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Flow cytometric identification of circulating neoplastic cells (Sezary cells) in patients with mycosis fungoides and Sezary syndrome is essential for diagnosis, staging, and prognosis. Although recent advances have improved the performance of this laboratory assay, the complex immunophenotype of Sezary cells and overlap with reactive T cells demand a high level of analytic expertise. We utilized machine learning to simplify this analysis using only 2 predefined Sezary cell-gating plots. We studied 114 samples from 59 patients with Sezary syndrome/mycosis fungoides and 66 samples from unique patients with inflammatory dermatoses. A single dimensionality reduction plot highlighted all TCR constant β chain-restricted (clonal) CD3 + /CD4 + T cells detected by expert analysis. On receiver operator curve analysis, an aberrancy scale feature computed by comparison with controls (area under the curve = 0.98) outperformed loss of CD2 (0.76), CD3 (0.83), CD7 (0.77), and CD26 (0.82) in discriminating Sezary cells from reactive CD4 + T cells. Our results closely mirrored those obtained by exhaustive expert analysis for event classification (positive percentage agreement = 100%, negative percentage agreement = 99%) and Sezary cell quantitation (regression slope = 1.003, R squared = 0.9996). We demonstrate the potential of machine learning to simplify the accurate identification of Sezary cells., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

T-lymphoblastic leukemia/lymphoma (T-ALL/LBL) is a rare acute leukemia that expresses cytoplasmic CD3 (cCD3) and frequently lacks surface CD3. Given that routine flow cytometric testing for cCD3 may not be feasible and cCD3 interpretation may be difficult, we investigate if surface CD2 and/or CD7 expression on blasts can be used by flow cytometry to screen for T-lineage acute leukemia. We retrospectively reviewed flow cytometric data from 233 acute leukemias (36 T-ALL/LBL, 8 mixed-phenotype acute leukemia T/myeloid, 80 acute myeloid leukemia, 97 B-ALL/LBL, 8 mixed-phenotype acute leukemia B/myeloid, and 4 acute undifferentiated leukemia cases). Uniform expression (≥75% of blasts) of CD2 and/or CD7 was seen in all 44 cCD3-positive cases but in only 11% (20/189) of cCD3-negative acute leukemias, thus demonstrating 100% sensitivity and 89% specificity in the identification of cCD3-positive (T-lineage) acute leukemia. To avoid selection bias, we prospectively studied 232 consecutive acute leukemias for which cCD3, CD2, and CD7 were automatically performed in all cases. Similar to the retrospective study, uniform expression of CD2 and/or CD7 on blasts showed 100% sensitivity and 88% specificity in the screening for cCD3-positive (T-lineage) acute leukemia. Therefore, acute leukemias with uniform expression of CD2 and/or CD7 warrant further testing for cCD3 to evaluate for T-lineage acute leukemia. Blasts that lack both uniform CD2 and CD7 expression do not require additional cCD3 testing. We propose that CD2 and CD7 could be utilized in a limited antibody flow cytometry panel as a sensitive, robust, and cost-effective way to screen for T-lineage acute leukemia., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objectives: The diagnosis of T-cell large granular lymphocytic leukemia (T-LGLL) is challenging because of overlapping immunophenotypic features with reactive T cells and limitations of T-cell clonality assays. We studied whether adding an antibody against T-cell receptor β constant region 1 (TRBC1) to a comprehensive flow cytometry panel could facilitate the diagnosis of T-LGLL., Methods: We added TRBC1 antibody to the standard T-cell and natural killer (NK) cell panel to assess T-cell clonality in 56 T-LGLLs and 34 reactive lymphocytoses. In addition, 20 chronic lymphoproliferative disorder of NK cells (CLPD-NKs) and 10 reactive NK-cell lymphocytoses were analyzed., Results: Clonal T cells were detected in all available T-LGLLs by monotypic TRBC1 expression and clonal/equivocal T-cell receptor gene rearrangement (TCGR) studies, compared with only 27% of T-LGLLs by killer-cell immunoglobulin-like receptor (KIR) restriction. Overall, 85% of T-LGLLs had a blood tumor burden greater than 500 cells/µL. Thirty-four reactive cases showed polytypic TRBC1 expression, except for 5 that revealed small T-cell clones of uncertain significance. All CLPD-NKs showed expected clonal KIR expression and negative TRBC1 expression., Conclusions: Addition of TRBC1 antibody to the routine flow cytometry assay could replace the TCGR molecular study and KIR flow cytometric analysis to assess clonality, simplifying the diagnosis of T-LGLL., (© American Society for Clinical Pathology, 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introducing a sensitive and specific peripheral blood flow cytometric assay for Sézary syndrome and mycosis fungoides (SS/MF) requires careful selection of assay design characteristics, and translation into a laboratory developed assay through development/optimization, validation, and continual quality monitoring. As outlined in a previous article in this series, the recommended design characteristics of this assay include at a minimum, evaluation of CD7, CD3, CD4, CD8, CD26, and CD45, analyzed simultaneously, requiring at least a 6 color flow cytometry system, with both quantitative and qualitative components. This article provides guidance from an international group of cytometry specialists in implementing an assay to those design specifications, outlining specific considerations, and best practices. Key points presented in detail are: (a) Pre-analytic components (reagents, specimen processing, and acquisition) must be optimized to: (i) identify and characterize an abnormal population of T-cells (qualitative component) and (ii) quantitate the abnormal population (semi/quasi-quantitative component). (b)Analytic components (instrument set-up/acquisition/analysis strategy and interpretation) must be optimized for the identification of SS/MF populations, which can vary widely in phenotype. Comparison with expert laboratories is strongly encouraged in order to establish competency. (c) Assay performance must be validated and documented through a validation plan and report, which covers both qualitative and semi/quasi-quantitative assay components (example template provided). (d) Ongoing assay-specific quality monitoring should be performed to ensure consistency., (© 2020 International Clinical Cytometry Society.)

