Splenic B cell lymphoma with lymphocyte clusters in peripheral blood smears

Abstract

EDTA induced clumping of lymphoid cells, both benign and malignant, in peripheral blood samples has been reported only rarely. Such clustering presents the laboratory and pathologist with unique diYculties in the accurate diagnosis of these disorders. A case of low grade B cell splenic lymphoma is presented with lymphocyte clumping in smears made from EDTA anticoagulated peripheral blood, the fourth case described in which neoplastic lymphoid cells form clusters in vitro in peripheral blood. The association with splenic lymphomas (three of the four cases) is intriguing but its significance remains uncertain. (J Clin Pathol 2000;53:228-230) Keywords: EDTA; leucoagglutination; lymphocytes; lymphoma In vitro agglutination of platelets or neutrophils and satellitosis of platelets around neutrophils can occur in blood exposed to ethylenediamine tetra-acetic acid (EDTA). Clumping of lymphocytes in the presence of EDTA has been reported in a single case of chronic lymphocytic leukaemia (CLL), two cases of splenic lymphoma with villous lymphocytes (SLVL), and in benign lymphocytes.1-4 Such clustering in peripheral blood samples introduces problems in the use of automated cell counters.We present a case of low grade B cell splenic lymphoma in which prominent clumping of lymphocytes was repeatedly observed in peripheral blood smears over a 30 month period, and review published reports to date concerning this phenomenon. MethodsSmears made from blood collected in tubes containing 5.4 mg K 2 EDTA and 0.129 M sodium citrate (Becton Dickinson Vacutainer Systems) were stained by Wright's method and examined by light microscopy. Bone marrow aspirate smears were stained by the WrightGiemsa method. All samples of peripheral blood collected were evaluated by a Coulter STKS haematology analyser. Flow cytometric evaluation of the peripheral blood was performed with the Coulter Epics-Profile II flow cytometer. Antibodies to B and T cell markers (Becton Dickinson) and immunoglobulin heavy and light chains (Dako) were used to characterise the phenotype of the circulating lymphocytes. Case reportA 65 year old white woman with a 25 year history of polycythaemia vera and stable splenomegaly presented with symptoms of increasing spleen size and subsequently underwent splenectomy. Preoperative examination of the peripheral blood showed a white count of 5.5 × 10 9 /litre, with 62% neutrophils and 27% lymphocytes; no clusters of lymphocytes were identified.The resected spleen weighed 3243 g. Microscopic examination showed diVuse infiltration of the red pulp by small, uniform appearing lymphocytes with round nuclei, inconspicuous nucleoli, and moderate amounts of cytoplasm, which expressed CD20 by immunohistochemical staining. Involvement of the white pulp was not identified, and no mass lesions were noted.Two days after surgery, a routine complete blood count showed marked leucocytosis (36 × 10 9 /litre). The automated diVerential count showed 85% neutrophils and 5% lymphocytes; however, a manual diVerential on a peripheral smear ...

Spurious counts and spurious results on haematology analysers: a review. Part II: white blood cells, red blood cells, haemoglobin, red cell indices and reticulocytes

“…Lymphocyte aggregation was also reported to occur in chronic lymphocytic leukaemia patients, spontaneously when lymphocyte counts are extremely high (>400 · 10 9 /l; O'Flaherty, Kreutzer & Ward, 1978), or not (Bizzaro & Piazza, 1991). Aggregates of three to 50 lymphoma cells were observed in two cases of Splenic Lymphoma with Villous Lymphocytes (SLVL; Juneja et al, 1992;Imbing et al, 1995) and in one case to non-Hodgkin lymphoma mimicking SLVL (Shelton & Frank, 2000). In these situations, the largest aggregates are overlooked by the HA and WBC count is spuriously but variably low.…”

“…Aggregates of three to 50 lymphoma cells were observed in two cases of Splenic Lymphoma with Villous Lymphocytes (SLVL; Juneja et al , 1992; Imbing et al. , 1995) and in one case to non‐Hodgkin lymphoma mimicking SLVL (Shelton & Frank, 2000). In these situations, the largest aggregates are overlooked by the HA and WBC count is spuriously but variably low.…”

“…The smallest aggregates may disturb WBC differential and may or may not generate a flag, depending on the type of HA used (Shelton & Frank, 2000). In all cases reported so far EDTA was implicated, although small clusters of cells were also observed on heparinized samples drawn as controls (Deol, Hernandez & Pierre, 1996) and sodium citrate did not appreciably change the clustering tendency of the lymphocytes in one case (Shelton & Frank, 2000). Fingerpricking and immediate dilution of blood seems the best way to avoid aggregates (Lesesve et al.…”

“…, 2001). Heating (37 °C) was reported to reduce clumping either partly (Shelton & Frank, 2000) or had no significant effect on the size of clumps (Lesesve et al. , 2001).…”

“…Fingerpricking and immediate dilution of blood seems the best way to avoid aggregates (Lesesve et al, 2001). Heating (37°C) was reported to reduce clumping either partly (Shelton & Frank, 2000) or had no significant effect on the size of clumps (Lesesve et al, 2001). As the number of cases reported is low, only hypotheses on the mechanism(s) leading to lymphocyte agglutination have been proposed, implicating various molecules such as adrenalin (epinephrin), arachidonic acid, or leukotrien B4 (Villa et al, 1984;Shelton & Frank, 2000).…”