Immunoflow cytometry and cell blockimmunohistochemistry in the FNA diagnosis oflymphoma: a review of 73 consecutive cases

Frederick Mayall, Michael Dray, Dianne Stanley, Barbara Harrison, Robin Allen

AbstractAims—To review the results of 73 consecu-tive fine needle aspirations (FNAs) thatwere collected by a pathologist and ana-lysed by immunoflow cytometry. Materialfor a cell block was also collected fromsome of these lesions.Methods—The setting was a large generalhospital in rural New Zealand. The FNAswere performed by a pathologist, or aradiologist for image guided localisations.Material for immunoflow cytometry wascollected into RPMI and, when required,material for a cell block was collected intoformalin.Results—Of the 73 samples collected byFNA nine were inadequate. Light chainrestriction could be demonstrated in mostFNA samples from B cell lymphomas (28of 30 adequate samples). The exceptionswere two cases of T cell rich B celllymphoma. Artefactual light chain re-striction was seen occasionally in T celllymphomas, presumably as a result ofautoantibodies binding to the cell sur-faces. It was possible to subtype most (18of 30 adequate samples) B cell lymphomasas chronic lymphocytic leukaemia (CLL),follicle centre cell lymphoma (FCCL), ormantle cell lymphoma. The CD4 to CD8ratio was not usually restricted in T celllymphomas and coexpression of CD4 andCD8 was not usually found. Loss of pan-Tcell antigens was seen in some T cell lym-phomas. Four of the six T cell lymphomasand three of the four non-lymphoid malig-nancies were diagnosed with the aid of cellblock immunohistochemistry. Only one ofthe four cases of Hodgkin’s lymphomashowed Reed-Sternberg cells in the FNAsmears.Conclusions—It is not always possible tocharacterise lymphomas as fully withFNA and immunoflow cytometry as ispossible with biopsy histology and a fullbattery of modern investigations.Nevertheless, in the setting of a large ruralgeneral hospital immunoflow cytometryon FNA samples is a highly eVectivemethod of diagnosing and typing B celllymphomas. Immunoflow cytometry is oflittle use for T cell lymphomas or Hodg-kin’s lymphomas. We advocate the use ofcell block immunohistochemistry in pref-erence to immunoflow cytometry forcases in which the cytological appearanceof the specimen is overtly malignant but

the diVerential diagnosis includes non-lymphoid malignancy.(J Clin Pathol 2000;53:451–457)

Keywords: lymphoma; flow cytometry; cell blocks;immunolabelling; fine needle aspiration

In the past, the cytological diagnosis oflymphomas from fine needle aspiration (FNA)samples has been particularly diYcult. Usually,one obtains a sample that is either obviouslymalignant but equivocally lymphoid or, con-versely, a sample that is obviously lymphoid butequivocally malignant. However, recently FNAdiagnosis of lymphoid lesions has been madeeasier by the arrival of immunoflow cytometryin most large pathology laboratories. Immuno-flow cytometry has been used mainly for theanalysis of haematological diseases, but in-creasingly it is being used by cytologists. Earlystudies of FNAs and immunoflow cytometryused a limited range of antibodies and were notable to perform dual staining. Recently, therehave been substantial advances in the sophisti-cation of the methods and equipment used forflow cytometry. It is accepted that FNA cytol-ogy with immunolabelled flow cytometry can,in some circumstances, serve as a replacementfor open biopsy and conventional histology andimmunohistochemistry.1 2 However, FNA withimmunoflow cytometry is not always success-ful. Scanty cellularity in the sample can preventa satisfactory analysis, and even with anadequate sample the results might be mislead-ing. In particular, non-lymphoid malignanciescan be hard to distinguish from lymphoidlesions if the sample also contains reactive lym-phoid cells; B cell lymphomas sometimes donot exhibit light chain restriction; and T celllymphomas can have a large population ofreactive B cells.

The aim of our study was to review theresults of 73 consecutive FNAs that werecollected by a pathologist and analysed byimmunoflow cytometry. Material for a cellblock was also collected from some of theselesions. The setting was a large general hospitalin rural New Zealand. The FNAs wereperformed over a two year period. In this time,approximately 800 FNAs of non-breast lesionswere performed together with approximately1400 breast FNAs.

