A Method for the Elution of Anti-CD20 With an EDTA/Glycine Acid Solution for Accurate Immunophenotyping of B Lymphocytes Sensitized With Rituximab. Issue 5 (26th October 2021)

• Record Type: Journal Article
• Authors: Fuentes-Chávez, Eli; Méndez-Ramírez, Nereida; Maltez, Marvin M; Salazar-Riojas, Rosario; Gómez-Almaguer, David; Jaime-Pérez, José C
• Is Part Of: American journal of clinical pathology. Volume 157:Issue 5(2022)
• Page Start: 685
• Page End: 690
• Publication Date: 2021-10-26
• Subjects: Flow cytometry -- B-cell malignancy -- EGA elution -- Rituximab -- Anti-CD20 -- Antigen masking -- Chronic lymphocytic leukemia -- Hairy cell leukemia -- Follicular lymphoma; Diagnosis, Laboratory -- Periodicals; Pathology -- Periodicals; 616.07
• DOI: 10.1093/ajcp/aqab176
• Languages: English
• ISSNs: 0002-9173

Abstract

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.