This review discusses the definition and major categories of cutaneous T-cell lymphoma, Sézary syndrome and mycosis fungoides, and the role of immunophenotyping in their diagnosis. The following key points are raised: (a) Sézary syndrome and mycosis fungoides cells most often have a characteristic CD3+ CD4+ CD7- and/or CD26- immunophenotype. (b) This immunophenotype is not specific, but can assist in the distinction from non-neoplastic T cells and other subtypes of mature T-cell neoplasm. (c) However, small subsets of normal and reactive T-cells can have an overlapping immunophenotype, and can be distinguished by evaluating for additional changes in antigen expression., (© 2020 International Clinical Cytometry Society.)

A peripheral blood flow cytometric assay for Sézary syndrome (SS) or circulating mycosis fungoides (MF) cells must be able to reliably identify, characterize, and enumerate T-cells with an immunophenotype that differs from non-neoplastic T-cells. Although it is also important to distinguish SS and MF from other subtypes of T-cell neoplasm, this usually requires information in addition to the immunophenotype, such as clinical and morphologic features. This article outlines the approach recommended by an international group with experience and expertise in this area. The following key points are discussed: (a) At a minimum, a flow cytometric assay for SS and MF should include the following six antibodies: CD3, CD4, CD7, CD8, CD26, and CD45. (b) An analysis template must reliably detect abnormal T-cells, even when they lack staining for CD3 or CD45, or demonstrate a phenotype that is not characteristic of normal T-cells. (c) Gating strategies to identify abnormal T-cells should be based on the identification of subsets with distinctly homogenous immunophenotypic properties that are different from those expected for normal T-cells. (d) The blood concentration of abnormal cells, based on any immunophenotypic abnormalities indicative of MF or SS, should be calculated by either direct enumeration or a dual-platform method, and reported., (© 2020 International Clinical Cytometry Society.)

