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Thrombocythemia (ET), polycythemia vera (PV), primary myelofibrosis (PMF), prefibrotic/early (pre-PMF), and overt fibrotic PMF (overt PMF) are classical Philadelphia-Negative (Ph-negative) myeloproliferative neoplasms (MPNs). Differentiating between these types based on morphology and molecular markers is challenging. This study aims to clarify the application of flow cytometry in the diagnosis and differential diagnosis of classical MPNs. This study retrospectively analyzed the immunophenotypes, clinical characteristics, and laboratory findings of 211 Ph-negative MPN patients, including ET, PV, pre-PMF, overt PMF, and 47 controls. Compared to ET and PV, PMF differed in white blood cells, hemoglobin, blast cells in the peripheral blood, abnormal karyotype, and WT1 gene expression. PMF also differed from controls in CD34 + cells, granulocyte phenotype, monocyte phenotype, percentage of plasma cells, and dendritic cells. Notably, the PMF group had a significantly lower plasma cell percentage compared with other groups. A lasso and random forest model select five variables (CD34 + CD19 + cells and CD34 + CD38 - cells on CD34 + cells, CD13 dim+ CD11b - cells in granulocytes, CD38 str+ CD19 +/- plasma, and CD123 + HLA-DR - basophils), which identify PMF with a sensitivity and specificity of 90%. Simultaneously, a classification and regression tree model was constructed using the percentage of CD34 + CD38 - on CD34 + cells and platelet counts to distinguish between ET and pre-PMF, with accuracies of 94.3% and 83.9%, respectively. Flow immunophenotyping aids in diagnosing PMF and differentiating between ET and PV. It also helps distinguish pre-PMF from ET and guides treatment decisions., (© 2024 International Clinical Cytometry Society.)

Multiparameter flow cytometry is widely used for acute myeloid leukemia minimal residual disease testing (AML MRD) but is time consuming and demands substantial expertise. Machine learning offers potential advancements in accuracy and efficiency, but has yet to be widely adopted for this application. To explore this, we trained single cell XGBoost classifiers from 98 diagnostic AML cell populations and 30 MRD negative samples. Performance was assessed by cross-validation. Predictions were integrated with UMAP as a heatmap parameter for an augmented/interactive AML MRD analysis framework, which was benchmarked against traditional MRD analysis for 25 test cases. The results showed that XGBoost achieved a median AUC of 0.97, effectively distinguishing diverse AML cell populations from normal cells. When integrated with UMAP, the classifiers highlighted MRD populations against the background of normal events. Our pipeline, MAGIC-DR, incorporated classifier predictions and UMAP into flow cytometry standard (FCS) files. This enabled a human-in-the-loop machine learning guided MRD workflow. Validation against conventional analysis for 25 MRD samples showed 100% concordance in myeloid blast detection, with MAGIC-DR also identifying several immature monocytic populations not readily found by conventional analysis. In conclusion, Integrating a supervised classifier with unsupervised dimension reduction offers a robust method for AML MRD analysis that can be seamlessly integrated into conventional workflows. Our approach can support and augment human analysis by highlighting abnormal populations that can be gated on for quantification and further assessment. This has the potential to speed up MRD analysis, and potentially improve detection sensitivity for certain AML immunophenotypes., (© 2024 International Clinical Cytometry Society.)

Presence of minimal residual disease (MRD), detected by flow cytometry, is an important prognostic biomarker in the management of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, data-analysis remains mainly expert-dependent. In this study, we designed and validated an Automated Gating & Identification (AGI) tool for MRD analysis in BCP-ALL patients using the two tubes of the EuroFlow 8-color MRD panel. The accuracy, repeatability, and reproducibility of the AGI tool was validated in a multicenter study using bone marrow follow-up samples from 174 BCP-ALL patients, stained with the EuroFlow BCP-ALL MRD panel. In these patients, MRD was assessed both by manual analysis and by AGI tool supported analysis. Comparison of MRD levels obtained between both approaches showed a concordance rate of 83%, with comparable concordances between MRD tubes (tube 1, 2 or both), treatment received (chemotherapy versus targeted therapy) and flow cytometers (FACSCanto versus FACSLyric). After review of discordant cases by additional experts, the concordance increased to 97%. Furthermore, the AGI tool showed excellent intra-expert concordance (100%) and good inter-expert concordance (90%). In addition to MRD levels, also percentages of normal cell populations showed excellent concordance between manual and AGI tool analysis. We conclude that the AGI tool may facilitate MRD analysis using the EuroFlow BCP-ALL MRD protocol and will contribute to a more standardized and objective MRD assessment. However, appropriate training is required for the correct analysis of MRD data., (© 2023 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals LLC on behalf of International Clinical Cytometry Society.)

