A
S i m p l e
for
M e t h o d
for
S c a n n i n g
Processing
Electron
Erythrocytes
M i c r o s c o p y
CATHARINE L. DEWAR, B . S C , M. W. WOLOWYK, P H . D . , AND J. R. HILL,
M.D.
Family of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Cana
ABSTRACT
Dewar, Catharine L., Wolowyk, M. W., and Hill, J. R.: A simple method for
processing erythrocytes for scanning electron microscopy. Am J Clin Pathol
66: 760-765, 1976. A simple method for preparing erythrocytes for scanning
electron microscopy by sequential fixation with glutaraldehyde and dehydration in a graded series of alcohols is presented. The method will allow
visualization of membrane defects not seen under the light microscope
and is therefore suitable for routine processing of erythrocytes for diagnosis
of pathologic states. (Key words: Scanning electron microscopy; Erythrocytes.)
SCANNING
ELECTRON
MICROSCOPY
Principle
has
evolved as a valuable method for the examination of cell-surface morphology.
With this method, a three-dimensional
image of the erythrocyte is presented with
increased depth of focus and resolution,
allowing visualization of surface features
not seen under the light microscope.
Salsbury and Clarke 7 presented a brief
review of the use of this method, while
Lessin and associates4 discussed the use of
additional ultrastructural methods of examining the erythrocyte membrane. T h e
time required to process erythrocytes for
scanning electron microscopy and the cost
have been major disadvantages preventing
routine use of the method in clinical
pathology. In this paper a simple inexpensive method of obtaining scanning electron
micrographs of erythrocytes is presented.
Erythrocytes must be dried and hardened to withstand the vacuum in the scanning electron microscope. 3 Drying artifacts
such as wrinkling, shrinking, and flattening
of cells can be overcome by sequentially
fixing the isolated cells in glutaraldehyde. 3
Dehydration after fixation is achieved by
rinsing the cells with increasing concentrations of alcohol (which also acts to fix
the cells further), followed by air drying.
Sample for Analysis
Venous blood from normal volunteers or
patients with hematologic disorders was
collected into Vacutainer tubes containing
dipotassium EDTA as anticoagulant (1.5
mg/ml blood). Blood can also be collected
Received August 18, 1975; accepted for publicainto heparinized glass capillaries. It is imtion November 19, 1975.
Supported by a grant from the University of portant that the processing of the sample
Alberta General Research Fund for electron microsbegin as soon as possible after collection.
copy.
Address reprint requests to Dr. Wolowyk: Division
We have also successfully used the presof Clinical Pharmacy Education. Faculty of Pharmacy ent method to process isolated lymphocytes
and Pharmaceutical Sciences, University of Alberta,
for scanning electron microscopy.
Edmonton. Canada T6G 2N8.
760
October 1976
SCANNING ELECTRON MICROSCOPY OE ERYTHROCYTES
Method
Approximately 1 ml. of blood was
centrifuged at 160 X g for 3 minutes and
the plasma and buffy layer were removed
with a Pasteur pipette. T h e erythrocytes
were then diluted with 2 ml of 0.1%
glutaraldehyde in Ca 2+ -free physiologic
saline solution at room temperature and
allowed to fix for half an hour. T h e tube
was periodically mixed by inversion during
this time. The cells were then centrifuged
at 100 x g for 3 minutes and the supernatant
discarded. The cells were then resuspended in 2 ml of 2% glutaraldehyde in
Ca 2+ -free physiologic saline solution at
room temperature and allowed to fix for
another half hour. Again, the tube was
gently mixed during this time. T h e cells
were then centrifuged at 50 X g for 2lA
minutes and the supernatant removed.
T h e cells were then washed with 2 ml of
the saline solution for 5 minutes to remove
the glutaraldehyde, and centrifuged at 50
X g for 2V'i minutes. The cells were resuspended into a second wash in the saline
solution for 5 minutes, then centrifuged at
30 x g for 2 minutes. After removal of the
supernatant the cells were ready for dehydration by washing with increasing concentrations of ethanol. The ethanol concentrations used were 1.8, 10, 20, 40, 50,
70, 80, 90 and 95% ethanol, in that order.
T h e cells were exposed to each alcohol
solution for 2 minutes and centrifuged at
15 Xg for 2 minutes in each case. Once
the cells had been resuspended in 95%
ethanol they were ready for examination
in the scanning electron microscope. One
milliliter of the final suspension was airdried on a glass coverslip glued to an
aluminum scanning electron microscope
stub. A coaling of carbon and gold was
applied before the cells were observed in
the microscope ata beam voltage of 20 K.V.
