Flow Cytometry Of Reactive T-Cells Differentiates Lymphoproliferative Diseases

By LabMedica International staff writers
Posted on 02 Sep 2022

Image: Bone marrow aspirate from a patient with Classic Hodgkin lymphoma showing large multinucleated Reed-Sternberg cells. “Hodgkin cells” are mononuclear while “Reed-Sternberg” cells are multinucleate forms (Photo courtesy of Nidia P. Zapata, MD and Espinoza-Zamora Ramiro).

Classic Hodgkin lymphoma (cHL) is an unusual form of lymphoma characterized by a small number of neoplastic Hodgkin and Reed–Sternberg (HRS) cells in an extensive inflammatory background and about 90% of all Hodgkin lymphomas are this type.

In light of the frequent non-neoplastic causes of lymphadenopathy and involvement of sensitive locations, lymph node fine-needle aspiration (FNA) or core needle biopsy (CNB) as minimally invasive procedures are frequently the first alternatives to obtain lymph node tissue to diagnose lymphoproliferative disorders.

Clinical Laboratorians at the Zhejiang University School of Medicine (Hangzhou, China) included in a study cohort consisting of 125 males and 31 females with a male-to-female ratio of 4:1. The patients' ages ranged from 1 to 16, with a median age of 7.7 years. Within this cohort, 25 cases of cHL and 44 cases of reactive lymphoid hyperplasia (RLH) were evaluated for their CD3+CD45RO+T-cell population and CD7 expression on T-cells. Of the reactive cases, 13 were Epstein–Barr virus (EBV) positive.

Lymph node biopsy specimens were obtained from the patients, fixed in 10% neutral buffered formalin, and immediately sent to the pathology laboratory. Samples were prepared for staining with hematoxylin and eosin (HE), periodic acid-Schiff (PAS), and immunohistochemistry (IHC) for histopathology analysis. All immunohistochemistry staining was performed using a two-step technique with the DAKO EnVision HRP System (Agilent Technologies, Santa Clara, CA, USA). Single-cell suspensions from each lymph node were prepared according to a standard protocol. The specimens were analyzed for a range of antigens on a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

The investigators reported that of the suspected lymphoma cases, 55.7% (87/156) were diagnosed with non-Hodgkin lymphoma (NHL) and 16.0% (25/156) with Hodgkin lymphoma based on the histopathological features and immunohistochemical results. The NHL group consisted of 27 cases of T-lymphoblastic lymphoma (T-LBL), 10 cases of B-lymphoblastic lymphoma (B-LBL), 20 cases of Burkitt's lymphoma, nine cases of diffuse large B-cell lymphoma (DLBCL), one follicular lymphoma (FL) case, 17 anaplastic large cell lymphoma (ALCL) cases, one NK case, and two cases of NK/T lymphoma.

The overall concordance of FCI data with the histopathologic results of these cases was 81.4%. A reactive expansion of T-cells with increased expression of CD45RO was present in the reactive infiltrate of cHL (CD45RO/CD3, 67.5%) and Epstein–Barr virus (EBV) infected RLH (62.7%) but not in EBV-negative RLH (28.0%). The mean fluorescence intensity (MFI) of CD7 was higher for cHL and differed significantly from EBV-positive RLH (138.5 versus 63.8). A proposed diagnostic algorithm markedly elevated the overall concordance rate from 81.4% to 97.4%.

The authors concluded that immunophenotyping the reactive infiltrate of lymphoid tissue using flow cytometry is a reliable supplement to histopathology for the rapid diagnosis of pediatric cHL. The study was published on August 21, 2022 in the Journal of Clinical Laboratory Analysis.