Journal of Clinical Pathology, 1979, 32, 162-1 67

A rapid method for electron microscopic examinationof blood cellsANTONIO COPPOLA

From the Department ofPathology, College of Medicine, Downstate Medical Center, State University ofNew York, Brooklyn, NY 11203, USA

SUMMARY A method is described whereby the time required for the processing of white blood cells forelectron microscopy can be shortened from 36 to 3- hours. Because cells of the peripheral bloodare not attached to each other, fixation, dehydration, and infiltration of the embedding mediumis more rapid. This makes it possible for clinicians to use fine structural data for diagnosis as

illustrated in three cases.

In haematology little emphasis has been placed onelectron microscopy for diagnostic purposes. One ofthe principal reasons may be that with standardmethods for electron microscopy the time requiredfor the fixation and embedding of the specimens isapproximately 36 hours (Bessis, 1973). In haema-tology the electron microscope has therefore beenconsidered primarily a tool for research purposes.

It has been proposed, however, that in the study ofthe bone marrow, in addition to smears, sections ofparticles, bone biopsy, and cytochemistry, finestructural study should also be done if light micro-scopic methods fail to reveal the nature of the cells orif it is necessary to define cells ultrastructurally(Coppola and Athanassiades, 1977). However, theaccepted procedures require 24-48 hours for regularprocessing for electron microscopy. So it is con-venient to have a rapid method for fine structuralanalysis of cells.

It is the purpose of this paper to present evidencethat haemopoietic cells can be processed for electronmicroscopy and examined in 31 hours. The clinicianwill thus have quick access to fine structural datawhich will aid in making the exact diagnosis indifficult haematological cases.

Material and methods

Electron microscopic examination of peripheralblood taken from patients with leukaemias was doneby both the quick method of fixation and embeddingand the standard method.

Received for publication 19 July 1978

Two 5-mi aliquots of peripheral blood from eachpatient were placed into test tubes containing EDTA.The blood was kept for 30 minutes in the verticalposition at 40C. The plasma layers rich in whiteblood cells (WBC) were then aspirated, placed intoother test tubes, and centrifuged at 2000 rpm for 10minutes in a Clay-Adams centrifuge. The plasmasupernatants were discarded from each tube, and thetwo pellets from each patient were labelled foreither the rapid experimental procedure or thecontrol procedure. They were then processed asdescribed below.

Experimental procedure

The cells were immediately resuspended in sodium-cacodylate buffered glutaraldehyde, pH 7*4 (Sabatiniet al., 1963) and kept for 10 minutes at 4VC. After theprimary aldehyde fixation, they were washed threetimes in sodium cacodylate buffer over six minutes,and then postosmicated in sodium-acetate-sodium-veronal-buffered osmium tetroxide, pH 7*4 (Palade,1952) for 15 minutes. The cells were subsequentlywashed for three minutes in 35%, 50%, 70%, and95% alcohol. The dehydration was further accom-plished by two washes in absolute alcohol and twochanges in propylene oxide washes over six minutes.After this last step the cells were suspended in apropylene-Epon mixture for 10 minutes, and thenplaced into Beam capsules and centrifuged at 3000rpm for five minutes. The propylene-Epon mixturewas then aspirated, and Epon was introduced intothe capsules (Luft, 1961). The Beam capsules con-taining packed cells at the tip were kept at 370C for

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A rapid method for electron microscopic examination of blood cells

45 minutes, and subsequently Epon polymerisationwas carried out at 1000C for one hour. After cooling,the blocks were cut with glass and diamond knives.Thick sections were stained with toluidine blue(Trump et al., 1961) and thin sections with meth-anolic uranyl acetate (Stempak and Ward, 1964).

CONTROL PROCEDUREWBC from the same patients were processedaccording to our routine method over a 24-hourperiod (Bessis, 1973; Coppola, 1973). In summary,the cell pellets were cut into blocks less than 1 mm3,fixed for one hour in buffered glutaraldehyde, andpostfixed in osmium tetroxide for an additional hour.Subsequently, the cell pellets were dehydrated ingraded alcohols and propylene oxide over 2 2 hours.Finally, the cell pellets were embedded in Epon 812and polymerisation was performed at 60'C for17-18 hours.