761
Table 1. Percentages of Abnormal Cells
Observed in a Normal Blood
Sample by Light and
Electron Microscopy
Mean ai id Standard
1j r o r
Capillary
Teclinic
Blood smear
Scanning electron
microscopy
Sample
Venous
Sample
1 1.8 ± 3.2
12.9 ± M.2
8.4 ± 2.2
11.7 ± 4.1
pi perazine-N'-2-etha nes ul fon ic acid),
Sigma Chemical Company, P.O. Box
14508, St. Louis, Mo. 63178. Dextrose
(anhydrous), Baker Chemical Company,
Phillipsburg, N.J. 08865. Glutaraldehyde
EM (25% aqueous solution), TAAB Laboratories, 52 Kid more End Road, Enimer
Green, Reading, England. Reagent grade
NaCl, KCl, and MgCl2 • 6 H 2 0 are also required. The alcohol solutions can be made
with either grain alcohol or 95% ethanol
(benzene-free). Distilled deionized water
should be used throughout the procedure.
Solutions
HEPES Buffer (58.5 »>M, pH 7.4). Dissolve 13.94 g of HEPES in approximately
900 ml of distilled deionized water, adjust
to/;H 7.4 with 1 N NaOH, and make up to
1 liter with distilled deionized water. Store
indefinitely at 4 C in Nalgene containers.
Dextrose (155 nm). Dissolve 13.94 g in
enough distilled deionized water to make
500 ml. Store at 4 C in Nalgene; make this
solution up fresh each time.
Deca Ca2+-free Physiologic Solution Saline.
NaCl 53.4 g, KCl 2.89g, and MgCL • 6
H 2 0 1.57 g are dissolved in enough distilled deionized water to make 500 ml.
This solution can be stored at 4 C indefinitely in Nalgene.
Ca2+-free Physiologic Saline
Solution.
Reagents
Dilute 10 ml of deca Ca 2+ -free physiologicHEPES (anhydrous, molecular weight saline solution with 90 ml of distilled
238.3), No. H-3375 (N-2-hydroxyethyl- deionized water, add 10 ml of 155 m,\i dcx-
762
DEWAR, WOLOWYK, AND HILL
A.J.C.P. —Vol. 66
FIG. 1 (left). Normal erythrocytes. Scanning electron micrograph. x2,850, reduced from X3.800.
FIG. 2 (right). Abnormal erythrocytes in paroxysmal nocturnal hemoglobinuria (transplant).
Scanning election micrograph. x2,250, reduced from x3,000.
trose and 20 ml of 58.8 mM HEPES buffer.
Aerate well with 0 2 prior to use; make up
fresh each time in Nalgene.
2% Glutaraldehyde in Ca2+-free Physiologic
Saline Solution. Dilute 10 ml of deca Ca 2+ free physiologic saline solution and 10.6
ml of glutaraldehyde EM (25% aqueous
solution) with enough distilled deionized
water to make 100 ml, then add 10 ml of
155 mM dextrose and 20 ml of 58.5 mM
HEPES buffer. Aerate well prior to use;
make up fresh each time in Nalgene.
0.1% Glutaraldehyde in Ca2+-free Physiologic Saline Solution. Dilute 5 ml of 2%
glutaraldehyde solution (above) with 95
ml of Ca 2+ -free physiologic saline solution
and aerate well with 0 2 before using.
Make up fresh each time in Nalgene.
Alcohol Solutions. 1.8, 10, 20, 40, 50, 70,
80, 90, and 95% (v/v) ethanol in distilled
deionized water. Store indefinitely at 4 C.
Quality Control
In the evolution of the technic the
reliability of the method for routine diag-
nosis was tested by comparing blood smears
and scanning electron micrographs of
freshly withdrawn blood from four normal
volunteers. Normal and abnormal bloods
processed for the scanning electron microscope immediately after collection were
compared with the same bloods processed
after storage overnight. At least one normal
sample was always processed for the scanning electron microscope concurrently
with diseased blood.
Results
In comparing blood smears and scanning electron micrographs from the normal volunteers, the numbers of abnormal
erythrocytes were counted (Table 1).
Almost all of the abnormal cells were
poikilocytes or anisocytes, although a few
cells with small holes in the membranes
were seen under scanning electron microscopy and counted as abnormal. The normal venous blood smear had 12.9% abnormal erythrocytes, while the same
venous blood processed for the scanning
October 1976
SCANNING ELECTRON MICROSCOPY OF ERYTHROCYTES
763
FIG. 3. Dimpled (rough-surfaced) cell seen in paroxysmal nocturnal hemoglobinuria (transplant)
blood. Scanning electron micrograph. X23.000.
electron microscope had 11.7% abnormal
erythrocytes. The difference between the
two methods was not statistically significant at the 5% level (t test). Capillary blood
from the finger tip showed 11.8 and 8.4%
abnormal erythrocytes in the smears and
micrographs, respectively. Again, the
difference was not statistically significant
(5% level, t test). Whether the source of
blood results in a significant difference in
the number of abnormal erythrocytes seen
was not tested because the anticoagulants
for capillary and venous blood samples
(heparin and EDTA, respectively) were
different.
Freshly withdrawn venous blood from a
normal healthy volunteer (Fig. 1) and blood
from a paroxysmal nocturnal hemoglobinuria patient who has had a bone
marrow transplant were processed for the
scanning electron microscope. Many of
the erythrocytes from the patient showed
deformed, wrinkled or dimpled (roughsurfaced) membranes (Figs. 2 and 3).
