MULTICENTRIC MFI30 STUDY: STANDARDIZATION OF CD30 EXPRESSION BY FLOW CYTOMETRY IN NON-HODGKIN LYMPHOMA

Objectives: Flow cytometry (FC) has become a useful support for cytomorphologic evaluation (CM) of fine-needle aspirates (FNA) and serous cavity effusions (SCE) in cases of suspected non-Hodgkin lymphoma (NHL). FC results may be hampered by the scarce viability and low cellularity of the specimens. Study Design: We developed a single-tube FC assay (STA) that included 10 antibodies cocktailed in 8-color labeling, a cell viability dye, and a logical gating strategy to detect NHL in hypocellular samples. The results were correlated with CM and confirmed by histologic or molecular data when available. Results: Using the STA, we detected B-type NHL in 31 out of 103 hypocellular samples (81 FNA and 22 SCE). Of these, 8 were not confirmed by CM and 2 were considered to be only suspicious. The FC-negative samples had a final diagnosis of benign/reactive process (42/72), carcinoma (27/72), or Hodgkin lymphoma (3/72). Conclusions: The STA approach allowed obtainment of maximum immunophenotyping data in specimens containing a low number of cells and a large amount of debris. The information obtained by STA can help cytomorphologists not only to recognize but also to exclude malignant lymphomas.

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Flow Cytometric Detection of the Double-Positive (CD4+CD8+)/PD-1bright T-Cell Subset Is Useful in Diagnosing Nodular Lymphocyte-Predominant Hodgkin Lymphoma

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2020-0726-oa ◽

2021 ◽

Author(s):

Zhongchuan Will Chen ◽

Juanita Wizniak ◽

Chuquan Shang ◽

Raymond Lai

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

B Cell ◽

Hodgkin Lymphoma ◽

B Cell Lymphoma ◽

The Other ◽

Non Hodgkin Lymphoma ◽

Flow Cytometric ◽

Large B Cell

Context.— Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is characterized by neoplastic lymphocyte-predominant cells frequently rimmed by CD3+/CD57+/programmed death receptor-1 (PD-1)+ T cells. Because of the rarity of lymphocyte-predominant cells in most cases, flow cytometric studies on NLPHL often fail to show evidence of malignancy. Objective.— To evaluate the diagnostic utility of PD-1 in detecting NLPHL by flow cytometry, in conjunction with the CD4:CD8 ratio and the percentage of T cells doubly positive for CD4 and CD8. Design.— Flow cytometric data obtained from cases of NLPHL (n = 10), classical Hodgkin lymphoma (n = 20), B-cell non-Hodgkin lymphoma (n = 22), T-cell non-Hodgkin lymphoma (n = 5), benign lymphoid lesions (n = 20), angioimmunoblastic T-cell lymphomas (n = 6) and T-cell/histiocyte–rich large B-cell lymphomas (n = 2) were analyzed and compared. Results.— Compared with the other groups, NLPHL showed significantly higher values in the following parameters: CD4:CD8 ratio, percentage of T cells doubly positive for CD4 and CD8, percentage of PD-1–positive T cells, and median fluorescence intensity of PD-1 expression in the doubly positive for CD4 and CD8 subset. Using a scoring system (0–4) based on arbitrary cutoffs for these 4 parameters, all 10 NLPHL cases scored 3 or higher, as compared with only 3 cases from the other groups, producing an overall sensitivity of 100% and a specificity of 96% (72 of 75). Two of the 3 outliers were non-Hodgkin lymphoma, and both showed definitive immunophenotypic abnormalities leading to the correct diagnosis. The remaining outlier was a case of T-cell/histiocyte–rich large B-cell lymphoma. Conclusions.— The inclusion of anti–PD-1 in flow cytometry is useful for detecting NLPHL in fresh tissue samples, most of which would have otherwise been labeled as nondiagnostic or reactive lymphoid processes.

