A Method for the Elution of Anti-CD20 With an EDTA/Glycine Acid Solution for Accurate Immunophenotyping of B Lymphocytes Sensitized With Rituximab

• Published in: American journal of clinical pathology 2022-05, Vol.157 (5), p.685-690
• Main Authors: Fuentes-Chávez, Eli, Méndez-Ramírez, Nereida, Maltez, Marvin M, Salazar-Riojas, Rosario, Gómez-Almaguer, David, Jaime-Pérez, José C
• Format: Article
• Language: English
• Subjects: Antibodies, Monoclonal, Murine-Derived - therapeutic use; Antigens; Antigens, CD20 - analysis; Antigens, CD20 - therapeutic use; Antineoplastic Agents - therapeutic use; B cells; B-Lymphocytes - pathology; CD20 antigen; Centrifugation; Chronic lymphocytic leukemia; Edetic acid; Edetic Acid - pharmacology; Elution; Ethylenediaminetetraacetic acid; Flow cytometry; Glycine; Glycine - therapeutic use; Hairy cell leukemia; Humans; Immunophenotyping; Immunoreactivity; Immunotherapy; Leukemia; Leukemia, Lymphocytic, Chronic, B-Cell - pathology; Leukocytes (mononuclear); Lymphocytes B; Lymphoma; Methods; Monoclonal antibodies; Non-Hodgkin's lymphomas; Rituximab; Rituximab - therapeutic use; Targeted cancer therapy
• ISSN: 0002-9173; 1943-7722
• DOI: 10.1093/ajcp/aqab176

Abstract Objectives To assess the efficacy of a method to circumvent CD20-positive antigen masking by rituximab for flow cytometry analysis of B-cell malignancies in hematology patients. Methods Mononuclear cells (MNCs) from 10 healthy individuals and 5 untreated patients with B-cell malignancies were sensitized with rituximab. Patients’ diagnoses included chronic lymphocytic leukemia, hairy cell leukemia, and follicular lymphoma. MNCs were isolated by gradient density centrifugation. An EDTA/glycine acid (EGA) elution method was used to dissociate CD20-rituximab complexes; afterwards, CD20-positive immunoreactivity was assessed by flow cytometry. A saturation curve was built based on serial dilutions of rituximab. Median fluorescent intensities of CD20-positive signals were obtained before sensitization with rituximab and after its elution with EGA. Results CD20-positive signals were not detectable by flow cytometry after rituximab sensitization of B cells. CD20-sensitized vs CD20-unsensitized, CD20-sensitized vs CD20-eluted, and CD20-eluted vs CD20-negative control (NC) MNC populations exhibited statistical differences (P = .001), while CD20-sensitized vs CD20-NC populations did not (P = .499), confirming CD20 antigen masking by rituximab. Conclusions Rituximab interfered with the flow cytometry protocol for CD20 determination on normal and neoplastic B cells. The EGA method efficiently eluted rituximab, allowing for accurate identification of CD20-positive B cells.