Background: Mycosis fungoides (MF) and Sézary Syndrome (SS) are clinically distinct cutaneous T-cell lymphomas with strikingly similar morphologic and phenotypic features. Prior studies have suggested phenotypic differences based on markers of antigen experience, suggesting a different cell of origin., Methods: Seventy-nine involved peripheral blood or bone marrow samples from 33 patients with SS and 19 patients with MF were studied by 10-color flow cytometry, including CD62L, CD45RA, CCR4, and PD-1. Gated tumor events were classified as naïve (T N ), central memory (T CM ), effector memory (T EM ), or effector memory with reacquired CD45RA (T EMRA ); based on CD62L + /CD45RA + , CD62L + /CD45RA - , CD62L - /CD45RA - , or CD62L - /CD45RA + phenotype, respectively. Sequential specimens were compared to assess for phenotypic stability., Results: The naïve/memory phenotype of the neoplastic T-cells was markedly heterogeneous, with a dominant T N , T CM , T EM , or T EMRA subset on 11 (14%), 32 (41%), 30 (38%), and 6 (8%) cases, respectively. There was no correlation between the diagnosis of MF or SS and putative cell of origin (P = 0.4). Overexpression of CCR4 and PD1 was observed in most cases, with higher intensity in SS compared to MF. The naïve/memory phenotype remained the same for 10 patients up to 273 days after the initial analysis; while on six patients, the naïve/memory phenotype was different from the original phenotype., Conclusions: Both SS and MF can have phenotypic features of any of the major naïve/memory T-cell subsets, which questions the current principle of "cell-of-origin" distinction between SS and MF. Phenotypic shifts within these subsets are common, suggesting a functional state rather than a cell-of-origin surrogate. © 2018 International Clinical Cytometry Society., (© 2018 International Clinical Cytometry Society.)

Objectives: To assess the ability of flow cytometry (FC) to detect putative neoplastic T-cell subsets on skin shave biopsy (SSB) specimens from patients with mycosis fungoides (MF) and to study the immunophenotype of skin-infiltrating tumor cells in MF., Methods: SSB specimens from patients with suspected MF were bisected and submitted for both FC and routine histopathology. Six-dimensional gating strategies were applied to identify putative neoplastic cells, independently from their expected immunophenotype., Results: Aberrant T cells were detected by FC in 18 of 33 SBB specimens, of which all had clinicomorphologic features of MF. Of the remaining 15 SSB specimens, six had clinicomorphologic features of MF and nine were diagnosed with benign inflammatory dermatoses. Unexpectedly, CD26 was aberrantly overexpressed in 11 (73%) and lost in three (20%) of 15 SSB specimens from patients with MF where this antigen was evaluated. Other detected aberrancies included CD3 dim- (13/18 [72%]), CD7 dim- (15/18 [83%]), and CD4-/CD8- (3/18 [17%])., Conclusions: FC is capable of identifying putative neoplastic cells on SSB specimens from patients with MF. Bright homogeneous CD26 expression is a common and previously undescribed immunophenotypic aberrancy on MF skin infiltrates., (Copyright© by the American Society for Clinical Pathology.)

Aims: Assessment of peripheral blood tumour burden for staging of cutaneous T cells lymphoma is most often accomplished by flow cytometry (FC) using various non-standarised strategies. We report the results of calculating absolute Sezary cell counts (SCCs) by FC, based on the identification of aberrant T cell clusters on a virtual 6-dimensional space and independently of the expected immunophenotype (6D-FC SCC)., Methods: 6D-FC SCCs were calculated on 65 peripheral blood specimens from 28 patients with erythrodermic cutaneous T cells lymphoma (stage III or IV). Comparisons were made with recommended FC strategies and correlations with overall mortality were studied., Results: At first visit, 17 of 28 patients (61%) had 6D-FC SCCs meeting current criteria for Stage IV disease (≥1000 SC/μL); while only 2 patients (7%) met Stage IV criteria on other tissues. As defined by comprehensive staging using clinicomorphological criteria and 6D-FC SCCs, Stage IV disease identified a subgroup of patients with worse overall survival (p=0.0227). Residual non-aberrant CD4 T cells were markedly decreased in Stage IV disease (p=0.018). Among 65 specimens, discrepancies were observed between 6D-FC SCCs and usual FC thresholds for Stage IV disease, namely a CD4:CD8 ratio ≥10:1 (9 discrepancies, 14%), and ≥40% aberrant CD4 T cells (4 discrepancies, 6%). Surprisingly, 8 cases (12%) from 6 patients exhibited two distinctively separate clusters of aberrant CD4 T cells with different CD7 and/or CD26 expression., Conclusions: Visual 6-dimensional identification of aberrant T cell clusters by FC allows for the calculation of clinically significant SCCs. Simplified gating strategies and relative quantitative values might underestimate the immunophenotypical complexity of Sezary cells.