Multiparameter flow cytometry data is visually inspected by expert personnel as part of standard clinical disease diagnosis practice. This is a demanding and costly process, and recent research has demonstrated that it is possible to utilize artificial intelligence (AI) algorithms to assist in the interpretive process. Here we report our examination of three previously published machine learning methods for classification of flow cytometry data and apply these to a B-cell neoplasm dataset to obtain predicted disease subtypes. Each of the examined methods classifies samples according to specific disease categories using ungated flow cytometry data. We compare and contrast the three algorithms with respect to their architectures, and we report the multiclass classification accuracies and relative required computation times. Despite different architectures, two of the methods, flowCat and EnsembleCNN, had similarly good accuracies with relatively fast computational times. We note a speed advantage for EnsembleCNN, particularly in the case of addition of training data and retraining of the classifier., (© 2024 International Clinical Cytometry Society.)

The monocyte subset partitioning by flow cytometry, known as "monocyte assay," is now integrated into the new classifications as a supporting criterion for CMML diagnosis, if a relative accumulation of classical monocytes above 94% of total circulating monocytes is observed. Here we provide clinical flow cytometry laboratories with technical support adapted for the most commonly used cytometers. Step-by-step explanations of the gating strategy developed on whole peripheral blood are presented while underlining the most common difficulties. In a second part, interpretation recommendations of circulating monocyte partitioning from the dedicated French working group "CytHem-LMMC" are shared as well as the main pitfalls, including false positive and false negative cases. The particular flow-defined inflammatory profile is described and the usefulness of the nonclassical monocyte specific marker, namely slan, highlighted. Examples of reporting to the physician with frequent situations encountered when using the monocyte assay are also presented., (© 2024 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals LLC on behalf of International Clinical Cytometry Society.)

Background: Measurable residual disease (MRD) is an important prognostic indicator of chronic lymphocytic leukemia (CLL). Different flow cytometric panels have been developed for the MRD assessment of CLL in Western countries; however, the application of these panels in China remains largely unexplored., Methods: Owing to the requirements for high accuracy, reproducibility, and comparability of MRD assessment in China, we investigated the performance of a flow cytometric approach (CD45-ROR1 panel) to assess MRD in patients with CLL. The European Research Initiative on CLL (ERIC) eight-color panel was used as the "gold standard.", Results: The sensitivity, specificity, and concordance rate of the CD45-ROR1 panel in the MRD assessment of CLL were 100% (87/87), 88.5% (23/26), and 97.3% (110/113), respectively. Two of the three inconsistent samples were further verified using next-generation sequencing. In addition, the MRD results obtained from the CD45-ROR1 panel were positively associated with the ERIC eight-color panel results for MRD assessment (R = 0.98, p < 0.0001). MRD detection at low levels (≤1.0%) demonstrated a smaller difference between the two methods (bias, -0.11; 95% CI, -0.90 to 0.68) than that at high levels (>1%). In the reproducibility assessment, the bias was smaller at three data points (within 24, 48, and 72 h) in the CD45-ROR1 panel than in the ERIC eight-color panel. Moreover, MRD levels detected using the CD45-ROR1 panel for the same samples from different laboratories showed a strong statistical correlation (R = 0.99, p < 0.0001) with trivial interlaboratory variation (bias, 0.135; 95% CI, -0.439 to 0.709). In addition, the positivity rate of MRD in the bone marrow samples was higher than that in the peripheral blood samples., Conclusions: Collectively, this study demonstrated that the CD45-ROR1 panel is a reliable method for MRD assessment of CLL with high sensitivity, reproducibility, and reliability., (© 2024 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals LLC on behalf of International Clinical Cytometry Society.)

The assessment of T-cell clonality by flow cytometry has long been suboptimal, relying on aberrant marker expression and/or intensity. The introduction of TRBC1 shows much promise for improving the diagnosis of T-cell neoplasms in the clinical flow laboratory. Most laboratories considering this marker already have existing panels designed for T-cell workups and will be determining how best to incorporate TRBC1. We present this comprehensive summary of TRBC1 and supplemental case examples to familiarize the flow cytometry community with its potential for routine application, provide examples of how to incorporate it into T-cell panels, and signal caution in interpreting the results in certain diagnostic scenarios where appropriate., (© 2024 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals LLC on behalf of International Clinical Cytometry Society.)