MethodsThe FNAs were performed by one of twomobile pathologists with an interest in FNAcytology, except for image guided FNAs, in

J Clin Pathol 2000;53:451–457 451

Department ofPathology, WaikatoHospital, Private Bag3200, Hamilton, NewZealandF MayallM DrayD StanleyB HarrisonR Allen

Correspondence to:Dr Mayallemail: mayallf@hwl.co.nz

Accepted for publication4 November 1999

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which case one of these pathologists preparedthe slides after the radiologist had aspirated thelesion. The FNAs performed by the patholo-gists were done with a needle only technique,using a 23 or 25 gauge 1.25 inch (3 cm) needle.The slides were air dried, often aided by a smallhair dryer, and DiV-Quik® stained (DadeBehring Diagnostics, Newmarket, Auckland,New Zealand). The slides were then examinedwith a portable Olympus CHK microscopeand a hand written report was issued within afew minutes. If this examination of thespecimen suggested that the lesion might belymphoid and lymphoma seemed possible,then a second FNA was performed, still using aneedle only technique, to collect a second sam-ple. This sample was collected quickly and theneedle was washed through with 3 ml ofheparin RPMI (12.8 mg of heparin ammo-nium in 45 ml of RPMI) within a few secondsof being collected. The reason for the urgencywas that a clotted sample reduces the yield ofcells for flow cytometry. Early in our experi-ence with collecting specimens for flow cyto-metry we washed needles through with sterileheparin saline before the FNA was performed,but we found that this was not necessary. Theslides were taken back to the laboratory to bemounted and examined again. The specimenin RPMI was taken to the haematology labora-tory, transferred to a 5 ml tube and centrifugedfor two minutes at approximately 400 ×g. Thesupernatant was discarded and the cells wereresuspended in 2 ml of ammonium chloridelysis solution. The cells were gently vortexed,incubated at room temperature for 10 minutes,and then centrifuged again for two minutes.The leucocytes were then resuspended in 2 mlof phosphate buVered saline (PBS). If debriswas present, it was removed with a nylon Swissscreen filter. An approximate cell count wasperformed and a panel of directly conjugatedmonoclonal antibodies was selected (table 1)(Immunotech Inc, Westbrook, Maine, USA).The cells and the antibodies were incubated for20 minutes in the dark at room temperature.These were then washed once in PBS, spundown, and then washed again. They were thenanalysed with the flow cytometer. Material forê and ë light chain analysis was treated diVer-ently. The centrifuged sample in RPMI wasincubated with prewarmed PBS for 20–30minutes at 37°C, to remove the cytophilicimmunoglobulin, and then centrifuged. Thesupernatant was discarded and if red bloodcells were present they were lysed with ammo-

nium chloride as above. Cells were thenincubated with antibodies (against CD19/êlight chain or CD19/ë light chain) as above.

The “first run” panel of antibodies evolvedas our experience developed and with theacquisition of a new immunoflow cytometerapproximately half way through our study. Atthe start of the study, a Coulter PROFILE IIwas used but later the laboratory obtained aCoulter EPICS® XL (Beckman Coulter Inc,Fullerton, California, USA). In some cases weperformed a “second run” with a more special-ised panel to investigate a specific diagnosis.The panel of antibodies used was sometimesrestricted by the scarcity of cells. In these cases,antibodies against CD19/ê light chain andCD19/ë light chain were given priority.

In our laboratory we have adopted lightchain ratio limits from previous studies.2–4 A êto ë ratio of greater than 3 or a ë to ê ratio ofgreater than 2 was accepted as evidence ofmonoclonality.

For some cases, cell blocks for immunohisto-chemistry were collected as describedpreviously.5 These were usually taken after theinitial FNA showed overtly malignant cytologybut it was not certain that the lesion waslymphoid in nature.

ResultsTable 2 shows our results—the cases are set inchronological order within each diagnostic cat-egory. It can be seen that the range of cellmarkers expanded during the course of ourstudy. The panel of markers was sometimestailored a little in view of the initial diagnosis,so as to focus on a particular issue and to avoidwasting cells.

DiscussionNine of the 73 samples (12%) had inadequatecells for analysis. Four of these were frombenign lymph nodes. It was felt that adequatesamples were easier to obtain from malignantlesions simply because of their larger size.Young et al found that five of 107 (4.7%) oftheir specimens were inadequate in a similarstudy.2 A highly cellular lymphoma aspiratemight exhibit uniform negativity on immuno-flow cytometry if the cells are necrotic, as incase 3.