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Flow Cytometry of B-Non Hodgkin Lymphoma

Diagnostic Flow Cytometry in Cytology ◽

10.1007/978-981-16-2655-5_11 ◽

2021 ◽

pp. 117-142

Author(s):

Pranab Dey

Keyword(s):

Flow Cytometry ◽

Hodgkin Lymphoma ◽

Non Hodgkin Lymphoma

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The Value of Fluorescence In Situ Hybridization and Polymerase Chain Reaction in the Diagnosis of B-Cell Non-Hodgkin Lymphoma by Fine-Needle Aspiration

Archives of Pathology & Laboratory Medicine ◽

10.5858/2004-128-1395-tvofis ◽

2004 ◽

Vol 128 (12) ◽

pp. 1395-1403 ◽

Cited By ~ 4

Author(s):

Anne M. Safley ◽

Patrick J. Buckley ◽

Andrew J. Creager ◽

Rajesh C. Dash ◽

Leslie G. Dodd ◽

...

Keyword(s):

Flow Cytometry ◽

B Cell ◽

Hodgkin Lymphoma ◽

Fine Needle Aspiration ◽

Cell Lymphoma ◽

Molecular Genetic ◽

Needle Aspiration ◽

Low Grade ◽

Fine Needle ◽

Non Hodgkin Lymphoma

Abstract Context.—Molecular genetic analyses have been predicted to improve the diagnostic accuracy of fine-needle aspiration of B-cell non-Hodgkin lymphoma. Objective.—To determine the value of routine molecular genetic assays, polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH), in the diagnosis of B-cell non-Hodgkin lymphoma by fine-needle aspiration (FNA). Design.—A multiparametric method, including cytology, flow cytometry, PCR, and FISH, was prospectively evaluated in the diagnosis of B-cell non-Hodgkin lymphoma by FNA. Aspirates from 30 consecutive patients with suspected hematolymphoid malignancies were collected. All aspirates were triaged through a uniform program including cell-size analysis, B- and T-cell clonality studies, flow cytometric immunophenotyping, and bcl-1 and bcl-2 gene rearrangements by PCR and FISH. After completion of FNA evaluations, FNA results were compared with diagnoses from prior or subsequent surgical biopsies. Results.—Monoclonal B-cell populations were detected in 18 of 20 B-cell non-Hodgkin lymphomas by flow cytometry and PCR. bcl-1 gene rearrangement was detected in 2 of 2 cases of mantle cell lymphoma. bcl-2 rearrangement was detected in 5 cases including 4 of 4 low-grade follicular lymphomas and 1 transformed follicular lymphoma. By incorporating the results of molecular genetic and ancillary diagnostics, a definitive classification was reached in 12 cases of B-cell non-Hodgkin lymphoma by FNA, including all cases of low-grade follicular lymphoma (4/4) and mantle cell lymphoma (2/2) and approximately 50% of small lymphocytic lymphoma (2/4) and large B-cell lymphoma (4/8). Ten of the 12 cases with a final classification reached by FNA had either prior or follow-up surgical biopsies, and all 10 cases showed agreement between the diagnoses rendered on FNA and surgical biopsies. Conclusions.—With proper handling and management of specimens, FNA can routinely provide samples adequate for molecular genetic studies, in addition to cytomorphology and flow cytometry, making it possible to consistently render accurate and definitive diagnoses in a subset of B-cell non-Hodgkin lymphomas. By incorporating FISH and PCR methods, FNA may assume an expanded role for the primary diagnosis of B-cell non-Hodgkin lymphoma.

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Flow Cytometric Analysis of Lymphomas: Current Status and Usefulness

Archives of Pathology & Laboratory Medicine ◽

10.5858/2006-130-1850-fcaolc ◽

2006 ◽

Vol 130 (12) ◽

pp. 1850-1858

Author(s):

Zahid Kaleem

Keyword(s):

