Flow Cytometric Immunophenotyping for Minimal Residual Disease Analysis in Multiple Myeloma
Authors: Gupta Ritu , Bhaskar Archana , Jain Paresh , Sharma Atul , Kumar Lalit 
Published: April 2020

Abstract
Recent studies using multi-parametric flow cytometry (MFC) have shown frequent immunophenotypic aberrations of plasma cells (PCs) and their utility in minimal residual disease (MRD) detection in multiple myeloma (MM). Presence of normal PCs and hemodilution of the bone marrow aspirate are important considerations in MRD assessment by MFC. The purpose of present study was to characterize PCs in MM to determine the incidence of aberrant antigen expression on myeloma PCs and its role in estimation of minimal residual disease. A total of 108 patients of MM were evaluated to study the immunophenotype of PCs and 37 patients for estimation of MRD using pre-titrated volumes of the following monoclonal antibodies: CD19 FITC, CD20 FITC, CD45 FITC, CD117 PE, CD56 PE, CD38 PE-Cy5.5, CD138 APC (BD Biosciences, San Jose, CA, USA), CD52 PE, Kappa (κ) FITC and Lambda (λ) PE (Serotec). CD38 and CD138 were added to all the tubes for specific identification of PCs. 2ml of bone marrow aspirate was collected from all the subjects as per the guidelines of the institute ethics committee, processed for immunophenotyping studies using standard whole blood lysis technique and analyzed with 4-color flow cytometry. At least two antigens were aberrantly expressed in all and three in 92.6% of MM with CD19 being most frequent followed by CD56, CD45, CD52, CD117 and CD20. Using aberrant immunophenotype for identification of neoplastic PCs, MRD by MFC was detectable in all the patients of MM with ≤5% PC on bone marrow smears. The neoplastic PCs as percentage of total bone marrow cells could not differentiate protein electrophoresis (PE) + from PE− samples (Figure 1A). Assuming that in a hemodiluted bone marrow aspirate both the neoplastic and normal PCs would be proportionately reduced and normal PCs would outnumber the neoplastic clone after successful therapy; when we analyzed the tumor load i.e. the neoplastic PC as percentage of total PCs, a cut-off of 50% neoplastic PCs of total PCs could differentiate PE + samples from PE− ones (Figure 1B). To conclude, MRD detection by aberrant antigen expression is useful and evaluation of tumor load i.e. neoplastic PCs as percentage of total PC may help in better assessment of response to therapy and circumvent the problem of hemodilution in MFC based MRD assays in MM.
Figure 1: Tumor load i.e. neoplastic plasma cells (NPCs) as % of total PCs (A) & of total bone marrow cells (B) in samples evaluated for minimal residual disease. The NPCs constituted ≤50% of the total PCs in protein electrophoresis (PE) & immunofixation negative samples and &gt;60% of the total PCs in PE+ samples (B). Arrow indicates two cases which were PE- but positive on immunofixation and had relatively low numbers of neoplastic PCs (B) &lt;&gt; Figure 1:. Tumor load i.e. neoplastic plasma cells (NPCs) as % of total PCs (A) & of total bone marrow cells (B) in samples evaluated for minimal residual disease. The NPCs constituted ≤50% of the total PCs in protein electrophoresis (PE) & immunofixation negative samples and &gt;60% of the total PCs in PE+ samples (B). Arrow indicates two cases which were PE- but positive on immunofixation and had relatively low numbers of neoplastic PCs (B) &lt;&gt;