Accurate knowledge of expression patterns/levels of commonly used MRD markers in regenerative normal-B-cell-precursors (BCP) is highly desirable to distinguish leukemic-blasts from regenerative-BCP for multicolor flow cytometry (MFC)-based measurable residual disease (MRD) assessment in B-lymphoblastic leukemia (B-ALL). However, the data highlighting therapy-related immunophenotypic-shift in regenerative-BCPs is scarce and limited to small cohort. Herein, we report the in-depth evaluation of immunophenotypic shift in regenerative-BCPs from a large cohort of BALL-MRD samples. Ten-color MFC-MRD analysis was performed in pediatric-BALL at the end-of-induction (EOI), end-of-consolidation (EOC), and subsequent-follow-up (SFU) time-points. We studied normalized-mean fluorescent intensity (nMFI) and coefficient-of-variation of immunofluorescence (CVIF) of CD10, CD19, CD20, CD34, CD38, and CD45 expression in regenerative-BCP (early, BCP1 and late, BCP2) from 200 BALL-MRD samples, and compared them with BCP from 15 regenerating control (RC) TALL-MRD samples and 20 treatment-naïve bone-marrow control (TNSC) samples. Regenerative-BCP1 showed downregulation in CD10 and CD34 expression with increased CVIF and reduced nMFI (p < 0.001), upregulation of CD20 with increased nMFI (p = 0.014) and heterogeneous CD45 expression with increased CVIF (p < 0.001). Immunophenotypic shift was less pronounced in the BCP2 compared to BCP1 compartment with increased CVIF in all but CD45 (p < 0.05) and reduced nMFI only in CD45 expression (p = 0.005). Downregulation of CD10/CD34 and upregulation of CD20 was higher at EOI than EOC and SFU time-points (p < 0.001). Regenerative-BCPs are characterized by the significant immunophenotypic shift in commonly used B-ALL-MRD markers, especially CD10 and CD34 expression, as compared to treatment-naïve BCPs. Therefore, the templates/database for BMRD analysis must be developed using regenerative-BCP., (© 2020 International Clinical Cytometry Society.)

Background: Daratumumab is an anti-CD38 immunotherapeutic drug that has increasingly been used to treat patients with heavily pre-treated and relapsed/refractory multiple myeloma. In so doing, the detection of CD38 antigen on plasma cells by flow cytometry is impeded. We hypothesized that alternative markers can be used in place or in addition to CD38 when detecting plasma cells post-treated with daratumumab., Methods: A total of 16 alternative markers were tested using 22 bone marrow aspirates from patients with plasma cell neoplasm. The ability of selected markers to discern plasma cells from other hematopoietic cells were evaluated. The stability of tested markers when stored at 4 or 25°C after T = 0, 24, 48, and 72 h was also established. Finally, selected markers were incorporated into a panel used for monitoring multiple myeloma measurable residual disease to test their utility to identify plasma cells in the presence of daratumumab and/or elotuzumab (anti-CD319) drugs., Results: Out of the 16 tested markers, CD319, CD54, CD229, CD317, and p63 were expressed by >90% of the plasma cells. Only CD319, CD54, and CD229 achieved 100% detection sensitivity. Further analysis showed that CD319 was better than CD229 and CD54 at resolving plasma cells from background hematopoietic cells, with CD54 being the worst (resolution metric, mean ± SD: CD319 [2.04 ± 0.86]; CD229 [1.47 ± 0.45]; and CD54 [1.22 ± 0.60]). CD229 was expressed by >90% of T lymphocytes, whereas CD319 was expressed preferentially by the CD8+ T cells and less frequently in CD4+ T cells. Additionally, CD229 was found on >60% of B and NK cells, as well as minor subsets of monocytes and granulocytes. CD319 was expressed on most NK cells and a minor subset of B cells, granulocytes, and monocytes. Even though CD229 and CD319 were expressed by different leukocyte subsets, their expression levels were highest on plasma cells. The expression of CD138 on plasma cells was significantly lower after storage at 4°C, while the expression levels of CD38, CD229, and CD319 remained stable at 4 or 25°C. Using limiting dilution experiments, the treatment of cells with daratumumab severely impeded the detection of CD38 antigen on plasma cells, whereas elotuzumab treatment did not block detection of CD319 on plasma cells., Conclusions: CD319 is a suitable alternative to CD38 for identifying plasma cells. Our results showed that a panel used for monitoring multiple myeloma measurable residual disease could be modified by using CD319 alone or in combination with CD38 to detect PCs in daratumumab or elotuzumab treated patients., (© 2020 International Clinical Cytometry Society.)