Twenty eight cases exhibited light chainrestriction (defined above), allowing an un-equivocal diagnosis of B cell lymphoma to bemade on the FNA sample alone. On review ofthe notes at the end of our study, we could findno evidence that any of these diagnoses wereincorrect. Case 48 had been thought to be sus-picious of lymphoma with a ê to ë ratio of 1.95.However, a review of the notes about a yearafter the FNA was performed revealed no clearevidence that the patient had developedlymphoma. In particular, there was no evi-dence of lymphoma on a computed tomogra-phy scan. Case 73 also had a marginal lightchain ratio but did not have clinically evidentlymphoma eight months later.

Five of the 33 B cell lymphomas did notexhibit light chain restriction. For three casethis was because of inadequate cellularity. The

Table 1 Antibodies and their fluorochrome labels

Antibody Fluorochrome

CD2 FITCCD3 FITCCD4/CD8 FITC/PECD5 PECD10 FITCCD19 FITCCD20 FITCCD23 FITCFMC7 FITCSm IgM FITCCD19/ê chain PE/FITCCD19/ë chain PE/FITC

FITC, fluorescein isothiocyanate; PE, phycoerythrin.

452 Mayall, Dray, Stanley, et al

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Flow cytometry and immunohistochemistry in lymphoma diagnosis 453

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other two (cases 9 and 33) were particularlyconfusing because the cytology showed amalignant tumour with highly atypical cellsmixed with benign looking lymphoid cells. Theflow cytometry showed no evidence of lightchain restriction. Biopsies were taken and theflow cytometry results from these were similarto those from the FNA. However, paraYn waxembedded sections and immunohistochemis-try showed that the large highly atypical cellswere CD45 and CD20 positive and negativefor cytokeratin, S100, and CD3. Thus, a diag-nosis of T cell rich B cell lymphoma was made.We assumed that the neoplastic B cells werenot expressing light chains strongly or were“swamped” by a population of reactive B cells.

Except for cases 9 and 33, the lowest lightchain ratio of any B cell lymphoma was a ë to êratio of 3.1 (case 16). Case 37 was remarkablein that it showed artefactual light chain restric-tion in a T cell lymphoma (fig 1). We assumethat this was as a result of specific binding of êto the surface of the tumour cells, perhaps as anautoimmune response to the tumour cells. Thelymphoma occurred in a patient with AIDS.On biopsy, the tumours cells were stronglypositive for CD3 but negative for CD20.Fortunately, this artefact did not confuse thediagnosis, because coexpression of light chainwith CD19 was not found.

For 18 cases it was possible to make a diag-nosis of B cell lymphoma and also subtype thelymphoma as either CLL (chronic lymphocyticleukaemia/small lymphocytic lymphoma),FCCL (follicle centre cell lymphoma), or man-tle cell lymphoma. Most of the other cases thatexhibited light chain restriction were called “Bcell lymphoma, exact type uncertain” or“diVuse large B cell lymphoma” if they werecomposed of overtly malignant large cells.Uncertainty as to the exact type of B celllymphoma was not considered suYcient reasonto perform a biopsy. This was usually becausethe patient had other medical problems thatwere a more immediate risk to the patient’shealth or because exact typing would not altertreatment.

Four of the six T cell lymphomas were diag-nosed with a combination of cytology, flowcytometry, and cell block immunohistochemis-try, but for two it was not possible to collect acell block. Both of these then had biopsies. Theparticipation of the pathologist in the collec-tion of the specimen and the immediate assess-ment of the cytology meant that the need forcell block histology had been anticipated. Theratio of CD4 to CD8 (or CD8 to CD4) wasexamined but this was not usually useful in therecognition of T cell lymphoma. The benignlesions in cases 59 and 65 had CD4 to CD8ratios of 4.15 and 4.29, respectively. Only twoof the six T cell lymphomas had CD4 to CD8(or CD8 to CD4) ratios greater than these. Jef-fers et al describe one case of T cell lymphomain their series of 46 FNA sampled lesions.6 Thislymphoma showed subset restriction, withmore than 90% of T cells expressing CD8.Coexpression of CD4 and CD8 was examinedfor four of the six T cell lymphomas but wasonly found in case 34. Loss of pan-T cell anti-Ta

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454 Mayall, Dray, Stanley, et al

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gens was seen in cases 34 and 37. Zardawi et alhave stated that the loss of one or more pan-Tcell antigen allows a firm diagnosis of T celllymphoma to be made (in the correct clinicalcontext).4 It should be remembered thatimmature thymic T cells exhibit loss of pan-Tcell antigens, as in case 44.

