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J Clin Pathol 1997;50:985-990

A review of 50 consecutive cytology cell blockpreparations in a large general hospital

Frederick Mayall, Bridget Chang, Ann Darlington

Department ofPathology, WaikatoHospital, Hamilton,New Zealand

Correspondence to:Dr Mayall, Department ofPathology, Waikato Hospital,Private Bag 3200, Hamilton,New Zealand; email:mayallf(hwl.co.nz

Accepted for publication2 September 1997

AbstractAims-To review consecutive cell blockpreparations of cytological specimens in alarge general hospital.Methods-50 cell blocks were made overan 18 month period in which about 1900fine needle aspirations (FNAs) were per-formed. The aspirator was a cytologist or,for image guided FNAs, a radiologist witha cytologist at hand to collect the speci-men. Forty eight cell blocks were fromFNAs and two were from serous fluids.Results-The cellularity of the cell blockswas inadequate in only four preparations.The main motive for making cell blockswas to obtain tissue for immunohisto-chemistry. This was performed on 28cases and a total of 107 immunostainedsections were produced. The most com-mon diagnostic dilemma was betweencarcinoma and melanoma, and the secondmost common between carcinoma andlymphoma. Consequently cytokeratin,S-100, and LCA were the most frequentlyused antibodies. At least one ofthese threeantibodies was positive in 17 cases. Fivecases were immunostained only for prog-nostic breast markers.Conclusions-The use of cell block immu-nohistochemistry is a reliable and techni-cally unsophisticated aid in the cytologicalexamination of tumours other than lym-phomas. Success depends on havinghighly experienced aspirators that reliablyobtain sufficiently cellular material.(C Clin Pathol 1997;50:985-990)

Keywords: cell block; immunohistochemistry; fine nee-dle aspiration cytology; audit

Cytology cell blocks are made by centrifugingcytology specimens to form solid pellets. Paraf-fin wax embedded sections can be cut for hae-matoxylin and eosin histology and specialstains, including immunohistochemistry. Thisstudy reviewed 50 consecutive cell blockpreparations of cytological specimens in a largegeneral hospital.

Patients and methodsThe vast majority of cell block specimens wereobtained from fine needle aspirations (FNAs).These were performed as part of a near-patientrapid diagnosis FNA service. A second FNAwas used to collect the specimen for the cellblock after the initial diagnosis had been madeon the first specimen. The image guided FNAs(17 cases) were performed by a consultantradiologist with a cytologist at hand to collectthe specimen. A variety of needles was useddepending on the site. The manually guidedFNAs (31 cases) were performed with a 23gauge 3.75 cm needle using a "needle only"technique and the operator was always aconsultant pathologist with a special interest inFNA cytopathology. Maximum material wascollected by extensive agitation of the needle inthe lesion. Ten per cent formal saline wasdrawn up though the needle into an attachedsyringe and then gently ejected back into aspecimen container.

Table 1 Primary antibodies. Their sources, dilutions, and enhancement methods

Antibody Source Dilution Enhancement

Cytokeratin (CAM 5.2) Becton Dickinson Prediluted MicrowaveS100 Dako 1/1500 MicrowaveCarcinoembryonic antigen (CEA) Dako 1/200 MicrowaveBer EP4 Dako 1/100 TrypsinProstate specific antigen (PSA) Dako 1/30 MicrowaveHMB-45 Dako 1/25 MicrowaveLeucocyte common antigen (LCA) Dako 1/400 MicrowaveCD20 Dako 1/200 MicrowaveCD43 Dako 1/200 MicrowaveKappa light chain (K) Novocastra 1/100 TrypsinLambda light chain (L) Dako 1/50 Microwavep53 Dako 1/25 None & microwaveOestrogen receptor (ER) Novocastra 1/50 MicrowaveProgesterone receptor (PR) Novocastra 1/40 Microwavec-erb-B2 Dako 1/900 MicrowaveThyroglobulin (TG) Dako 1/100 Microwavea Feto protein (aFP) Dako 1/500 MicrowaveDesmin Dako 1/400 MicrowaveSmooth muscle actin (SMA) Dako 1/40 MicrowaveCD34 Novocastra 1/25 MicrowaveFactor VIII (F VIII) Dako 1/1000 Microwave

