar to human erythrocytes from patients
with the elliptocytic trait (Figs. 3a and 4a).
The shadow cast at right angles to the long
axis of the cell by the llama's elliptocytes
(Figs. 36, 46) differs significantly from hu-
man elliptocytes, however. The expected
elongated semilunar shadow is cast by the
llama elliptocyte, whereas a projection at
either pole, giving the shape of a canoe to the
shadow, is cast by the human elliptocyte
(Figs. 1, 26, c). This finding implies that
the human elliptocyte is in reality a biconcave, dumbbell-shaped structure in one plane
and an ellipse in another plane. Furthermore,
our morphologic study demonstrates a bipolar massing of hemoglobin in human
elliptocytes which could logically account for
this structural abnormality (Figs. 9 and 10).
Our concept of the three-dimensional appearance of the human elliptocyte is depicted in Figure 11.
Jan. 1968
ULTRASTRUCTURAL F A U L T I N H U M A N
3a
F I G . 3. Llama elliptocytes. A, direct
microscopy; IS, semilunar shadow.
Of further interest is the fact that llama
elliptocytes do not lyse when they are suspended in distilled water, but human elliptocytes do undergo stromatolysis and lysis
(Fig. S, a and 6) induced by distilled water.
Our studies also show that the elliptocytes of
the homozygote (Fig. 7) were more susceptible to lysis than were either heterozygous
elliptocytes or normal erythrocytes. During
the phase of crenation in these studies, it was
noted that the polar contours of the elliptocytes were preserved, while the sides of the
elliptocyte folded, or crumpled, like those of
an accordion (Fig. 10).
DISCUSSION
Motulsky^and co-workers11 in 1954
theorized that, because hereditary ellipto-
F I G . 4. Llama elliptocytes. A,
shadows.
ELLIPTOCYTES
21
cytosis and thalassemia had many features
in common, perhaps a hidden, unknown
factor or factors, as in thalassemia, rendered
one heterozygote's elliptocytes more susceptible to destructive forces than another's.
Since then, Ozer and Mills12 have reported
three isolated instances of elliptocytosis
with hemolytic anemia, associated in one
with cirrhosis and decreased erythrocyte
glutathione, in another with glucose-6-phosphate dehydrogenase deficiency, and in the
last with no demonstrable enzymatic defect.
They suggested that the presence of frank
hemolysis and elliptocytosis should stimulate a search for a coincidental biochemical
abnormality.
Hereditary elliptocytosis has been sporadically described in association with the
heterozygous occurrence of hemoglobin
g;5.6.9,"i8,19 hemoglobin C,2 and /3-thalassemia.1, 14 One instance of homozygous S disease with mild "ovalocytosis" has been
reported.17 In the majority of the heterozygotes, significant hemolysis was not apparent, although, in the case reported by Van
Slyck and Rebuck18 it was.
No abnormality of the globin amino acid
chains peculiar to the elliptocyte hemoglobin
has been found. Of interest, however, is a
finding reported by Breuer and associates4
in 1956. By using the moving boundary
electrophoresis method to examine various
congenital hemolytic anemias, they found
two components of hemoglobin in the descending limb in one case of elliptocytosis, as
direct microscopy; IS, semilunar
FIG. 5. Human elliptocytes of the father of the patient whose cells are depicted in
Figure 7. X 8500 (reduced to 4250).
FIG. 6. Human elliptocytes of the mother of the patient whose cells are depicted in
Figure 7. X 8500 (reduced to 4250).
FIG. 7. Jluman elliptocytes, homozygous state. Notestromatolysis. X 6500 (reduced to
3250).
22
8a
10
F I G . 8. A, human elliptocyte partial hemolysis; note retention of elliptocytic outline.
B, human elliptocyte, complete hemolysis with retention of elliptocytic outline. C, sickle
cell, complete hemolysis with reversion to disk form. X 8500 (reduced to 4250).
F I G . 9. H u m a n elliptocytes. Note densities at poles. Stages in schistocvlosis. X 8500
(reduced to 4250).
F I G . 10. H uman elliptocytes. Crenated forms. Note bipolar absence of excrescences. X 8500
(reduced to 4250).
23
24
REBUCK AND VAN SLYCK
Vol. 49
&**•'
11
Fici. 11. Artist's drawing of bipolar hemoglobin aggregation in the human elliptocyte
well as in hereditary spherocytosis. This
phenomenon of "splitting" of the protein in
the descending limb can be demonstrated
with normal adult hemoglobin, but only by
raising the temperature or by lowering the
ionic strength. This finding does not necessarily indicate a different arrangement of the
amino acids in the globin chains, but may
reflect a weakening of the binding forces
between the protein subunits in the hemoglobin molecule.
It is possible that the peculiarity of shape
of the spherocyte or elliptocyte also contributes a physical stress factor toward
intermolecular disassociation of normal
hemoglobin within the cell, since Ponder 15
has shown that normal hemoglobin has both
a structural and respiratory function. Indeed, Ponder equates the intermolecular
forces of normal hemoglobin with those
found within molten metal. The findings in
the present report lend support to this latter
concept, since the bipolar massing of hemo-
globin seen in human elliptocytes might well
be expected to weaken the intermolecular
binding forces, as well as to render the polar
surfaces more resistant to crenation. The
physiologic true elliptocyte (a biconcave
ellipse) seen in the species Camelidae shows
a remarkable resistance to the lytic forces of
a hypotonic environment. This suggests an
advantageous arrangement in desert creatures who, because they must imbibe huge
quantities of liquid at infrequent intervals
for survival, could be expected to have a
wider range of osmolality in their plasma.
One might expect human elliptocytes to behave in a similar fashion, but on the contrary, they lyse promptly on exposure to
hypotonicity, the homozygous elliptocyte
lysing more readily than the heterozygous
elliptocyte.
After evacuation of the intracellular contents by lysis, human elliptocytes maintain
their elliptical outline (Fig. 86), in contrast
to sickled sickle cells, which revert to bicon-
Jan. 1968
ULTRASTRUCTURAL F A U L T I N H U M A N
cave disks after lysis15 (Fig. Sc). This observation implies that one basic defect in
hereditary elliptocytosis resides in the cell
membrane; however, the further aberration
in the shape of the elliptocyte, brought out
by shadow-casting, is the result of hemoglobin massing at the poles.
•SUMMARY
By using electron microscopy and a
shadow-casting technic, we have shown a
constant bipolar hemoglobin massing in the
erythrocytes from patients with hereditary
elliptocytosis. This is not present in the
naturally-occurring elliptical erythrocytes of
the llama. This is most readily appreciated
by viewing the unexpected canoe-shaped
shadows cast by the human elliptocytes.
Further studies to delineate the surface
structure in a more complete manner are
contemplated.
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ELL1PT0CYTES
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