Multiple Parameter Flow Cytometric Immunophenotyping Is Helpful in the Diagnosis of Patients with Chronic Lymphoproliferative Diseases.
Authors: Sun Xuemei , Chen Junhao , Xu Jingyan , Zhang Yifen , Xie Wei 
Published: October 2019

Abstract
The new WHO classification of lymphoid malignancies can become a big challenge to pathologists, owing to technical limitations of conventional pathologic diagnosis and immunohistochemistry. Multiple parameter analysis by flow cytometry (FCM) could provide a potent tool in improving the diagnostic status of chronic lymphoproliferative diseases(CLPDs). To evaluate its role in improving the accuracy and efficacy of diagnosis, we studied the immunophenotype by FCM in a total of 214 samples, including lymph nodes, peripheral blood, bone marrow and cerebrospinal fluid samples, in 186 patients with suspected mature B or T cell neoplasmas. A total of 102 male and 84 female patients were included in this study. The medium age was 49 years old, ranging from 3 to 82. Samples were prepared in routine fashion for morphology, pathology and immunohistochemistry, as LCA, L26, UCHL-1, CD5, Cyclin D1, CD10, Bcl-2, CD20, CD3, TdT and CD79α stains were performed by traditional APAAP method. A diagnosis was made according to the new WHO classification for lymphoid malignancies. Meanwhile, suspended single cells were also prepared by direct immunophenotyping with fluorescent conjugated antibody to CD3, CD5, CD7, CD19, CD20, CD56, CD57, CD38, CD138, CD22 on cell surface and CD3, CD22, Kappa, Lambda in cytoplasm. Immunophenotype of the gated lymphocytes were analyzed with multiple parameter cytometric analysis to come to a diagnosis. The accuracy and efficacy of the diagnosis was compared between methods of conventional and conventional combined with FCM immunophenotyping. Nine out of 38 lymph nodes failing to get a correct diagnosis by conventional methods were accurately diagnosed when FCM immunophenotyping was performed (P< 0.01), but there still remained 3 cases that did not get a clear pathological diagnosis. A total of 102 out of 104 peripheral blood or bone marrow samples were diagnosed according to the new WHO classification by flow cytometric immunophenotype analysis, compared to 90 out of 104 samples by conventional immnohistochemistry (P< 0.01). There remained 2 cases without clear diagnosis, including a patient with CD5 positive B cells. He was finally diagnosed as splenic marginal zone lymphoma, after cyclinD1 immunochemistry and FISH detection of translocation between chromosome 11 and 14 were performed to exclude mantle cell lymphoma. Multiple parameter phenotype analysis in cerebrospinal fluid by FCM was also informative for the diagnosis of the infiltration of leukemia or lymphoma cells in central nervous system. A patient with Burkit lymphoma was diagnosed as diffused infiltration of lymphoma cell in center nerves system after cerebrospinal fluid examination. The same patient was considered brain infection by CT scanning before FCM immunophenotype analysis was conducted. We concluded that multiple parameter phenotypic analysis by FCM can be used in the diagnosis, differentiated diagnosis and detection of minimal residue diseases in CLPDs. It provides a useful tool to improve the accuracy and efficacy of diagnosis in lymphoid malignancies.