Utility of CD200 Expression and CD20 Antibody Binding Capacity in Differentiating Chronic Lymphocytic Leukemia from Other Chronic Lymphoproliferative Disorders
Authors: Naseem Shano , R. Poongodi , Varma Neelam , Malhotra Pankaj , Varma Subhash 
Published: November 2019

Abstract
Introduction: B-chronic lymphoproliferative disorders (CLPD) are a heterogeneous group of clonal proliferations of mature B lymphoid cells with variable clinical presentations and outcomes. Therefore, accurate classification is crucial for deciding therapy. At present flow cytometry immunophenotyping is a useful tool for diagnosing these diseases. However overlapping immunophenotypes do exist. Recently few studies have highlighted the differential expression of CD200 in various CLPDs. Studies have also shown that quantitation of CD20 antibody binding capacity (ABC) varies in different CLPDs.
In this study we therefore, planned to evaluate the utility of CD200 expression and CD20 quantitation by flow cytometry in categorizing various CLPDs and to analyse whether CD200 expression and quantitation of CD20 can be used to differentiate various CLPDs.
Methods: A total of 77 patients were recruited in the study. CD200 expression was evaluated semi-quantitatively by comparing with the isotype control; and designated as 1+, 2 + and 3+, when there was <1 log, 1-2 log and >2 log shift in mean fluorescence intensity (MFI) compared with isotype control respectively.
For CD20 ABC quantitation, 4 sets of precalibrated quantibrite beads and quantibrite CD20 phycoerythrin antibody (Becton Dickinson, San Jose) were used. ABC was calculated using geometric means on a statistical spread sheet.
Results: Out of the 77 patients, 54 (70.1%) were of chronic lymphocytic leukemia (CLL), it being the most common type of CLPD in our group of patients. Other types of CLPDs in our study included 6 (7.8%) patients of mantle cell lymphoma (MCL); 5 (6.5%) of hairy cell leukemia (HCL); 3 (3.9%) each of diffuse large B-cell lymphoma (DLBCL) and unclassified B-cell lymphomas; 2 (2.6%) each of follicular lymphoma (FL) and splenic marginal zone lymphoma (SMZL); and 1 each of small lymphocytic lymphoma (SLL) and lymphoplasmacytic lymphoma (LPL).
CD200 expression was seen in 100% CLL cases with a homogenous bright (2+) intensity. On the contrary CD200 was uniformly negative in all MCL cases except in 1, however, the intensity was dim (1+). The difference of CD200 expression between CLL and MCL was found to be statistically significant (p<0.001). All cases of HCL and 1 case of FL were also positive for CD200. Other CLPD did not express CD200.
Analysis of CD20 ABC showed that CD20 ABC of the lymphoid cells differ among various subtypes and it was significantly lowest in CLL and highest in HCL both on peripheral blood and bone marrow samples. The highest mean CD20 ABC of HCL in comparison to the lowest mean CD20 ABC of CLL was statistically significant (p<0.001). However, there was no statistically significant differences among the CD20 ABC of LPL, MCL, DLBCL, SMZL, SLL and B cell lymphomas unclassified (p>0.05). CD20 ABC of lymphoid cells was also performed in 36 normal controls. A higher CD20 ABC compared to normal controls was seen in HCL and LPL cases only. CLL and other remaining CLPD cases showed CD20 ABC lower than normal controls. Statistical analysis showed that the higher CD20 ABC of normal B lymphocytes in comparison to CLL lymphocytes was statistically significant (p=0.001).
Conclusion: In this study, we found that CD200 expression and CD20 quantitation aided in differentiating various CLPDs, especially CLL from MCL. All cases of CLL showed CD200 positivity and low CD20 ABC, compared to MCL where CD200 was predominantly negative and CD20 ABC was high.
In addition, amongst all CLPD, HCL had a distinct characteristic pattern for CD200 expression and CD20 ABC, being brightest and highest respectively.
Our results indicate that CD200 can be added to routine CLPD panel and is particularly useful in differentiating CLL from MCL.
Although, CD20 ABC is useful in differentiating CLPD, adding it to routine panel does not seem practical and cost effective; however, it may be tested in difficult diagnostic cases or where anti-CD20 therapy is planned.

Disclosures
No relevant conflicts of interest to declare.