Becton Dickinson, Mountain View, California, USA; Dako, High Wycombe, Bucks, UKiNovocastra, Newcastle upon Tyne, UK

CELL BLOCKSSerous fluids were prepared by centrifuging at1270 xg for seven minutes, discarding thesupernatant, and resuspending in formalin.After at least an hour of fixation the suspensionof the FNA specimen in formalin or the resus-pension of the serous fluid was centrifuged at1270 xg for seven minutes. The supernatantformalin was decanted off. The deposit wasresuspended in molten 0.9% agar at 50°C andquickly placed in a centrifuge again. It wasspun at 1270 xg for two minutes and thenrefrigerated at 4°C for 30 minutes to hardenthe agar. The excess agar was trimmed to leavea cell button. This was wrapped in tissue paperand placed in a plastic cassette for paraffinprocessing as for a routine surgical biopsyspecimen. A haematoxylin and eosin stainedsection was cut from each cell block and exam-ined. Any further stains were then requestedand performed.

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Mayall, Chang, Darlington

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Figure 1 A cell block section from a spinal myeloma deposit showing strongimmunostaining for lambda. There was no positivity for kappa.

Figure 2 A cell block section of a thymoma showing immunostaining for cytokeratin(CAM 5.2) in the epithelial cells but not in the smaller lymphocytes.

IMMUNOHISTOCHEMISTRY

Immunohistochemistry was performed on

3 gm paraffin sections cut on to APES coatedslides. Microwave pretreatment was in citratebuffer at pH 6 for 10 minutes at boilingtemperature. The sources, dilutions, and incu-bation times of the primary antibodies areshown in table 1. The incubation time for theprimary antibodies was 10 minutes at room

temperature. The sections were then developedusing a streptavidin-biotin-immunoperoxidasekit (Dako, High Wycombe, Bucks, UK). Thechromogen was DAB.

ResultsThe results are displayed in table 2. These 50cell blocks were from an 18 month period inwhich about 1900 FNAs were performed.Forty eight of the 50 cases were FNAs and twowere serous fluid specimens. Seventeen of theFNAs were image guided. Most of these were

computed tomography guided. Some were

fluoroscopically guided FNAs of lung, and afew were ultrasound guided FNAs of the neck.The cell blocks contained sufficient cells in

46 cases. In 18 cases the cell blocks were notused for immunohistochemistry. In most of

these the motive for taking the cell block hadbeen to collect material for immunohistochem-istry but then a confident diagnosis was madefrom conventional cytology alone and immuno-staining was not performed. In a few cases, inparticular some possible small cell carcinomasof the lung, the cell blocks were taken to see thehaematoxylin and eosin stained histology ofthecells.Immunostaining was performed in 28 cases.

A total of 107 sections were immunostainedand in only one (case 38) were there insuffi-cient cells. None of these sections was inad-equate for any other reason. The two mostcommon diagnostic dilemmas for which im-munostaining was employed were betweencarcinoma and melanoma (10 cases) andbetween carcinoma and lymphoma (ninecases). Cell block immunohistochemistry wasperformed solely for breast carcinoma prog-nostic markers (oestrogen receptor, progester-one receptor, and c-erb-B2) in five cases.Breast markers were performed in conjunctionwith other immunostains in five further cases.

Cytokeratin was used for 21 of the 28immunostained cases, S-i 00 on 11 cases, andleucocyte common antigen (LCA) for ninecases. At least one of these three antibodies waspositive for 17 cases. Cytokeratin was used onevery occasion that one or both ofthe other twowere used. These three antibodies were usedfar more than any others except for prognosticbreast markers (10 cases). The largest numberof immunostains done on one cell block waseight. Twenty one different antibodies wereused in total; seven being used only once. Adiastase periodic acid-Schiff stain was alsoused on two cases.

Cell blocks were not usually performed ontumours that appeared to be lymphoid in thesmears as the material obtained from thesecond aspirate was usually used for immu-nolabelled flow cytometry instead. There were24 FNA samples taken for lymphoid markerflow cytometry during the period studied.Consequently the cell block cases for whichlymphoid immunostains were positive tendedto be unusual lymphoid neoplasms or othertumours. These included a myeloma deposit inthe spine (fig 1), a high grade T cell lymphoma,a thymoma (fig 2), and a high grade B celllymphoma.

