INTEGRATION OF CD200, CD43 AND ROR1 IN MULTIPARAMETER FLOW CYTOMETRY (MFC) ROUTINE PANELS FOR THE DIFFERENTIAL DIAGNOSIS OF B-CELL LYMPHOPROLIFERATIVE DISORDERS (B-LPDS)
Authors: Laganà Alessandro , Maglione Raffaele , Costa Alessandro , Mandelli Biancamaria , Bisegna Maria Laura , Milani Maria Laura , Filipponi Valeria , Nardacci Maria Grazia , Soriano Tania , Santacroce Eugenio , Petrucci Luigi , Giordano Carla , Martelli Maurizio , De Propris Maria Stefania 
Published: January 2025

Background: Clonal mature B-cell lymphoproliferative disorders (B-LPDs) are a heterogeneous group of neoplasia characterized by the proliferation of mature B lymphocytes in the peripheral blood, bone marrow and/or lymphoid tissues. B-LPDs classification into different subtypes and their diagnosis are based on a multiparametric approach, however accurate diagnosis may be challenging, especially for cases of ambiguous interpretation. Multiparameter flow cytometry (MFC) represents an extensively used technique to detect the presence of different cellular lines in immunology and hematology. MFC results provide an essential contribution to B-LPDs diagnostic process, even more considering that panels are constantly integrating novel markers to improve diagnostic accuracy.
Objectives: The aim was to evaluate the contributing role of MFC routinary studies analyzing the expression and the mean fluorescence intensity (MFI) of CD200, ROR1 and CD43 in various B-LPDs, to evaluate their usefulness in differential diagnosis of these diseases.
Methods: We retrospectively evaluated a total of 2615 consecutive cases of newly samples (collected mostly from patients with lymphocytosis) analyzed by MFC carried out in B-LPDs diagnostic process referred to the Division of Hematology of ‘Sapienza’ University of Rome. We compared results of CD200, ROR1 and CD43 expression percentage and their MFI between different subtypes of B-LPDs.
Results: In chronic lymphocytic leukemia (CLL) CD200, ROR1 and CD43 were always expressed with bright intensity. CLL samples presented CD200 expression and MFI [CD200%, mean: 100 (range, 24-100); positivity rate: 100%; MFI, mean = 168 (range, 10-1200)] and highly expressed statistically higher than mantle cell lymphoma (MCL) (p<0.001), which is usually negative for CD200, and variant hairy cell leukemia (vHCL, according to 2022 ICC) (p<0.001), but comparable with classic HCL (cHCL) (p>0.9). ROR1 resulted expressed in all CLL [ROR1%, mean: 100 (range, 52-100), positivity rate: 100%; MFI, mean 53 (range, 10-202)] and MCL cases with comparable MFI (p>0.9). CD43 expression and MFI were significantly higher in CLL [CD43%, mean: 99 (range, 59-100); positivity rate: 100%; MFI, mean = 218 (range, 41-980)] than in MCL, vHCL, cHCL and all the others mature B-cell neoplasia (p<0.001). CD200 and CD43 expression and MFI were significantly higher in cHCL compared to vHCL. Among the others mature B-cell neoplasia, CD200 was variably expressed in follicular lymphoma (FL), marginal zone lymphoma (MZL), diffuse large B-cell lymphoma (DLBCL) and lymphoplasmacytic lymphoma (LPL). ROR1 and CD43 presented a very low expression percentage in this latter group, being mostly negative. Persistent polyclonal B-cell lymphocytosis (PPBL) resulted uniformly positive for CD200 and negative for ROR1 and CD43.
Conclusions: Our data suggest that evaluation of CD200, ROR1 and CD43 antigens and their intensity of expression, along with commonly used markers in MFC routine panels for B-LPDs, might be extremely useful for a prompt diagnostic evaluation in the differential diagnosis of these diseases.