The diagnosis of leukemic T-cell malignancies is often challenging, due to overlapping features with reactive T-cells and limitations of currently available T-cell clonality assays. Recently developed therapeutic antibodies specific for the mutually exclusive T-cell receptor constant β chain (TRBC)1 and TRBC2 isoforms provide a unique opportunity to assess for TRBC-restriction as a surrogate of clonality in the flow cytometric analysis of T-cell neoplasms. To demonstrate the diagnostic utility of this approach, we studied 164 clinical specimens with (60) or without (104) T-cell neoplasia, in addition to 39 blood samples from healthy donors. Dual TRBC1 and TRBC2 expression was studied within a comprehensive T-cell panel, in a fashion similar to the routine evaluation of kappa and lambda immunoglobulin light chains for the detection of clonal B-cells. Polytypic TRBC expression was demonstrated on total, CD4+ and CD8+ T-cells from all healthy donors; and by intracellular staining on benign T-cell precursors. All neoplastic T-cells were TRBC-restricted, except for 8 cases (13%) lacking TRBC expression. T-cell clones of uncertain significance were identified in 17 samples without T-cell malignancy (13%) and accounted for smaller subsets than neoplastic clones (median: 4.7 vs. 69% of lymphocytes, p < 0.0001). Single staining for TRBC1 produced spurious TRBC1-dim subsets in 24 clinical specimens (15%), all of which resolved with dual TRBC1/2 staining. Assessment of TRBC restriction by flow cytometry provides a rapid diagnostic method to detect clonal T-cells, and to accurately determine the targetable TRBC isoform expressed by T-cell malignancies. [ABSTRACT FROM AUTHOR]

Objectives: Patients with clonal cytopenia of undetermined significance (CCUS) are at increased risk of developing myeloid neoplasia (MN). We evaluated whether a simple flow cytometry immunophenotyping (FCIP) assay could differentiate the risk of development of MN in patients with CCUS.Methods: Bone marrow aspirates were assessed by FCIP panel in a cohort of 80 patients identified as having CCUS based on next-generation sequencing or cytogenetics from March 2015 to May 2020, with available samples. Flow cytometric assay included CD13/HLA-DR expression pattern on CD34-positive myeloblasts; CD13/CD16 pattern on maturing granulocytic precursors; and aberrant expression of CD2, CD7, or CD56 on CD34-positive myeloblasts. Relevant demographic, comorbidity, and clinical and laboratory data, including the type and extent of genetic abnormalities, were extracted from the electronic health record.Results: In total, 17 (21%) patients with CCUS developed MN over the follow-up period (median survival follow-up, 28 months [95% confidence interval, 19-31]). Flow cytometry immunophenotyping abnormalities, including the aberrant pattern of CD13/HLA-DR expression, as detected at the time of the diagnosis of CCUS, were significantly associated with risk of developing MN (hazard ratio, 2.97; P = .006). Additional FCIP parameters associated with the development of MN included abnormal expression of CD7 on myeloblasts and the presence vs absence of any FCIP abnormality.Conclusions: A simple FCIP approach that includes assessment of CD13/HLA-DR pattern on CD34-positive myeloblasts can be useful in identifying patients with CCUS at higher risk of developing MN. [ABSTRACT FROM AUTHOR]

Treated patients with multiple myeloma (MM) and PC leukemia (PCL) who were considered measurable residual disease (MRD) testing-eligible were identified and the proportion of patients in whom MRD testing was avoided by a positive PCPRO was calculated. The S-phase fraction of abnormal PCs was estimated by dividing the number of abnormal PCs in the S-window by the total number of abnormal PCs after manually gating G0-G1 and G2-M peaks [[7]]. B TO THE EDITOR: b Multiple myeloma (MM) is the second most common hematological malignancy in the United States predicted to cause 34,470 new cases and 12,640 deaths in 2022 [[1]]. [Extracted from the article]