Results

Complete polymerisation of the Epon blocks wasobtained within one hour at 1000C. Thick sectionswere easily cut and were well stained with toluidineblue. No difficulties were encountered in cutting thinsections with a diamond knife. When the thinsections were cut, stained with uranyl acetate, andexamined with the electron microscope we could seelittle difference between the rapid and the conven-tional methods. There was a slight decrease in thecytoplasmic translucency and the cytoplasm oftenappeared of increased density in the rapidly pro-cessed material. Cytoplasmic granules were darkerand somewhat better defined in the conventionallyfixed cells. However, with the rapid method therewas very good preservation of both organelles andgranules. The morphology of the granules wasadequate for identification of cell types and theirstage of maturation. Three illustrative cases follow.

PATIENT 1A 34-year-old woman with a two-year history ofchronic myeloid leukaemia was admitted to hospital.While the patient was still in the emergency room,examination of the peripheral blood revealed a WBCof 79 x 109/l. The smear showed a total spectrum ofgranulocyte maturity, which was compatible with thediagnosis of chronic granulocytic leukaemia, butthere were also 24% blast cells that were peroxidase-negative. The peripheral blood was prepared forelectron microscopy in order better to define thenature of the blast cells. Ultrastructurally, theseblast cells appeared to have an agranular cytoplasm,displaying the general appearance of lymphoblasts(Fig. 1). The occurrence of lymphoid cells in the

blastic crisis of chronic granulocytic leukaemia hasrecently been stressed (Rosenthal et al., 1977). Thestructure of the other leukaemic cells was not unusual:their granules, mitochondria, Golgi apparatus, ribo-somes, plasma membranes, nuclear envelopes,chromatin, and nucleoli were similar to those usuallyseen in such cases (Figs 2, 3, 4). The diagnosis ofchronic granulocytic leukaemia in blastic crisis wasconfirmed.

PATIENT 2A 64-year-old woman was admitted with purplemacular lesions on the chest. In the emergency roomher WBC was 10-5 x 109/l. The differential countshowed 39% monocytoid cells, some of which wereimmature. The peripheral blood was then studied byelectron microscopy. Ultrastructurally, mature andimmature monocytoid cells were seen (Fig. 5). Thesewere characterised by kidney-shaped or irregularlyshaped nuclei, which contained a variable amount ofchromatin which rarely was seen attached and con-densed against the nuclear envelope. Approximately5% of them were markedly convoluted. The cyto-plasm contained the usual organelles as seen inmonocytes. Some cells displayed a variable numberof pleomorphic granules and vacuoles. Also in thiscase on high power magnification there was anexcellent resolution of the cytoplasmic organelles(Fig. 6). The diagnosis of acute monocytic leukaemiawas made from this material, substantiating thediagnosis made on the light microscopic charactersof the cells in the peripheral blood.

PATIENT 3A 67-year-old man was admitted to hospital withprogressive pruritic dermatitis of four years' duration.The WBC in the emergency room was 120 x 109/l.Sezary's syndrome was suspected because 30% oflymphocytes had cerebriform, convoluted nuclei andcytoplasmic vacuoles. Ultrastructurally, the cells ofthis patient had the characteristic features ofSezary's cells, that is, cerebriform-shaped nuclei withcondensed chromatin adjacent to the nuclearenvelope. Occasionally large nucleoli were seen(Figs 7 and 8). Thus we were able to provide theclinician with accurate diagnostic data in a veryshort period of time.

Discussion

Fixatives and embedding media for electron micro-scopy have been greatly improved over the past 20years. In our experience, glutaraldehyde-osmiumfixation gives the best cellular detail. The best em-bedding medium is the epoxy resin suggested byLuft (1961). The suggested time for fixation and

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Fig. I An agranular malignant cell with morphological Fig. 2 A myelocyte displaying normal cytoplasmiccharacteristics of a lymphoblast. M = mitochondria. granules and nuclear chromatin. Ag = azurophilicUranyl acetate x 12 054. granules; N = nucleus. Uranyl acetate x 13 560.

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Fig. 3 Note excellent preser vation of the nucleus, nucleolus, and cytoplasmic organelles of this leukaemicmtyelocyte. ER = rough endoplasmic reticulum; N = nucleus. Uranyl acetate x 23 985.

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Fig. 4 The structure of the mitochondria and profiles of the rough endoplasmic reticulum are clearly delineatedin this myeloid cell. ER = rough endoplasmic reticulum;M = mitochondria. Uranyl acetate x 55 965.