Introduction: Pre-analytical and analytical errors can threaten the reliability of flow cytometry (FC) results. A potential solution to some of these is the use of dry, pre-mixed antibodies, such as the ClearLLab 10C system. The purpose of the present study was to compare the diagnostic performance of the ClearLLab 10C B cell tube with that of our standard laboratory practice., Methods: We compared the diagnoses made with the ClearLLab 10C B cell tube (experimental strategy) with those made with standard laboratory practice (standard strategy). Samples were selected aiming for representation of the full spectrum of B cell disorders, with an emphasis on mature B cell malignancies, as well as healthy controls., Results: We included 116 samples (34 normal controls, 4 acute lymphoblastic leukemias, 54 mature lymphoproliferative disorders in peripheral blood and bone marrow, 3 myelomas, 6 bone marrow samples with involvement by lymphoma and 1 with elevated hematogone count, 14 lymph node samples, 1 cerebrospinal fluid, and 1 pleural effusion). There were two diagnostic errors (1.7%). The agreement between the two strategies in the percentage of CD19 cells and fluorescence intensity of CD5, CD19, CD20, CD200, and CD10 was very good., Conclusions: In this study, the ClearLLab 10C B cell tube performed similarly to our standard laboratory practice to diagnose and classify mature B cell malignancies., (© 2020 International Clinical Cytometry Society.)

Introduction: Myeloid neoplasm with blasts showing mast cell (MC)-differentiation and MC-component less than 10% of all nucleated cells but not fulfilling the criteria for systemic mastocytosis with associated hematological neoplasm (SM-AHN) or myelomastocytic leukemia (MML) has not been described in the literature. Herein, we report a study of diverse myeloid malignancies with blasts showing MC-differentiation but not meeting the criteria for SM-AHN or MML. We also evaluated the utility of flow-cytometric immunophenotyping (FCI) in the characterization of immature-MCs (iMCs)., Methods: We identified nine patients of myeloid neoplasms and studied their morphological, FCI, immunohistochemistry, cytogenetic and molecular characteristics. We also compared the immunophenotypic features of MCs from patient samples with control samples., Results: The study included patients with newly-diagnosed acute myeloid leukemia (n = 4), chronic myelomonocytic leukemia (n = 1), and chronic myeloid leukemia on follow-up (n = 4) showing MC differentiation in leukemic-blasts. These patients had mildly increased MCs (range, 0.5%-3%) in bone-marrow morphology, including immature-forms and did not meet the criteria for either SM-AHN or MML. On FCI, iMCs were positive for bright-CD117, heterogeneous-CD34, dim-to-negative-HLADR, and moderate-CD203c expression. Expression-levels of CD123 and CD38 were higher (p < 0.001) but CD33 and CD45 were lower in iMCs compared to mature-MC from control samples (p = 0.019 and p = 0.0037)., Conclusion: We reported a rare finding of MC differentiation of leukemic blasts in diverse myeloid neoplasms and proposed it as a potential pre-myelomastocytic leukemia condition. We described the distinct immunophenotypic signature of immature-MCs using commonly used markers and highlighted the utility of FCI for the diagnosis of this entity., (© 2020 International Clinical Cytometry Society.)

Background: Lymphocytic variant of hypereosinophilic syndrome (L-HES) is a subtype of HES driven by cytokines produced by clonal T-cells. Due to the rarity of its occurrence and challenges in diagnosis, this subtype of HES is under recognized., Methods and Results: We report seven patients with L-HES, diagnosed from a group of 136 patients who were referred to our institution for the work-up of hypereosinophilia. The clinical presentation, symptoms and signs were heterogeneous and uncharacteristic; indistinguishable from idiopathic HES. Flow cytometry immunophenotypic analysis revealed aberrant T-cells in all patients, with a Th2 immunophenotype, CD2 + CD3-CD4 + CD5 + CD7dim+/-CD8- in six of seven (86%) cases. CD10 was partially expressed in one of seven (14%) cases, and clonal TCR gene rearrangement was detected by PCR in five of seven (71%) patients. All patients were treated with corticosteroids and two of seven (29%) patients received anti-IL5 antibody therapy. With a median follow-up time of 7.5 years (2.3-14.1 years), one (11%) patient developed peripheral T-cell lymphoma 6.1 years after the initial diagnosis of L-HES and responded well to chemotherapy. All patients were alive at the last follow-up., Conclusion: In conclusion, a combination of flow cytometry immunophenotyping and molecular analysis allows the identification of aberrant T-cells, facilitating a diagnosis of L-HES in patients with eosinophilia. A correct diagnosis is essential for the proper management of these patients., (© 2020 International Clinical Cytometry Society.)

