J Clin Pathol 1992;45:538-540

Clumping of lymphoma cells in peripheral bloodinduced by EDTA

S Juneja, M Wolf, R McLennan

AbstractA peripheral blood smear from a patientwith probable splenic lymphoma withvillous lymphocytes (SLVL) showedclumping oflymphoma cells. The clump-ing was not seen in films made fromunanticoagulated blood, and has not beenpreviously described in lymphomas. Thepatient also had metastatic prostaticadenocarcinoma for 30 months beforelymphoma was diagnosed and the clum-ped cells posed diagnostic problems.

Evidence of lymphoma in peripheral blood atpresentation is uncommon and the incidencevaries according to histological subtype.' Cir-culating tumour cells of non-haematologicalmalignancies are rarely found when peripheralblood is examined.2 When present, the lym-phoma cells are seen singly whereas the carcin-oma cells tend to occur in clumps.2 We describea patient with a known adenocarcinoma for30 months who posed diagnostic problemsbecause he presented at one stage with clumpsof tumour cells in the peripheral blood. Theseturned out to be the cells of an unusual type oflymphoma showing clumping induced byEDTA.

Department ofHaematology, PeterMacCallum CancerInstitute, Melbourne,AustraliaS JunejaM WolfGeelong Hospital,Victoria, AustraliaR McLennanCorrespondence to:Dr S Juneja, Department ofHaematology, PeterMacCallum Cancer Institute,481 Little Lonsdale Street,Melbourne 3000, AustraliaAccepted for publication2 October 1991

MethodsThe blood was collected in K2EDTA and a fullblood examination (FBE) was performed withthe Coulter S-Plus IV and the differentialcount was done manually. Immunologicalmarker studies were carried out on mononu-clear cell suspensions separated from blood andbone marrow on Ficoll/hypaque. Cells were

washed twice in phosphate buffered saline andstained for surface immunoglobulin usingF(ab') fragments of goat anti-human Ig sera

conjugated to fluorescein isothiocyanate(FITC), polyvalent and y, pi, ax, (5, K and Aspecific antisera. Sheep erythrocyte, mouse

erythrocyte antibody rosette tests were per-formed according to previously describedmethods.3 The bone marrow biopsy specimenwas stained with monoclonal antibodies usingthe four-layer immunoperoxidase (PAP) tech-nique.

Case reportA 66 year old man presented in October 1987with a lytic lesion in the right lower tibia. Bonescans showed additional hot spots in the righthumeral shaft, the transverse process of thefifth thoracic vertebra and ischium and anextensive periosteal reaction in the right lowertibia. A biopsy specimen of the right tibial

osteolytic lesion showed a well differentiatedadenocarcinoma. The tumour was negative forprostate specific stains. Rectal examinationshowed some irregularity in the right lobe ofthe prostate which was not thought to bemalignant. A prostatic biopsy specimen con-tained benign tissue only and a limited trans-urethral resection of the prostate was carriedout. Investigation of the gastrointestinal tractand lung did not indicate the presence of anyprimary tumour. Metastatic adenocarcinomaof unknown primary site was diagnosed.He was treated with a course of radiotherapy

to the right shoulder and right lower leg inDecember 1987, to the face and neck inFebruary 1988, lumbar spine in May 1988,thoracic and lumbar vertebrae in July 1988,and pelvis in October 1988. In 1989 he receivedsix courses ofCMF (cyclophosphamide, meth-otrexate, and 5-fluorouracil) chemotherapy fortwo cycles, with no response. Furtherchemotherapy with vincristine and mitoxan-trone, followed by radiotherapy to the right leg,was given for relief of pain. In March 1990 hehad a pathological fracture of right lower ribsthat required local radiotherapy. One monthlater he was admitted with severe pain in theleft foot and bilateral L4 and L5 nerve rootpain. A myelogram showed cauda equina syn-drome and he was admitted for urgent radio-therapy.On physical examination he had a soft mobile

submandibular lymph node but no other palp-able lymphadenopathy. The spleen and liverwere enlarged. There was tenderness over thelower lumbar and sacroiliac joints. Investiga-tions showed the serum calcium to be 2-11mmol/l (2 10-2 60), bilirubin 14 mmol/l(0-17), alkaline phosphatase 584 IU/1 (30-120),gammaGT 69 IU/l (15-162), aspartate trans-aminase 17 IU/l (5-40), total protein 62 g/l(60-80), and albumin 24 g/l (34-47). A fullblood picture showed that haemoglobin was119 g/l, the white cell count was 8- 1 x 109/l andplatelets were 209 x 109/l. A differential countshowed that 30% were abnormal cells whichwere present singly and in clumps of4-50 cells(fig 1): these cells were 10-15 pm in diameterwith moderate nuclear:cytoplasmic ratio. Thenuclei were rounded in most cells withoccasional irregular forms. The nuclearchromatin was relatively condensed but wasfiner than that seen in cells of chronic lym-phocytic leukaemia. There were occasionallymphoplasmacytic cells but this was not astriking feature. The cytoplasm was pale blueand in many cells showed hairy projectionswhich were often seen at one pole rather than

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Clumping of lymphoma cells in peripheral blood induced by EDTA

Figure I Peripheralblood smear showing aclump of lymphoma cells(Inset individuallymphoma cell).