The cytological diagnosis of Hodgkin’slymphoma depends on the presence of typical

Reed-Sternberg cells. Flow cytometry does notcontribute except to exclude a monoclonal Bcell population. There were no definite Reed-Sternberg cells in the FNA samples of three ofour four cases. Because a diagnosis oflymphoma was suspected clinically, the nodeswere biopsied. One case did show abundantReed-Sternberg cells in the FNA sample and adiagnosis was made cytologically. The node

Figure 1 A scatter plot showing ê light chain restriction in a T cell lymphoma from a patient with AIDS (case 37). Therewas no coexpression of CD19 and ê, exemplifying the importance of dual staining in assessing light chain restriction.

% Count Mn X Mn Y PkPosX PkPosY PkCnt FPCVX FPCVY

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Flow cytometry and immunohistochemistry in lymphoma diagnosis 455

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was excised before treatment and the diagnosiswas confirmed. We would not advocate thediagnosis of Hodgkin’s lymphoma being madeon FNA alone except in exceptional circum-stances. Young et al studied the cytology andflow cytometry of 107 aspirates of suspectedlymphoid lesions.2 Three of these were Hodg-kin’s lymphoma but none showed Reed-Sternberg cells in the FNA samples. Flowcytometry showed a polyclonal population.

There were four cases that had a finaldiagnosis of a non-lymphoid neoplasm. Theinitial diVerential diagnosis favoured a non-lymphoid tumour in each case but lymphomawas also thought to be possible. In one of thesepatients, a core biopsy was performed by aradiologist once the cytology had been exam-ined. Cell blocks were taken for the others.

There were 28 cases that seemed to bebenign on cytology and flow cytometry.However, two of these were clinically suspi-cious and had biopsies that showed them to beHodgkin’s lymphoma. None of the otherpatients had a biopsy and none had evidence oflymphoma when the notes were reviewed at theend of our study.

Before the widespread availability ofimmunoflow cytometry, immunofluorescentcytochemistry on cytospin preparations wasused in some large laboratories for the charac-terisation of lymphomas from FNA samples.This has the advantage that it can beperformed with fewer cells and it is not diYcultto appreciate the relation between the size ofthe cells and their antigen expression. The lat-ter makes it easier to diagnose some conditionssuch as T cell rich B cell lymphomas.Immunocytochemistry has some major disad-vantages. The technique is highly labour inten-sive and requires specially trained technicalstaV. Dual expression of markers is less easy todemonstrate. A result would usually takeconsiderably longer than the four hour turnaround time that we have for immunoflowcytometry. The number of cells that can becounted and the number of markers that can beused are restricted by the time it takes toperform manual counting. A higher light chainratio (about 6 : 1) is required to provemonoclonality,7 although in practice this rarelyalters the detection of monoclonality in asample.8 For these reasons, immunoflow cyto-metry is probably the preferred method exceptin a few specialist centres.

Young et al graded the follicle centre celllymphomas in their study2 according to theproportion of “transformed cells” in the smear.Their method was validated in a previousstudy.9 We did not attempt to do this because itwould rarely change the way in which thepatient was treated.

Eight of the 47 malignant lesions were diag-nosed using cell blocks. These comprised onecase of myeloma, four of the six T cell lympho-mas, and three of the four non-lymphoidmalignancies. This demonstrates the role ofcell block immunohistochemistry in the diag-nosis of suspected lymphoid neoplasms. Al-though immunoflow cytometry is highly eVec-tive in the diagnosis of B cell lymphomas,

particularly CLL, FCCL, and mantle celllymphoma, cell block immunohistochemistryis probably more useful for “high gradelymphomas”, especially peripheral T cell lym-phomas, and also for non-lymphoid neo-plasms. If a sample from a suspected lymphoidlesion is overtly malignant on cytological crite-ria then more information is likely to beobtained from a cell block, but if the sample isequivocally benign/malignant then immuno-flow cytometry is probably more useful.Ideally, tissue should be obtained for immuno-flow cytometry and a cell block, although inpractice this is often not possible.