Background: Flow cytometric detection of T-cell clonality is challenging. The current available methodology for T-cell receptor (TCR) Vβ repertoire evaluation is a complex assay and has limited sensitivity especially for detecting low levels of disease. Therefore, there is an unmet need for a reliable, simple, and rapid assay to identify T-cell clonality. The rearrangement of the TCRB gene involves the random and mutually exclusive expression of one of two constant β chain genes (TRBC1 and TRBC2), analogous to the kappa and lambda gene utilization by B cells., Methods: Here, we used a single TRBC1 antibody, in conjunction with other T-cell associated markers, to detect T-cell clonality in tissue biopsies and body fluids. A total of 143 tissue/body fluid specimens from 46 patients with a definitive diagnosis of a T-cell neoplasm and 97 patients with no T-cell malignancy were analyzed with a cocktail of monoclonal antibodies including CD2/CD3/CD4/CD5/CD7/CD8/CD45/TCRγδ/TRBC1., Results: We examined TRBC1 expression on neoplastic T-cell populations identified based on their immunophenotypic aberrancies, and monotypic TRBC1 expression was identified in all 46 known T-cell lymphoma cases. We applied a similar gating strategy to the 97 cases without T-cell neoplasms, and arbitrarily dissected T-cell populations into immunophenotypically distinct subsets; in this group, we found that all cases revealed an expected polytypic TRBC1 expression in all subsets., Conclusions: Single TRBC1 antibody detection of T-cell clonality by flow cytometry is a simple, rapid, and robust assay that could be routinely utilized in flow cytometric laboratories., (© 2020 International Clinical Cytometry Society.)

Background: The Matutes score (MS) was proposed to differentiate chronic lymphocytic leukemia (CLL) from other B-cell non-Hodgkin lymphomas (B-NHLs). However, ambiguous immunophenotypes are common and remain a diagnostic challenge. Therefore, we evaluated the diagnostic benefit of measuring CD200 and CD43 expression together with the standard MS antigens., Methods: 138 lymphoma patient samples and a validation cohort of 138 additive samples were classified according to the standard MS and further assigned with one or two additional points, for high CD200 and/or CD43 expression levels. The "classical" MS and the "Matutes score-extended" (MS-e) were categorized as high (4-5/6-7), intermediate (2-3/4-5), and low (0-1/0-3). Samples were reclassified into the MS-e with focus on ambiguous cases with an intermediate "classical" MS., Results: A total of 35 of 138 (25.4%) patient samples were assigned to the intermediate MS group and confirmed by histopathological reports as CLL (14/40.0%) and B-NHLs other than CLL (21/60%). MS-e analysis identified 13 of 14 (92.9%) of CLL cases (MS-e 4-5) and 18/21 (85.7%) non-CLL cases (MS-e ≤ 3) correctly. Overall, the sensitivity of the CLL diagnosis was significantly increased by application of MS-e compared to the "classical" MS (98.8% vs. 82.7%; p = 0.0009), while specificity of both methods was almost equal (94.7% vs. 98.3%; p = 0.4795). Of note, sole measurement of CD43 and CD200 on B-cells sufficiently differentiated CLL from non-CLL with a test accuracy superior to the "classical" MS (F1 score 96.2 vs. 93.6)., Conclusion: CD200 and CD43 have a high informative value in diagnostic immunophenotyping and facilitate the separation of CLL from other B-NHLs particularly in ambiguous cases., (© 2020 International Clinical Cytometry Society.)