Storage of the same samples overnight at
4 C. as whole blood in EDTA resulted in
echinocyte formation (Figs. 4 and 5).
T h e echinocytes in the normal sample were
echinocytes II, while those in the sample
from the patient were echinocytes 111, ac-
764
DEWAR, WOLOWYK, AND HILL
AJ.C.P.—Vol.
66
FIG. 4 (left). Normal blood fixed after storage overnight. Scanning electron
micrograph. X2.850, reduced from x3,800.
FIG. 5 (right). Paroxysmal nocturnal hemoglobinuria (transplant) blood fixed after storage overnight.
Scanning electron micrograph. x2,550, reduced from X3.400.
cording to Bessis.2 In addition, the wrinkled and deformed membranes in the fresh
sample from the patient (Fig. 2) were not
observed when the sample was fixed after
an overnight delay (Fig. 5).
Discussion
The present method of preparing erythrocytes for scanning electron microscopy utilizes sequential fixation in glutaraldehyde. Osmium tetroxide has been
used by Bessis and Weed 1 in place of the
higher concentration of glutaraldehyde,
but this chemical adds gready to the cost
of processing the cells, and prevents the
worker from stopping the procedure at a
convenient time. We have found that we
can leave the cells in 2% glutaraldehyde
in Ca 2+ -free saline solution for 24 hours
without danger of overfixation, a problem
present with osmium. Delicate fixation first
in the low concentration of glutaraldehyde
preserves the initial shape best, and subsequent fixation in a higher concentration of
glutaraldehyde protects the cell from the
osmotic gradients during dehydration. 3
Also, coating the cells with both carbon and
gold once dried ensures good electrical
conductivity of the sample and thus, clear
micrographs. 3
Erythrocytes having pitted deformed
membranes under the scanning electron
microscope have been described by Lewis,
Danon, and Marikovsky. 5 T h e exact mechanism underlying the membrane disorder
is not understood, but it is thought to be
due to a susceptibility of the lipid component of the membrane to autoxidation. 6
T h e rough-surfaced membrane (Fig. 3)
seen in the freshly prepared cells from the
patient with paroxysmal nocturnal hemoglobinuria and a bone marrow transplant
has also been observed in micrographs
from prosthetic heart valve patients undergoing intravascular hemolysis. 8 This type
of membrane abnormality in an otherwise
normal erythrocyte cannot be resolved with
the light microscope. Furthermore, the
October 1976
SCANNING ELECTRON MICROSCOPY OF ERYTHROCYTES
present study indicates that the roughsurfaced erythrocytes are unstable and will
not be observed in the scanning electron
microscope if the blood is not fixed soon
after collection (compare Figs. 2 and 5).
On the other hand, a morphologic difference between the stored normal and
paroxysmal nocturnal hemoglobinuria
samples can still be observed. Echinocytes
111 were seen in the sample from the
patient, while only echinocytes II were
observed in the equivalent stored control
(Figs. 4 and 5). This could indicate
that the echinocytogenic factor as described by Bessis2 was slightly more active
in the sample from the patient.
765
References
1. Bessis M, Weed RI: In Advances in Biological
and Medical Physics. Edited by Lawrence JH,
Gofman JVV. New York and London. Academic Press, 1973, pp 61-63
2. Bessis M: Living Blood Cells and their Ultrastructure. Translated by Weed RI. New
York, Heidelberg, and Berlin, SpringerVerlag, 1973, pp 146-149
3. Cohen AL: In Principles and Techniques of
Scanning Electron Microscopy. Edited by
Hayat MA. New York, Van Nostrand Reinhold
Company, 1974, pp 45-105
4. Lessin LS, Jensen WN, Klug P: L'ltrastructure
of the normal and hemoglobinopathic red
blood cell membrane. Arch Intern Med 129:
306-319, 1972
5. Lewis SM, Danon D, Marikovsky Y: Electronmicroscope studies of the red cell in paroxysmal
nocturnal haemoglobinuria. Br J Haematol 1 1:
689-695, 1965
6. Mengel CE, Kami HE Jr. Meriwether WD:
We have found that when the processing
Studies of paroxysmal nocturnal hemoglobinuria erythrocytes: Increased lysis
of the sample is begun as soon as possible
and lipid peroxide formation by hydrogen
after collection, as suggested, the present
peroxide. J Clin Invest 46:1715-1723. 1967
method can provide reliable micrographs 7. Salsbury AJ, Clarke JA: New method for detecting changes in the surface appearance of
of blood within three hours.
human red blood cells. J Clin Pathol 20:
603-610, 1967
8. Wolowyk MW. Eraser RS: Red Cell Structural
Changes in Patients with Prosthetic Heart
AcknmiMgments. Mrs. A. Biscoe provided technical
Valves. In Proceedings. The Association of
assistance during the evolution of the teclmic, and
Faculties of Pharmacv of Canada. Ottawa.
Mr. G. Braybrook provided technical assistance
May 21. 1974
with the scanning electron microscope.
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