Flow Cytometry ◽

Hodgkin Lymphoma ◽

Flow Cytometric Analysis ◽

Molecular Diagnostic ◽

Routine Practice ◽

Becton Dickinson ◽

Color Flow ◽

Non Hodgkin Lymphoma ◽

Flow Cytometric ◽

Diagnosis And Classification

Abstract Context.—Immunophenotyping has become a routine practice in the diagnosis and classification of most cases of non-Hodgkin lymphoma, and flow cytometry is often the method of choice in many laboratories. The role that flow cytometry plays, however, extends beyond just diagnosis and classification. Objective.—To review and evaluate the current roles of flow cytometry in non-Hodgkin lymphoma, to compare it with immunohistochemistry, and to discuss its potential future applications in the molecular diagnostic era. Data Sources.—The information contained herein is derived from peer-reviewed articles on the subject published in the English-language medical literature during the years 1980 to 2005 that were identified using PubMed (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi, 1980–2005) search, various books and other sources on flow cytometry, and the author's personal experience of more than 10 years with flow cytometric analysis of lymphomas and leukemia using Becton-Dickinson (San Jose, Calif) and Beckman-Coulter (Miami, Fla) flow cytometers. Study Selection.—Studies were selected based on adequate material and methods, statistically significant results, and adequate clinical follow-up. Data Extraction.—The data from various sources were compared when the methods used were the same or similar and appropriate controls were included. Most of the studies employed 2-color, 3-color, or 4-color flow cytometers with antibodies from Becton-Dickinson, Beckman-Coulter, or DakoCytomation (Carpinteria, Calif). Results were evaluated from studies utilizing the same or similar techniques and flow cytometers. Only objective data analyses from relevant and useful publications were included for reporting and discussion. Data Synthesis.—Flow cytometry serves a variety of roles in the field of lymphoma/leukemia including rapid diagnosis, proper classification, staging, minimal residual disease detection, central nervous system lymphoma detection, evaluation of prognostic markers, detection of target molecules for therapies, ploidy analysis of lymphoma cell DNA, and evaluation of multidrug-resistance markers. It offers many advantages in comparison to immunohistochemistry for the same roles and provides uses that are either not possible or not preferable by immunohistochemistry such as multiparameter evaluation of single cells and detection of clonality in T cells. Conclusions.—By virtue of its ability to evaluate not only surface but also cytoplasmic and nuclear antigens, flow cytometry continues to enjoy widespread use in various capacities in lymphoma evaluation and treatment. Additional roles for flow cytometry are likely to be invented in the future and should provide distinctive uses in the molecular era.

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Signal-Regulatory Protein-α (SIRP- α) Expression Delineates Distinct Subsets in Monocytes/Macrophages in Normal Tissue and in B-Cell Non-Hodgkin Lymphoma

Blood ◽

10.1182/blood.v128.22.2515.2515 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2515-2515

Author(s):

Ya-Ping Chen ◽

Zhi-Zhang Yang ◽

Jose C. Villasboas ◽

Tammy Price-Troska ◽

Hyo Jin Kim ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

B Cell ◽

Hodgkin Lymphoma ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Regulatory Protein ◽