Background: There are no immunophenotypic guidelines for the investigation of MYC-rearranged lymphomas. We aimed to identify simple immunophenotypic features that would help to differentiate between MYC-rearranged lymphomas and guide cytogenetic analysis., Methods: We reviewed diagnostic samples from patients diagnosed with Burkitt lymphoma (BL), double-hit lymphoma (DHL), MYC-rearranged diffuse large B-cell lymphoma (MYC-DLBCL), and standard (non-MYC-rearranged) DLBCL over the last decade in our Institution. Using flow cytometry (with antibodies CD20, CD10, CD38, bcl-2, Ki-67, FMC-7, CD43, CD27, CD79b, CD23, and CD22) we determined antigen% expression and median-fluorescence intensity ratios (MFIR). The forward scatter (FS) and side scatter (SS) characteristics of tumor B-cells were compared with normal T-cells (B/T ratios) for patients with MYC-rearranged lymphomas., Results: We identified 51 patients of whom 14 had BL, 10 had DHL (6 MYC+/BCL2+; 4 MYC+/BCL6+), 8 MYC-DLBCL, and 19 standard DLBCL. The significant differences (p <.05) were: higher CD38% in BL than standard DLBCL; higher CD10% in BL and DHL versus MYC-DLBCL and standard DLBCL; higher CD10MFIR in BL than MYC-DLBCL and standard DLBCL; higher Ki-67% in BL than DHL and MYC-DLBCL; higher bcl-2% in DHL than BL; higher FMC-7% in BL than MYC-DLBCL and standard DLBCL; and lower SS (B/T) ratio in DHL than MYC-DLBCL., Conclusions: The combination of CD38% > 90, CD10% > 80, CD10MFIR > 10, bcl-2% < 30, and Ki-67% > 70 was characteristic of BL. "Deviation" from these cut-offs should raise suspicion for DHL and, therefore, BCL2 and/or BCL6 FISH is required. We also found that a diagnosis of DHL rather than of MYC-DLBCL was significantly associated with CD10% > 60, Ki-67% > 50, and SS (B/T) <1.5., (© 2020 International Clinical Cytometry Society.)

Background: The role of CD200 in the differential diagnosis of chronic lymphocytic leukemia (CLL) and classical mantle cell lymphoma (MCL) is well established. Its role in the differential diagnosis of CLL and other lymphoproliferative disorders (LPD) is less clear, in particular its positive predictive value (PPV)., Materials and Methods: We conducted a systematic review of the use of CD200 in the differential diagnosis of CLL, MCL, and other predominantly leukemic, typically CD103-negative LPD. With the results, we then derived a curve to determine the PPV based on the prevalence of the disorders included in the differential diagnosis., Results: Of 43 publications screened, 27 were included in the systematic review (5,764 patients). The median CD200 positivity rate in all studies and the percentage of CD200-positive (pooled) patients was 100% and 95% (3,061/3,208) in CLL, 4 and 8% (86/1112) in MCL and 56 and 62% (425/689) in other LPD., Conclusion: CD200 is suboptimal for the differential diagnosis of CLL and disorders other than nodal MCL., (© 2019 International Clinical Cytometry Society.)

Indolent T lymphoblastic proliferations have been reported rarely in extramedullary and extrathymic tissues. Recent work has identified these indolent T lymphoblast populations to be mostly CD4+/CD8+ (characterized by immunohistochemistry), with only limited immunophenotypic evaluation of these populations by flow cytometry (FC). We retrospectively reviewed our institutional FC archives and identified 12 samples from 10 patients with incidental T lymphoblastic populations. Samples were characterized with respect to expression of T-cell antigens, CD45, TdT, CD1a, and T/NK antigens, and light scatter properties. Overall, the proportion of T lymphoblasts was small (range 0.01-8.8% of white cells; mean, 1.7%). Histologic correlation showed scattered immature T lymphoblasts in samples without overt distortion of underlying architecture. T lymphoblasts were identified most frequently in association with Castleman disease (four) or tissues with Castleman features (four), marginal zone B-cell lymphoma (one), or tissue with reactive/atypical changes (three cases). Although three cases were composed predominantly of CD4+/CD8+ T cells, the majority of cases in our cohort (eight) included a major subset of CD4-/CD8- T lymphoblasts by FC (one case CD8+/CD4-), which has not been described previously. There was no evidence of subsequent progression to T lymphoblastic leukemia. Incidental, indolent T lymphoblastic proliferations may be detected by clinical FC. In contrast to reports, we find that these proliferations in clinical samples may contain not only CD4+/CD8+ immature T cells but also CD4-/CD8- immature T cells, expanding the immunophenotypic spectrum of this entity., (© 2019 International Clinical Cytometry Society.)

