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Multiparametric flow cytometry is a powerful diagnostic tool that permits rapid assessment of cellular antigen expression to quickly provide immunophenotypic information suitable for disease classification. This chapter describes a general approach for the identification of abnormal lymphoid populations by flow cytometry, including B, T, NK, and Hodgkin lymphoma cells suitable for the clinical and research environment. Knowledge of the common patterns of antigen expression of normal lymphoid cells is critical to permit identification of abnormal populations at disease presentation and for minimal residual disease assessment. We highlight an overview of procedures for processing and immunophenotyping non-Hodgkin B- and T-cell lymphomas and also describe our strategy for the sensitive and specific diagnosis of classic Hodgkin lymphoma, nodular lymphocyte predominant Hodgkin lymphoma, and T-cell/histiocyte-rich large B-cell lymphoma., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: Flow cytometry has not been widely used in routine clinical practice for the diagnosis of gastrointestinal lymphoma; this is mainly because of the absence of an appropriate protocol. Here, we established a protocol for flow cytometric analysis of a single biopsy specimen from the gastrointestinal mucosa and investigated its sensitivity and specificity., Design: In this prospective study, we enrolled patients with previously diagnosed gastrointestinal lymphoma and patients with gastrointestinal lesions that were suspected to be lymphoma., Results: Overall, 15 patients with gastric extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (N=8), duodenal follicular lymphoma (grade 1; N=5), and benign lymphoid hyperplasia (ileum, N=1, and rectum, N=1) were included in this study. Of these, lymphocytes were isolated from 14 patients (93.3%). There were 200,000-1,500,000 viable cells per patient. Biopsy specimens from 10 out of the 12 patients with lymphoma were positive for light chain restriction; the two patients with benign lymphoid hyperplasia showed negative results., Conclusions: An adequate number of lymphocytes for flow cytometry could be isolated from a single specimen of endoscopic mucosal biopsy from 93.3% of the patients. Overall, the sensitivity of flow cytometric analysis of light chain expression for the diagnosis of B-cell lymphoma was 83.3%, and the specificity was 100%. Although further investigation is required as the sample size of the present study was small, our study suggests a potential option for diagnosing B-cell lymphoma in the gastrointestinal mucosa., (© 2020 by the Association of Clinical Scientists, Inc.)

Tonsillar marginal zone hyperplasia may mimic mucosa-associated lymphoid tissue lymphoma, a rare diagnosis in children. Histologically, both entities can demonstrate expansion of the marginal zone with disruption of follicular architecture. However, marginal zone hyperplasia may appear polyclonal by flow cytometry. We present two pediatric tonsillectomy cases with tonsillar marginal zone hyperplasia and discuss the diagnostic challenges this poses in the pediatric population. Both tonsillectomies demonstrated expansion of marginal zones with partial architectural effacement, and flow cytometric analysis of both cases detected lambda light chain restricted, CD20(bright) B cells without CD38. Authors have suggested that the lambda restricted B cells in this setting represent naïve, unmutated B cells with preferential, but polyclonal, lambda expression. Our cases are in line with this thought. While robust, BIOMED-2 primer PCR can show dominant IgK peaks, which may be misinterpreted. This presents a diagnostic pitfall in the workup of pediatric tonsils that community pathologists must consider. [ABSTRACT FROM AUTHOR]