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Fig. 5 A malignant monocyte showing a bizarre shape of the nucleus and abundance ofazurophilic granulesalong with other cytoplasmic organelles. Ag = azurophilic granules; N = nucleus. Uranyl acetate x 15 590,

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Fig. 6 At this magnification ribosomes, centriole, anid membranous structures of the Golgi apparatus (G) areclearly demonstrated. C = centriole; Uranyl acetate x 55 965.

Fig. 7 A lymphoid cell of Sezary type with character- Fig. 8 A high-power view ofa Sezary cell. C =istic cerebriform-shaped nucleus. A large nucleolus is centriole; N = nucleuis. Uranyl acetate x 31 21!also seen. Nu = nucleolus. Uranyl acetate x 18 282.

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A rapid method for electron microscopic examination of blood cells 167

dehydration is approximately 7 hours and the timerequired for the Epon infiltration and polymerisationis about 24 hours (Bessis, 1973). With the presentmodified procedure, we have shown that the timerequired for the entire processing for electron micro-scopy can be shortened to 31 hours provided thatthe temperature for the Epon polymerisation is raisedto 100°C. In our experience, using this method inmany cases, no appreciable cell distortion norartifacts have been noted. The only inconvenienceis that, because of the centrifugation that followsevery step, only one specimen can be processed ata time. We have therefore limited our procedure tothose cases in which electron microscopy is ofimmediate diagnostic value.An analogous method has already proved to be

excellent for tissue biopsies (Rowden et al., 1974).Since we are dealing not with solid tissue but withbuffy coat, the fixation, dehydration, and embeddingsteps can be rapidly achieved even when there is alarge pellet. Despite the high temperature used for thepolymerisation of the resin, there is no detectablecellular distortion nor shrinkage of cells (Rowdenet al., 1974). We recommend the use of the rapidprocedure so that the clinical haematologist may addevaluation of the fine structural detail to the otherinformation he has available before making the finaldiagnosis.

I am indebted to Thomas J. Athanassiades andDr Joanna Sher for their critical review of the manu-script. The technical assistance of Alan Liebermanand Nilda Stahl is also appreciated. The generoussupport of the project by Dr John D. Broome,Professor and Chairman, Department of Pathology,is also gratefully acknowledged.

References

Bessis, M. (1973). Living Blood Cells and their Ultra-structure, translated by R. I. Weed, p. 717. Springer,New York and Berlin.

Coppola, A. (1973). A transplantable mouse lymphomawith unusual azurophilic granules. American Journal ofPathology, 73, 233-239.

Coppola, A., and Athanassiades, T. J. (1977). Therelative merits of bone marrow biopsy and particlesection technics (Letter). American Journal of ClinicalPathology, 67, 309.

Luft, J. H. (1961). Improvements in epoxy resin embed-ding methods. Journal of Biophysical and BiochemicalCytology, 9, 409-414.

Palade, G. E. (1952). A study of fixation for electronmicroscopy. Journal of Experimental Medicine, 95,285-298.

Rosenthal, S., Canellos, G. P., Whang-Peng, J., andGralnick, H. R. (1977). Blast crisis of chronic granu-locytic leukemia: morphologic variants and thera-peutic implications. American Journal of Medicine, 63,542-547.

Sabatini, D. D., Bensch, K.. and Barrnett, R. J. (1963).Cytochemistry and electron microscopy: the preser-vation of cellular ultrastructure and enzymatic activityby aldehyde fixation. Journal of Cell Biology, 17, 19-58.

Rowden, G., and Lewis, M. G. (1974). Experience with athree hour electron microscopy biopsy service. Journalof Clinical Pathology, 27, 505-510.

Stempak, J. G., and Ward, R. T. (1964). An improvedstaining method for electron microscopy. Journal ofCell Biology, 22, 697-701.

Trump, B. F., Smuckler, E. A., and Benditt, E. P. (1961).A method for staining epoxy sections for light micros-copy. Journal of Ultrastructure Research, 5, 343-348.

Requests for reprints to: Dr Antonio Coppola, Depart-ment of Pathology, Downstate Medical Center, StateUniversity ofNew York, 450 Clarkson Avenue, Brooklyn,New York 11203, USA.

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