Flow Cytometry Of Reactive T-Cells Differentiates Lymphoproliferative Diseases

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.

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).

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.