Cell blocks were taken from six possiblesmall cell carcinomas. In three of these casesonly haematoxylin and eosin stained sectionswere cut but immunostains for cytokeratin andLCA were performed on three, and were posi-tive for cytokeratin in two. The third (case 38)had insufficient cells in the immunostainedsections. In the other 27 cases the results of theimmunohistochemistry either confirmed thecytological diagnosis that had been suggestedin the provisional report issued at the time ofthe aspirate or, in a few cases, indicated thecorrect diagnosis from a choice of two or morethat were being considered. One example ofthelatter was lymphoma versus thymoma in case37. The immunolabelled flow cytometry forlymphoid markers showed this tumour to becomposed almost exclusively ofT cells but the

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Review of cytology cell block preparations

Figure 3 A cell block section of a leiomyosarcoma showing immunostaining for smoothmuscle actin.

strong cytokeratin positivity in a minority ofcells in the cell block allowed the correct diag-nosis to be made. A second example (case 34)was the diagnosis of metastatic signet ring car-

cinoma in the neck of a woman with a previousangiosarcoma of the head. A third example(case 43) was the diagnosis of metastatic breastcarcinoma in the axilla of a middle aged womanwho already had metastatic melanoma in thelymph nodes of her neck.

All lymphomas were positive for LCA. Allcarcinomas were positive for cytokeratin exceptcase 38. All melanomas were positive for S-100except case 11 and it was positive for HMB-45.These results are indicative of the high qualityof antigen expression in the cell block tissue.The strong positivity for lambda light chain inthe spinal deposit of myeloma is another exam-ple (fig 1). None of the diagnoses made in the50 cases was contradicted by subsequentinvestigations or follow up.

DiscussionThe high cell block adequacy rate can beattributed to a cytologist performing or super-vising a re-aspiration of the lesion for the solepurpose of collecting material for a cell blockrather than dividing the first aspirate in two. Aprevious study reported satisfactory division ofsingle aspirates into smears and cell blockmaterial when a relatively large (21 gauge)needle is used.' It could be argued that one

large gauge needle is no more painful than twosmall ones except that we only made a secondpass to collect cell block material in 50 of about1900 FNAs. The ability to perform a secondaspirate for a cell block in appropriate cases

depends on a cytologist being available fornear-patient reporting of the first aspirate.Success depends on the high cellularity of theaspirate.

In general the cell blocks that did not haveany further special stains did not contribute tothe diagnosis. This appears to conflict withprevious reports. Brown et al described a studyin which the examination of 84 FNAs revealed55 malignancies with cytology alone but a fur-ther nine malignancies were identified on

examining the cell blocks.2 This was not ourexperience but our study was fundamentallydifferent in that we examined the cytologyspecimens immediately and if they did notcontain sufficient material for a definitediagnosis the FNA was repeated and moresmears examined. In the study by Brown et althe cytology smears and the material for thecell blocks seem to have been sent away to thelaboratory for examination and there was noopportunity to repeat the aspirate if the smearswere not diagnostic. A study by Flint on cytol-ogy and cell blocks from 111 bronchialwashings found 52 malignancies in the cyto-logical preparations and 43 in the cell blocks.3Four of the "malignant" cell blocks were nega-tive for malignancy on cytology. We did notexamine any bronchial washing cell blocks inour study.The dominance of cytokeratin, S-100, and

LCA use reflects the importance of cell blockimmunostaining in distinguishing between car-cinoma and melanoma and between carcinomaand lymphoma. These were by far the mostcommon diagnostic dilemmas to be encoun-tered.One of the principal advantages of immu-

nostaining cell blocks over immunostainingsmears is that a large battery of stains can beeasily done on the former. This is exemplifiedby case 41 (fig 3) in which eight differentimmunostains were used. It would be difficultto make eight spare smears for immunocyto-chemistry from a single FNA sample.Another advantage of cell block immunohis-

tochemistry is that it requires no specialtechniques or equipment and so it may bereadily adopted by small laboratories that areable to perform immunohistochemistry onparaffin wax sections of histology specimensbut would only occasionally need to immuno-stain cytological specimens.Kung et al reviewed some other advantages