Background: The assessment of minimal residual disease (MRD) by flow cytometry (FC) has a prognostic impact in acute myeloid leukemia (AML), despite the low sensitivity in predicting relapse. Nonetheless, the role of leukemic-associated immunophenotypes (LAIPs)-related specificity on the sensitivity of MRD has not been clarified yet. In this respect, we accomplished this study., Methods: LAIP-frequencies of bone marrow samples from healthy donors and patients after treatment were quantified and subdivided in "categories of specificity" named as: "strong," "good," and "weak." At the following, the diagnostic performance of MRD was investigated in terms of sensitivity, specificity, predictive values, likelihood ratio (LR)., Results: "Strong" LAIPs were identified by CD7, CD2, CD4, and CD56 markers while "weak" LAIPs, independently of coexpressed markers, were mainly observed in CD33+ cells. MRD identified patients with significantly low DFS and OS but showed a low sensitivity in predicting relapse. Interestingly, majority of recurrences was noticed in patients with two LAIPs and lacking of "strong" LAIPs or only with one "good" LAIP. Thus, only patients showing one "strong" or two "good" LAIPs were considered suitable for MRD monitoring and selected to be further investigated. In this subset, positive MRD predicted a poor prognosis. Moreover, a higher sensitivity, negative predictive value (NPV) and LR- were observed after comparison with the previous series., Conclusions: These data highlight the relevant role of LAIP classification in "categories of specificity" in improving the sensitivity of MRD as assessed by FC., (© 2019 International Clinical Cytometry Society.)

Background: Recent advances in therapeutic interventions have dramatically improved complete response rates in patients with multiple myeloma (MM). The ability to identify residual myeloma cells (e.g., measurable residual disease [MRD]) can provide valuable information pertaining to patient's depth of response to therapy and risk of relapse. Multiparametric flow cytometry is an excellent technique to monitor MRD and has been demonstrated to correlate with patient outcome post-treatment. To achieve the high sensitivity (one abnormal cell in 10 5 -10 6 cells) required for MRD evaluation, millions of cells have to be acquired and conventional immunophenotyping protocols are unable to attain these numbers, indicating the needs for alternative flow cytometric staining procedures. A bulk, "Pre-lysis" method is the consensus approach for staining large number of cells, requires two red blood cell lysis steps, and can adversely affect epitope density. In this study, we tested the "Pooled-tube" and "Dextran Sedimentation" staining procedures and correlated them with the "Pre-lysis" method as potential alternative approaches., Methods: A total of 22 bone marrow aspirates from patients with plasma cell (PC) dyscrasia were processed in parallel using the "Pre-lysis," "Pooled-tube," and "Dextran Sedimentation" techniques. Stain indices were calculated and compared to assess their impacts on staining performance for each antibody used in the consensus panel. The recovery of normal and abnormal PCs, mast cells, and B cell precursors was enumerated and compared after their counts were normalized using fluorescent beads. The limit of blank, limit of detection, and lower limit of quantification were established using serial dilution experiments., Results: The staining performances of CD19 PECy7, CD27 BV510, CD81 APCH7, and CD138 BV421 were improved using the "Pooled-tube" method when compared to "Pre-lysis." "Pre-lysis" was better at resolving CD56 using clone C5.9 but our results demonstrated similar improvement can also be achieved by "Pooled-tube" when alternative CD56 PE clones were used. "Dextran sedimentation" yielded similar staining results when compared to "Pre-lysis" for all the markers analyzed. The "Pooled-tube" method, when normalized to "Pre-lysis," recovered higher numbers of total PCs (1.2 ± 0.2 times higher; p = .049), normal PCs (1.4 ± 0.26; p = .007), mast cells (1.46 ± 0.27; p = .003), and B cell precursors (1.42 ± 0.3; p = .011), but not abnormal PCs (1.09 ± 0.2; p = .352). There was no evidence that the recovery of cells was different between "Pre-lysis" versus "Dextran Sedimentation." All three flow cytometric assays achieved a minimum sensitivity of 10 -5 and approached that of 10 -6 for detecting rare events., Conclusion: Both "Pooled-tube" and "Dextran Sedimentation" staining procedures were comparable to the "Pre-lysis" method and are suitable high sensitivity flow cytometric approaches that can be used to process bone marrow samples for MM MRD testing., (© 2019 International Clinical Cytometry Society.)