Objectives: Flow cytometric detection of T-cell clonality is challenging, particularly in differential diagnosis of immature T-cell proliferations. Studies have shown utility of TRBC1, in conjunction with other T-cell markers, as reliable means to identify T-cell clonality by flow cytometry. One limitation of surface TRBC1 (sTRBC1) evaluation is it cannot be detected in surface CD3 (sCD3)-negative T cells, such as normal or abnormal immature T-cell precursors. Here, we assess surface and cytoplasmic TRBC1 expression patterns in the differential diagnosis of T-lymphoblastic leukemia/lymphoma (T-ALL) vs normal thymocyte expansions.Methods: Forty-three samples containing T-ALL, thymoma, normal thymus, and/or indolent T-lymphoblastic proliferation (i-TLBP), were evaluated.Results: All 24 cases with normal thymocytes or i-TLBPs revealed a characteristic and reproducible sCD3/sTRBC1 expression pattern indicative of polytypic T-cell maturation. In contrast, all 19 T-ALLs lacked this polytypic maturation pattern and were either completely negative for sCD3/sTRBC1 or showed a minor sCD3-positive subset with a monotypic TRBC1 expression pattern. Cytoplasmic TRBC1 evaluation in 9 T-ALLs demonstrated a monotypic intracellular TRBC1-positive (n = 4) or TRBC1-negative (n = 5) expression, indicative of clonality.Conclusions: Our findings demonstrate flow cytometric evaluation of surface and cytoplasmic TRBC1 expression can aid detection of T-cell clonality and differential diagnosis of immature T-cell proliferations. [ABSTRACT FROM AUTHOR]

Objectives: Distinguishing between T-cell and natural killer (NK)-cell neoplasms could be difficult given their overlapping immunophenotype. In this study, we investigated whether a flow cytometry assay with cytoplasmic staining for CD3 could be used for this purpose.Methods: Flow cytometry immunophenotyping was performed on 19 surface CD3 (sCD3)-negative mature T-cell neoplasms, 10 sCD3-positive mature T-cell neoplasms, 13 mature NK-cell neoplasms, and 19 normal controls. In addition to routine antibody panels (CD2, sCD3, CD4, CD5, CD7, CD8, CD16, CD45, CD56, CD57, CD94, CD158a, CD158b, CD158e, NKG2A TCRγ/δ), cytoplasmic staining for a monoclonal CD3 antibody (clone SK7/Leu-4) was assessed in all cases. A molecular study for T-cell receptor (TCR) gene rearrangement and an immunohistochemical study for TCRβ were performed.Results: Our data showed all T-cell neoplasms were uniformly positive for cytoplasmic CD3 (cCD3) regardless of sCD3 expression, whereas 85% of NK-cell neoplasms completely lacked cCD3 expression. The 2 cases with classic NK-cell immunophenotype but partial cCD3 expression showed no molecular genetic features of T-cell lineage by TCR gene rearrangement studies.Conclusions: Uniform cCD3 positivity and homogeneous cCD3 negativity highly suggest T-cell and NK lineage, respectively. When partial cCD3 expression is encountered, additional confirmatory studies should be pursued for the most accurate lineage assignment. [ABSTRACT FROM AUTHOR]

Objectives: To study the immunophenotypic changes of acute promyelocytic leukemia (APL) in patients who recently received all-trans retinoic acid (ATRA) and to assess the diagnostic utility of flow cytometry in this setting. Methods: Flow cytometry was performed on 29 newly diagnosed APLs and 93 other acute myeloid leukemias, including 25 HLA-DR- or CD34- cases. Clinical notes from referring institutions were reviewed to assess for recent ATRA administration. Results: Recent ATRA therapy was documented in 17 (59%) of 29 patients with APL. The main features of untreated APL were preserved with ATRA therapy, including CD34- (83% vs 82%), HLA-DR- (83% vs 100%), and CD 117+ (100% vs 77%). CD11b and CD11c were negative in all untreated APLs but positive in 76% and 88% of ATRA-treatedAPLs, respectively. Optimal diagnostic criteria for untreated APL (CD34- or HLA-DR- and CD11b- and CD11c-) showed 100% sensitivity and 98% specificity but were not useful after ATRA administration. The best interpretative approach to ATRA-treated APL (CD34- or HLA-DR-) showed 100% sensitivity but limited specificity (73%). Conclusions: Information about recent ATRA administration is critical for adequate interpretation of the flow cytometric findings in patients with suspected APL. [ABSTRACT FROM AUTHOR]