Evaluation of B-cell clonality can be challenging in the interpretation of lymphoid infiltrates on tissue sections. Clonality testing based on IG gene rearrangements analysis by PCR (IG-PCR) is the gold standard. Alternatively, B-cell clonality can be assessed by the recognition of immunoglobulin light chain (IgLC) restriction, by immunohistochemistry (IHC), chromogenic in situ hybridization (ISH) or flow cytometry (FC). IG-PCR requires molecular facilities, and FC requires cell suspensions, both not widely available in routine pathology units. This study evaluates the performance of B-cell clonality detection by IgLC-RNAscope® (RNAsc) in a group of 216 formalin-fixed, paraffin-embedded samples including 185 non-Hodgkin B-cell lymphomas, 11 Hodgkin lymphomas (HL) and 20 reactive samples. IgLC-RNAsc, performed in parallel with FC in 53 cases, demonstrated better performances (93% vs 83%), particularly in diffuse large B-cell lymphoma (98% vs 71%) and follicular lymphoma (93% vs 83%) diagnosis. IgLC-RNAsc was also superior to IHC and ISH especially in samples with limited tumor cell content, where IG-PCR was not informative. Performed for the first time on mediastinal lymphomas, IgLC-RNAsc identified monotypic IgLC transcripts in 69% of primary mediastinal large B-cell lymphoma (PMBCL) and 67% of mediastinal gray zone lymphomas (MGZL). IGK/L double-negative cells were detected in 1 PMBCL, 2 MGZL, and all classical HL, while monotypic IgLC expression appeared to be a hallmark in nodular lymphocyte-predominant HL. IgLC-RNAsc demonstrates to be a powerful tool in B-cell lymphoma diagnosis, above all in challenging cases with limited tumor cell content, ensuring in situ investigations on mechanisms of Ig regulation across lymphoma entities. [ABSTRACT FROM AUTHOR]

In this paper, we introduce a simplified, one-step procedure for lymphocyte isolation from an endoscopically biopsied fragment. For lymphocyte isolation, an endoscopically harvested specimen and 5 mL of normal saline solution were placed in a wire mesh strainer set in a porcelain bowl. To obtain the lymphocyte suspension, the solid specimen was crushed using the rubber portion of a plunger of a 10 mL injection syringe. Flow cytometry was performed using the lymphocyte suspension. For validating our methods, the one-step lymphocyte isolation technique was used to perform flow cytometry on samples from 23 patients with (n = 12) or without (n = 11) gastrointestinal lymphoma. Flow cytometry of light chain expression was performed in all patient samples (feasibility: 100%). Sensitivity was 83.3% (10/12) and specificity was 100% (11/11). In conclusion, lymphocytes isolated from a single endoscopic biopsy specimen using our simplified and quick procedure are suitable for flow cytometry. Considering that flow cytometry has an important advantage of providing the results on the examination day itself, the results of this study suggest that flow cytometric analysis using our single-step lymphocyte isolation technique can be potentially used to diagnose lymphoma in the gastrointestinal mucosa. • We introduce a simplified, one-step procedure for lymphocyte isolation from an endoscopically biopsied fragment. • Our technique is feasible for flow cytometric analysis in patients with gastrointestinal lymphoma as well as those with gastrointestinal lesions that are suspected to be lymphoma.

Background: The existence of immunoglobulin light chain restriction (LCR) strongly indicates B cell monoclonality. Although LCR-deficient B cell malignancies are often observed, their details are barely known. Methods: We retrospectively analyzed LCR-negative diffuse large B cell lymphoma (DLBCL) to elucidate their clinical features. Consecutive DLBCL patients (n = 119), whose histological diagnostic specimens were analyzed by flow cytometry (FCM), were divided into 2 groups: LCR-positive and LCR-negative DLBCL. Cases wherein FCM did not capture tumor cells were excluded. Results: There were 91 LCR-positive (76%) and 28 LCR-negative (24%) DLBCL. The 2 groups did not differ with regard to background, including the International Prognostic Index (IPI), each factor of IPI, gender, bulky mass and B-symptoms. FCM analysis showed that CD10-positive cases were less frequent in the LCR-negative DLBCL group than in the LCR-positive DLBCL group. CD5-positive cases were absent in the LCR-negative DLBCL group. Chromosomal analysis showed that the frequency of BCL2, BCL6 and MYC translocations did not differ between the groups. There was no survival difference in the groups. Conclusion: LCR-negative DLBCL accounts for about one fourth of all DLBCL and their prognosis is similar to that of LCR-positive DLBCL. CD10-negative status might characterize LCR-negative DLBCL. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]