of cell block immunostaining over immuno-cytochemistry of unstained or destainedsections.' Storage of cell blocks is easier thanunstained slides. Freezing is not necessary andthere is only minimal deterioration in the anti-genicity with time. Destaining cytology slides islabourious and results in the loss of what maybe the only conventionally stained cytologicalmaterial. In addition, the results from immu-nostaining smears tend to be poor unless theprocedure is frequently practised. In particularthere tends to be a high level of backgroundstaining. This is sometimes due to technicalartefact, but even with the best technique heavybackground staining often occurs, probablybecause of non-specific staining of thickclumps of cells and proteinaceous debris. It hasbeen claimed that ThinPrep (Cytyc Corp,Boxborough, Massachusetts, USA) typesmears reduce background staining by produc-ing an even deposit of cells that have beenwashed in buffer.4 Our own experience hasbeen that the use of an alkaline phosphatasestaining method rather than an immunoperoxi-dase method greatly reduces backgroundstaining in immunostained smears.5

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Immunostaining smears does have one nota-ble advantage over cell blocks in that far fewerare needed. This is some times cited as a reasonfor favouring smear immunostains rather thancell blocks but our study shows that it is almostalways possible to obtain sufficient cells froman FNA for cell block immunostaining. A pre-vious study found that 30 of333 cell blocks hadinadequate cellularity.6 However, most of thesewere thyroid cell blocks, which were not exam-ined in our study.

Fluorescent antibodies can be used asimmunolabels for unfixed cytospins and forflow cytometry. The effectiveness of these typesof labelling in the FNA diagnosis of lympho-mas has been examined and these techniquesare quite widely used.7 8 They are clearly moreeffective than cell block immunohistochemis-try for characterisation of lymphoid cells,mainly because many lymphoid markers do notsurvive fixation. However, flow cytometry isnot well suited for use with solid tumours as asuspension of single cells is needed. Immuno-staining unfixed cytospins of lymphoid lesionshas the advantage of requiring far fewer cellsthan for flow cytometry. However, there is agood deal of technical skill and manual labourinvolved and it is probably not appropriate foruse in centres in which it would be practisedonly infrequently. In contrast flow cytometryanalysis of lymphoid FNAs can be incorpo-rated as a small part of the routine flow cyto-metry work of a haematology laboratory thatconsists mainly of bone marrow aspirates andperipheral blood specimens. The high case vol-ume and level of automation makes the analy-sis cheap and rapid.

In conclusion, our study shows that FNAscan reliably obtain cell blocks with sufficientmaterial to be diagnostically useful. Theimportance of skilled aspirators is evident andwe plan to increase the availability of cytologistaspirators with an increase of 0.7 full timeequivalents in the consultant pathologist staffin the laboratory. This should help to cover theannual 1400 FNAs workload that has devel-oped since the introduction of the rapiddiagnosis FNA service two years ago. Werecognise that there are situations in which acell block is unlikely to contain sufficienttumour cells. In response, the laboratory hasrecently introduced an immunocytochemistrymethod that was previously used by one of theauthors in another hospital.5

1 Kung ITM, Chan S, Lo ESF. Application of theimmunoperoxidase technique to cell block preparationsfrom fine needle aspirates. Acta Cytol 1990;34:297-303.

2 Brown K, Fulbright RK, Avitabile AM, Bashist B. Cytologicanalysis in fine-needle aspiration biopsy. AmJ Radiol 1993;161:629-31.

3 Flint A. Detection of pulmonary neoplasms by bronchialwashings. Acta Cytol 1993;37:21-3.

4 Leung SW, Bedard YC. Immunocytochemical staining onThinPrep processed smears. Modern Pathology 1996;9:304-6.

5 Mayall F, Heryet A, Manga D, Kriegeskotten A. p53 immu-nostaining is a highly specific and moderately sensitivemarker of malignancy in serous fluid cytology. Cytopathol-ogy 1997;8:9-11.

6 Zito FA, Gadaleta CD, Salvatore C, Filotico R, Labriola A,Marzullo A, et al. A modified cell block technique for fineneedle aspiration cytology. Acta Cytol 1995;39:93-9.

7 Chernoff WG, Lampe HB, Cramer H, Banerjee D. Thepotential clinical importance of fine needle aspiration/flowcytometric diagnosis ofmalignant lymphoma.J Otolaryngol1992;21(suppl 1):1-15.

8 Hanson CA. Fine-needle aspiration and immuno-phenotyping: a role in diagnostic histopathology. AmJ ClinPathol 1994;101:555-6.

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