Diagnostic Utility of CD200 and CD43 Co-Expression by Flow Cytometry In Differentiating Chronic Lymphocytic Lymphoma From Other of Mature B Cell Non Hodgkin's Lymphoma.
Authors: Subramanian Papagudi Ganesan , Gadage Vijaya Siddharth , Kumar Ashok , Badrinath Yajamanam , Ghogale Sitaram , Shet Tanuja , AmareKadam Pratibha , Nair Reena , Gujral Sumeet 
Published: October 2019

Abstract
Abstract 4615
Diagnosis and subtyping of mature B cell Non-Hodgkin's lymphoma (NHL) in the bone marrow (BM) and peripheral blood in a leukemic phase may be challenging due to overlapping cell morphology and immunophenotypic features. This study aims to investigate the utility of CD200 and CD43 co-expression on lymphoid cells in differential diagnosis of mature B cell Non-Hodgkin's lymphoma by flow cytometry (FCM). CD200, known as OX-2 protein was first purified in 1982 as a type I membrane glycoprotein membrane of the Ig superfamily is postulated to play an immunoregulatory role in tumors. A prospective study of staging and diagnostic BM aspirates or peripheral blood samples for immunophenotyping from all consecutive cases of suspected Lymphomas referred to our center over a period of 3 months was done. Co-expression was determined by 3 color FCM. An additional tube with Fluorescein Isothiocyanate conjugated anti CD43, Phycoerythrin conjugated anti CD200 and Phycoerythrin cyanine5 conjugated CD19 was added to the routine panel of antibodies used for immunophenotyping in all the samples. Diagnostic utility of CD200 and CD43 co-expression was determined by comparison with Gold standard diagnosis made out of a combination of clinical features, morphology of tissue biopsies, FCM, immunohistochemistry and cytogenetics using Fluorescent insitu Hybridisation (FISH) for translocations involving IgH gene.
Total 116 patients of suspected cases of Lymphomas were referred to our laboratory during the period of study. In addition to Bone marrow and peripheral blood FCM evaluation was done in ascitic fluid (n=1) and pleural fluid (n=3) and FNAC of lymph node (n=1) and retro-orbital mass FNAC (n=1). Out of these, 60 patients showed involvement by mature B cell NHL. Age range of the patients was 26 years to 86 years (Male:Female = 48:12). Chronic Lymphocytic Leukemia (CLL) was the commonest subtype (43.3%, 26/60) followed by follicular lymphoma (16.6%, 10/60), Diffuse Large B-cell Lymphoma (DLBCL) (13.3%, 8/60), Mantle cell lymphoma (MCL) (8.3%, 5/60), Splenic Marginal Zone Lymphoma (SMZL) (5.0%, 3/60), Hairy cell leukemia variant (HCLv) and Waldenstrom's Macroglobulinemia (3.3%, 2/60 each), one patient each (3.3%, 1/60) of Chronic Lymphocytic Leukemia/Prolymphocytic leukemia (CLL/PL), Prolymphocytic leukemia (PL) and Burkitt's Lymphoma. One patient was of unclassifiable low grade B-cell NHL presenting with splenomegaly and pancytopenia with bone marrow involvement and no lymphadenopathy and absence of any trans locations involving IgH gene.This patient had the immunophenotype of CD19, CD22, CD23, CD25, CD79b, CD200 and Kappa positive, with CD20 dim+ and CD5, CD11c, CD103, CD123 and CD43 negative. Annexin A1 was negative in the bone marrow biopsy.
The detailed distribution of expression of CD200 and CD43 is given in Table 1.Table 1DiagnosisCD200 pos CD43 posCD200 pos CD43 negCD200 neg CD43 negCD200 neg CD43 posTOTALCLL2600026CLL/PL10001Follicular Lymphoma027110Splenic Marginal Zone Lymphoma02103Hairy cell leukemia variant00202Diffuse large B Cell Lymphoma12328Burkitts Lymphoma00101Small cell/Low grade B Cell Non-Hodgkins Lymphoma01001Mantle Cell Lymphoma00415Prolymphocytic Lymphoma10001Waldenstrom's macroglobulinemia02002Total29918460
Amongst all, CD200 and CD43 co-expression was noted in all cases of CLL, CLL/PL, PLL and only one case of DLBCL. This case of DLBCL was negative for CD5. The truth table for same is given in Table 2.Table 2CD43 and CD200CLL and related NHLNon CLL NHLTotalCoexpression present28129Coexpression absent03131Total283260
The sensitivity specificity data is given in Table 3.Table 3Specificity96.88%Sensitivity100%Positive predictive value96.55%Negative predictive value100%

Conclusion:
1. Absence of CD200 and CD43 co-expression strongly rules out a diagnosis of chronic lymphocytic leukemia and related neoplasms.
2. In the differential diagnosis CD5 positive NHL, CD200 positivity strongly suggests the diagnosis of CLL/PLL or PLL.

Disclosures:
No relevant conflicts of interest to declare.