Light chain (LC) expression by flow cytometry (FC) in B cell non-Hodgkin lymphomas (B-NHLs) can occasionally be detected with one anti-LC antibody but not with another. We retrospectively analyzed 564 four-color FC files from B-NHLs, assessing LC staining with monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs). Discrepancies in LC expression between mAbs and pAbs were present in 9.2% of cases, mainly in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL; 11.1%), diffuse large B-cell lymphoma (DLBCL; 10.2%), follicular lymphoma (9.5%), and mantle cell lymphoma (11.1%) and most frequently in body fluids. Equal proportions of cases were LC+ only with pAbs (4.8%) or mAbs (4.4%). Negative LC expression with both antibodies was present in 7.5% of cases, most frequently in DLBCL (21.6%) and body fluids (27.6%). Evaluation with both mAbs and pAbs increases the sensitivity for LC detection, with no single reagent outperforming the other, although CLL/SLL preferentially showed LC expression with pAbs. [ABSTRACT FROM AUTHOR]

T-cell clonality testing is integral to the diagnostic work-up of T-cell malignancies; however, current methods lack specificity and sensitivity, which can make the diagnostic process difficult. The recent discovery of a monoclonal antibody (mAb) specific for human TRBC1 will greatly improve the outlook for T-cell malignancy diagnostics. The anti-TRBC1 mAb can be used in flow cytometry immunophenotyping assays to provide a low-cost, robust, and highly specific test that detects clonality of immunophenotypically distinct T-cell populations. Recent studies demonstrate the clinical utility of this approach in several contexts; use of this antibody in appropriately designed flow cytometry panels improves detection of circulating disease in patients with cutaneous T-cell lymphoma, eliminates the need for molecular clonality testing in the context of large granular lymphocyte leukemia, and provides more conclusive results in the context of many other T-cell disorders. It is worth noting that the increased ability to detect discrete clonal T-cell populations means that identification of T-cell clones of uncertain clinical significance (T-CUS) will become more common. This review discusses this new antibody and describes how it defines clonal T-cells. We present and discuss assay design and summarize findings to date about the use of flow cytometry TRBC1 analysis in the field of diagnostics, including lymph node and fluid sample investigations. We also make suggestions about how to apply the assay results in clinical work-ups, including how to interpret and report findings of T-CUS. Finally, we highlight areas that we think will benefit from further research. [ABSTRACT FROM AUTHOR]

Hypereosinophilia (HE) is defined as persistently elevated absolute eosinophil count (AEC) ≥ 1.5 × 109/L, which can be due to a variety of underlying causes. In this study, we investigated the prevalence and spectrum of T-cell lymphoproliferative disorders in 124 consecutive patients with HE by flow cytometric immunophenotyping. Available medical records, pathology reports and T-cell receptor (TCR) gene rearrangement were reviewed. Fifteen patients (12%) with HE had abnormal T-cell populations that were initially detected by flow cytometry. The presence of immunophenotypically abnormal T cells was not associated with higher AEC or higher absolute lymphocyte count levels, in comparison to those without abnormal T cells. Molecular studies concordantly identified a clonal TCR gene rearrangement in 8 of 10 cases tested. Based on the combination of clinical presentation, morphologic findings and laboratory studies, seven patients were diagnosed with the lymphocytic variant of hypereosinophilic syndrome and five with overt T-cell lymphoma (4 peripheral T-cell lymphoma NOS, 1 primary cutaneous T-cell lymphoma). The remaining three had an unknown diagnosis due to lack of information and additional workup would be warranted. These findings underscore the importance of flow cytometry as a screening tool to identify T-cell lymphoproliferative disorders in patients with HE. • T-cell lymphoproliferations are an uncommon but important cause of hypereosinophilia. • T-cell lymphoma and the lymphocytic variant of hypereosinophilic syndrome are included in the differential diagnosis. • Flow cytometry can be used as a screening tool to identify T-cell abnormalities in patients with hypereosinophilia. [ABSTRACT FROM AUTHOR]