Normal Tissues ◽

Non Hodgkin Lymphoma ◽

Biopsy Specimens

Abstract BACKGROUND Monocytes and macrophages (mo/mΦ) are a key part of the composition of peripheral blood (PB) and tissues and increased numbers of mo/mΦ have been associated with patient outcome in non-Hodgkin lymphoma (NHL). Because CD14 is abundantly expressed on the surface of human mo/mΦ, it is often used to identify or isolate human mo/mΦ, and immunosuppressive CD14+HLA-DRlow monocytes have been shown to be increased in the peripheral blood of NHL patients. However, we have previously shown that CD14 expression on mo/mΦ is substantially lower than CD68 expression suggesting that many CD68+ mo/mΦ are CD14 negative, especially in spleen and lymph node tissues. To characterize both CD14+ and CD14- mo/mΦ in PB and tissues, we isolated all mo/mΦ from B-cell NHL specimens and normal controls and assessed their phenotype and function. METHODS Human mo/mΦ were isolated by negative selection from PB and tissue biopsy specimens (B-cell NHL and normal tissues) using the immunomagnetic isolation (monocyte enrichment kit). Morphological and immunophenotypic characteristics of isolated mo/mΦ were determined by Giemsa stain and flow cytometry. Phagocytosis and migration assays were used to determine the function of isolated mo/mΦ. T cells were co-cultured with mo/mΦ and T cell proliferation was evaluated by CFSE staining and detected by flow cytometry. RESULTS Using a monocyte enrichment kit to isolate all mo/mΦ, the purity of isolated mo/mϕ was 85~99%, which was defined by the percentage of lineage-negative cells (i.e. cells without expression of CD3,CD19, CD20, and CD56). We found that these isolated mo/mΦ constituted 2 populations: a more frequent population of larger cells and a less common population of smaller cells. In contrast to PB, CD14 positive mo/mΦ constituted less than 40% of the tissue mo/mΦ from the isolated population. Furthermore, we found that the cell size from CD14+ mo/mΦ were larger than CD14- mo/mΦ. Using CD14 and SIRP-α, we could identify 3 populations of mo/mΦ: CD14+SIRP-αhigh, CD14-SIRP-αdim and CD14-SIRP-α- cells. CD14+SIRP-αhigh cells and CD14-SIRP-αdim cells typically constituted the population of larger cells, while CD14-SIRP-α- cells constituted the population of smaller cells. CD14-SIRP-α- cells lacked the typical phenotypic markers and had decreased phagocytic and migratory ability compared to CD14+SIRP-αhigh and CD14-SIRP-αdim cells. Furthermore, we found that these 3 populations of mo/mΦ had a differential effect on activated T-cells and that the CD14-SIRP-α- cells appeared increased number in biopsy specimens from NHL when compared to normal tissues. CONCLUSIONS We have identified a unique population of small CD68+ mo/mΦ that lack expression of CD14, SIRP-α, and other FcγR markers. This subset of mo/mΦ is more prevalent in NHL tissues and has limited phagocytic and migratory functions. This CD14-SIRP-α- mo/mΦ subpopulation may play an inhibitory role in anti-cancer and inflammatory responses. Disclosures No relevant conflicts of interest to declare.

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Serological identification of HSP105 as a novel non-Hodgkin lymphoma therapeutic target

Blood ◽

10.1182/blood-2011-06-364570 ◽

2011 ◽

Vol 118 (16) ◽

pp. 4421-4430 ◽

Cited By ~ 21

Author(s):

Roberta Zappasodi ◽

Italia Bongarzone ◽

Gaia C. Ghedini ◽

Lorenzo Castagnoli ◽

Antonello D. Cabras ◽

...

Keyword(s):

Flow Cytometry ◽

Hodgkin Lymphoma ◽

Cell Lines ◽

Tumor Burden ◽

Direct Effects ◽

Immunodeficient Mice ◽

Non Hodgkin Lymphoma ◽

Serological Identification ◽

Sharp Band

Abstract We reported that the clinical efficacy of dendritic cell–based vaccination is strongly associated with immunologic responses in relapsed B-cell non-Hodgkin lymphoma (B-NHL) patients. We have now investigated whether postvaccination antibodies from responders recognize novel shared NHL-restricted antigens. Immunohistochemistry and flow cytometry showed that they cross-react with allogeneic B-NHLs at significantly higher levels than their matched prevaccination samples or nonresponders' antibodies. Western blot analysis of DOHH-2 lymphoma proteome revealed a sharp band migrating at approximately 100 to 110 kDa only with postvaccine repertoires from responders. Mass spectrometry identified heat shock protein-105 (HSP105) in that molecular weight interval. Flow cytometry and immunohistochemistry disclosed HSP105 on the cell membrane and in the cytoplasm of B-NHL cell lines and 97 diagnostic specimens. A direct correlation between HSP105 expression and lymphoma aggressiveness was also apparent. Treatment of aggressive human B-NHL cell lines with an anti-HSP105 antibody had no direct effects on cell cycle or apoptosis but significantly reduced the tumor burden in xenotransplanted immunodeficient mice. In vivo antilymphoma activity of HSP105 engagement was associated with a significant local increase of Granzyme B+ killer cells that very likely contributed to the tumor-restricted necrosis. Our study adds HSP105 to the list of nononcogenes that can be exploited as antilymphoma targets.

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