being distributed uniformly all around the cellsurface as in hairy cell leukaemia (fig 1; inset).The tumour cells did not stain with acidphosphatase or periodic acid Schiff (PAS)stains. Clumping of tumour cells was also seenin repeat blood specimens collected in EDTAbut was absent in smears made from directvenepuncture specimens without the addedanticoagulant. Immunological marker studieson cell suspensions showed that the tumourcells were monoclonal (u, 3, and A'). The surface

immunoglobulin was bright and only 1% cellsformed mouse rosettes. The lymphoid cellsstained with Ia, CD5, 1 la, 20, 22, 32B, 33, 37,38,40, FMC1 and FMC7. CD 1 IC stained only3% of the cells. Staining with monoclonalantibodies HC2 and CD25 was not carried out.The immunological markers were confirmed

on immunoperoxidase staining of the bloodsmears where the tumour cell clumps stainedpositively with the above B cell markers. Therewere low concentrations of monoclonal free

Figure 2 Bone marrowbiopsy specimen showingmetastatic carcinoma.

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Juneja, Wolf, McLennan

light chains (A) and oligoclonal IgG in theserum and severely depressed concentrationsof polyclonal IgG, polyclonal IgA, and poly-clonal IgM. A bone marrow biopsy specimenshowed that about 50% ofthe marrow had beenreplaced by metastatic adenocarcinoma (fig 2)which stained with antibody to prostate specificantigen (PSA) and epithelial membraneantigen (EMA). The bone marrow biopsyspecimen also showed an interstitial lymphoidinfiltrate the component cells of which stainedfor leucocyte common antigen (LCA). Cyto-genetic studies on the bone marrow aspirateshowed multiple and complex abnormalitiesincluding markers, rings, and double minutes,consistent with carcinoma. On the basis ofperipheral blood and bone marrow morpho-logy, cytochemistry, and immunologicalfeatures, it was considered to be a case ofmetastatic prostatic adenocarcinoma with aconcomitant B cell lymphoma/leukaemia-most likely splenic lymphoma with villouslymphocytes (SLVL). Computed tomographyscans of the abdomen showed an enlargedspleen. The patient was started on a course ofZoladex (Goserelin) for treatment of prostatecancer. Over the next few weeks the spleenbecame palpable below the costal margin with a

rising white cell count. He was given combina-tion chemotherapy consisting of vincristine,prednisolone, and mitoxantrone. He achieved a

partial response (50% or more reduction in thecirculating lymphoma cells or reduction inspleen size). Sepsis developed after the secondcourse ofthe above combination chemotherapyat which stage he abandoned treatment andsubsequently died in October 1990.

DiscussionTo our knowledge this is the first case oflymphoma in which the circulating lymphomacells in the peripheral blood showed EDTAinduced clumping. Moreover, this was a knowncase of metastatic adenocarcinoma, thus lead-ing to diagnostic confusion. In our experienceof several hundred lymphoma cases at our

institution we have never seen clumping oflymphoma cells in the peripheral blood. Cellclumping is a characteristic feature of meta-static carcinoma in bone marrow aspirates andhas been reported in very rare cases of carcino-

cythemia in which carcinoma cells are presentin the peripheral blood.2 In our case the tumourcells in the peripheral blood were clearly iden-tified as lymphoma cells and not carcinomacells. First, the cells marked as monoclonal Bcells and did not mark with antibodies toepithelial antigens. Second, there was a simul-taneous interstitial infiltration of the marrowby identical small mononuclear cells whichmarked as B cells. Lastly, carcinoma cells in theperipheral blood are extremely rare and inthese cases the patients usually present withpancytopenia as a preterminal event.Clumping of neutrophils4 and platelets5 is

sometimes seen in the peripheral blood and inmost of these cases it occurs in the presence ofEDTA or, rarely, other anticoagulants. Ittherefore disappears when the film is madefrom blood without added anticoagulant. Inour case, the clumping of lymphoma cells wasseen in the E-DTA blood specimen and disap-peared in the film made from unanticoagulatedblood.

Splenic lymphoma with villous lymphocytes(SLVL) had been recently described.6 Thesecases occur in middle aged to elderly persons,more commonly in men, and they usually havemassive splenic enlargement. The peripheralblood shows lymphocytes with villous projec-tions which are often localised to one pole of thecell. The cells are monoclonal B lymphocytesbut differ from hairy cell leukaemia in that theydo not react with HC2, CD25, Leu M5 orcytochemically with tartrate resistant acidphosphatase. Our case fulfilled many of theabove criteria.

1 Juneja SK, WolfM, Cooper IA. Value ofbilateral biopsies innon-Hodgkin's lymphoma. J Clin Pathol 1990;43:630-2.

2 Gallivan MVE, Libich JJ. Carcinocythemia (carcinoma cellleukaemia). Cancer 1984;53:1100-2.

3 Jose DG, Pilkington GR, Wolf MM, et al. Diagnosticinformation derived from immunophenotyping 1000patients with leukaemia and lymphoma. Pathology 1983;15:53-60.

4 Epstein HD, Kruskall MS. Spurious leukopenia due to invitro granulocyte aggregation. Am i Clin Pathol 1988:89:652-5.

5 George JN, Aster RH. Quantitative platelet disorders. In:William WJ, Butler E, Erslev RJ, Lichtman MA, eds.Hematology. 4th Edn. London: McGraw-Hill, 1990:1343.

6 Melo JV, Hegde V, Parreira A, et al. Splenic B cell lym-phoma with circulating villous lymphocytes: differentialdiagnosis of B cell leukaemias with large spleens. J ClinPathol 1987;40:642-51.

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