3-COLOR FLOW-CYTOMETRY IN THE DIAGNOSIS OF MALIGNANT-LYMPHOMA BASED ON THE COMPARATIVE CELL MORPHOLOGY OF LYMPHOMA-CELLS AND REACTIVE LYMPHOCYTES

This study examines the identification of unusual cell populations hig hly associated with lymphoma cells (UCP-L) in diagnostic biopsy specim ens using three-color flow cytometry (3-FCM), Patterns of surface anti gen expression were used to compare the morphology of distinct lymphoi d cell populations present in biopsy specimens and determine the prese nce or absence of UCP-L, UCP-L were identified by their larger size as compared to admired reactive lymphocytes, and the method is based on the concept that neoplastic lymphoma cells are larger than reactive ly mphocytes. The comparison of relative cell sizes was determined by ove rlaying forward scatter histograms by multicolor gating using PAINT-A- GATE software, In order for separate gates to be set on UCP-L and reac tive cell populations, UCP-L had to fulfill one or more immunophenotyp ic criteria. These included: (1) belonging to a subset of B cell antig en-positive cells showing restricted expression of kappa or lambda lig ht chains; (2) belonging to a subset of CD4-positive cells having dim or absent expression of CD45RA; (3) showing alterations in antigen exp ression (loss, dimmer or brighter); or (4) expressing an immunophenoty pe that is present on only rare cell populations or is absent from rea ctive lymph nodes. The immunophenotypic profiles of the respective cel l populations were demonstrated by cubic representations to assess mor e easily the co-expression of three antigens, The common morphology of UCP-L as defined by forward and side scatter grams was consistent wit h a 'lymphoid appearance' except in several cases of HTLV-l-positive T cell lymphoma and gamma delta T cell lymphoma. The immunophenotypic p rofiles of UCP-L were confirmed to correspond to the presumptive lymph oma cell population by use of a live gating procedure on the large cel ls, which eliminated interference by reactive cells or necrotic tissue fragments, Using this method, we identified UCP-L in 208 of 293 (71%) consecutive cases of non-Hodgkin's lymphomas, while no UCP-L were see n in 72 cases of non-specific hyperplasia of lymph nodes. Twenty-seven cases could not properly be examined about the existence of UCP-L bec ause of massive necrosis, extensive fibrosis or strong non-specific st aining reactions of unknown cause, When those cases were eliminated fr om the analysis, 80% of non-Hodgkin's lymphoma were found to contain U CP-L. In B cell lymphoma, the incidence of UCP-L in nodal lymphomas (8 0%) was much higher than in extranodal lymphomas (47%), Only one of 21 cases of Hodgkin's lymphoma was found to have UCP-L, The 3-FCM proced ure was validated by the combined use of immunohistochemistry, morphol ogic examination, cytogenetic and antigen receptor gene rearrangement analysis by Southern blot hybridization. Our findings indicate that de tection of UCP-L by 3-FCM is a reliable method to distinguish non-Hodg kin lymphomas from reactive hyperplasias in the majority of cases, eve n when the reactive cell population predominates over the malignant ce ll population.