Optimum diagnostic information might bebest obtained by biopsying every suspiciouslymph node and performing a battery of sophis-ticated investigations including oncogene ex-pression, cell kinetic studies, and detailedmolecular analysis. A biopsy might be particu-larly important if spare material is needed forresearch. However, only a few centres have thebudget, facilities, or expertise to do all of this. Wewould always advocate that an FNA diagnosedlymphoma in an otherwise fit young patientshould be followed by a biopsy. Most lympho-mas occur in elderly patients and are notcurable. In these patients, treatment and survivalare often influenced more by coexistent pathol-ogy, so that detailed analysis of the lymphomamight supply the clinician with a level of detailthat is superfluous. Despite the pitfalls that wereapparent in our study, FNA with immunoflowcytometry is a powerful technique for thediagnosis and typing of B cell lymphomas. Closecommunication between the oncologist andpathologist is needed to ensure that FNAsampling is performed on appropriate cases andthat the result and issues of uncertainty are con-veyed. In the setting of a large general hospital inrural New Zealand, the speed with which FNAcytology and immunoflow cytometry could beperformed (about four hours) was important. Itallowed patients who had travelled a longdistance to be given their result quickly, andallowed other investigations to be undertakenthe same day in preparation for treatment.

ConclusionsOur study shows that it is almost always possi-ble to obtain an adequate sample for immuno-flow cytometry from a fine needle aspirate.Light chain restriction could be demonstratedin almost all FNA samples from B cell lympho-mas. One notable exception was T cell rich Bcell lymphoma. Artefactual light chain restric-tion could occasionally be seen in T celllymphomas, presumably because of autoanti-bodies binding to the cell surfaces. It was pos-sible to subtype most B cell lymphomas asCLL, FCCL, or mantle cell lymphoma.Immunoflow cytometry is not particularlyhelpful in the diagnosis of T cell lymphoma orHodgkin’s lymphoma. The CD4 to CD8 ratiowas not usually restricted in T cell lymphomasand neither was coexpression of CD4 and CD8usually found. Loss of pan-T cell antigens wasseen in some T cell lymphomas. Four of the sixT cell lymphomas and three of the fournon-lymphoid malignancies were diagnosed

456 Mayall, Dray, Stanley, et al

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with the aid of cell block immunohistochemis-try. We advocate the use of cell block immuno-histochemistry in preference to immunoflowcytometry for cases in which the cytologicalappearance of the specimen is overtly malig-nant but the diVerential diagnosis includesnon-lymphoid malignancy.

1 Skoog L, Tani E. The role of fine-needle aspiration cytologyin the diagnosis of non-Hodgkin’s lymphoma. Diagn Oncol1991;1:12–18.

2 Young NA, Al-Saleem TI, Ehya H, et al. Utilization of fine-needle aspiration cytology and flow cytometry in the diag-nosis and subclassification of primary and recurrentlymphoma. Cancer 1998;84:252–61.

3 Zander DS, Iturraspe JA, Everett ET, et al. Flow cytometry:in vitro assessment of its potential application for the diag-nosis and classification of lymphoid processes in cytologicalpreparations from fine-needle aspirates. Am J Clin Pathol1994;101:577–86.

4 Zardawi IM, Jain S, Bennett G. Flow-cytometric algorithmon fine-needle aspirates for the clinical workup of patientswith lymphadenopathy. Diagn Cytopathol 1998;19:274–8.

5 Mayall F, Chang B, Darlington A. A review of 50 consecu-tive cytology cell block preparations in a large general hos-pital. J Clin Pathol 1997;50:985–90.

6 JeVers MD, Milton J, Herriot R, et al. Fine needle aspirationcytology in the investigation of non-Hodgkins lymphoma. JClin Pathol 1998;51:189–96.

7 Sneige N, Dekmezian RH, Katz RL, et al. Morphologic andimmunocytochemical evaluation of 220 fine needle aspi-rates of malignant lymphoma and lymphoid hyperplasia.Acta Cytol 1990;34:311–22.

8 Robins DB, Katz RL, Swan F, et al. Immunotyping oflymphoma by fine needle aspiration: a comparative study ofcytospin preparations and flow cytometry. Am J Clin Pathol1994;101:569–76.

9 Young NA, Al-Saleem TI, Al-Saleem Z, et al. The value oftransformed lymphocyte count in subclassification of non-Hodgkin’s lymphoma by fine needle aspiration. Am J ClinPathol 1997;108:143–51.

Flow cytometry and immunohistochemistry in lymphoma diagnosis 457

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doi: 10.1136/jcp.53.6.451 2000 53: 451-457J Clin Pathol

 Frederick Mayall, Michael Dray, Dianne Stanley, et al. casesof lymphoma: a review of 73 consecutiveimmunohistochemistry in the FNA diagnosis Immunoflow cytometry and cell block

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