Background: Several studies have used CD157 in white blood cells with or without proaerolysin (fluorescein-labeled proaerolysin [FLAER])-based flow cytometry assays in the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH)., Methods: We designed a seven-color CD marker panel comprising FLAER, CD15, CD64, CD24, CD14, CD157, and CD45 to verify CD157's clinical applicability and diagnostic performance in a clinical setting., Results: A total of 356 samples were tested. These included 43 PNH-positive samples and 313 PNH-negative samples. PNH clones confirmed by the CD157/FLAER combination were almost identical in size to the clones detected by the CD24/CD14/FLAER combination, and the accuracy of the CD157/FLAER combination was 100% in granulocytes and 99.7% in monocytes. Substitution of FLAER with CD157 resulted in 1.9% and 3.5% false-positives in granulocytes and monocytes, respectively. The accuracy was 98.3% and 96.9% in granulocytes and monocytes, respectively. Moreover, the loss of CD157 expression in granulocytes and monocytes was commonly observed in non-PNH patients. Some monocytes in non-PNH patients had weak expression of CD14 but normal expression of FLAER. In this study, PNH clones in granulocytes were always lower than those in matched monocytes., Conclusions: We performed the first prospective exploration of the clinical usefulness of FLAER and CD157 in simultaneously recognizing PNH clones in granulocytes and monocytes and verified the applicability of CD157 in substitute for both CD14 and CD24. In the conditions where FLAER is not available, substitution of FLAER with CD157 is acceptable for the identification of PNH clones under the premise of giving full attention to the potential for false-positives., (© 2019 International Clinical Cytometry Society.)

Background: The diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) relies on flow cytometric demonstration of loss of glycosyl-phosphatidyl inositol (GPI)-anchored proteins from red blood cells (RBC) and white blood cells (WBC). High-sensitivity multiparameter assays have been developed to detect loss of GPI-linked structures on PNH neutrophils and monocytes. High-sensitivity assays to detect PNH phenotypes in RBCs have also been developed that rely on the loss of GPI-linked CD59 on CD235a-gated mature RBCs. The latter is used to delineate PNH Type III (total loss of CD59) and PNH Type II RBCs (partial loss of CD59) from normal (Type I) RBCs. However, it is often very difficult to delineate these subsets, especially in patients with large PNH clones who continue to receive RBC transfusions, even while on eculizumab therapy., Methods: We have added allophycocyanin (APC)-conjugated CD71 to the existing CD235aFITC/CD59PE RBC assay allowing simultaneous delineation and quantification of PNH Type III and Type II immature RBCs (iRBCs)., Results: We analyzed 24 medium to large-clone PNH samples (>10% PNH WBC clone size) for PNH Neutrophil, PNH Monocyte, Type III and Type II PNH iRBCs, and where possible, Type III and Type II PNH RBCs. The ability to delineate PNH Type III, Type II, and Type I iRBCs was more objective compared to that in mature RBCs. Additionally, total PNH iRBC clone sizes were very similar to PNH WBC clone sizes., Conclusions: Addition of CD71 significantly improves the ability to analyze PNH clone sizes in the RBC lineage, regardless of patient hemolytic and/or transfusion status., (© 2019 International Clinical Cytometry Society.)

Background: We sought to enhance the cytometric analysis of myelodysplastic syndromes (MDS) by performing a pilot study of a single cell mass cytometry (MCM) assay to more comprehensively analyze patterns of surface marker expression in patients with MDS., Methods: Twenty-three MDS and five healthy donor bone marrow samples were studied using a 34-parameter mass cytometry panel utilizing barcoding and internal reference standards. The resulting data were analyzed by both traditional gating and high-dimensional clustering., Results: This high-dimensional assay provided three major benefits relative to traditional cytometry approaches: First, MCM enabled detection of aberrant surface maker at high resolution, detecting aberrancies in 27/31 surface markers, encompassing almost every previously reported MDS surface marker aberrancy. Additionally, three previously unrecognized aberrancies in MDS were detected in multiple samples at least one developmental stage: increased CD321 and CD99; and decreased CD47. Second, analysis of the stem and progenitor cell compartment (HSPCs), demonstrated aberrant expression in 21 of the 23 MDS samples, which were not detected in three samples from patients with idiopathic cytopenia of undetermined significance. These immunophenotypically abnormal HSPCs were also the single most significant distinguishing feature between clinical risk groups. Third, unsupervised clustering of high-parameter MCM data allowed identification of abnormal differentiation patterns associated with immunophenotypically aberrant myeloid cells similar to myeloid derived suppressor cells., Conclusions: These results demonstrate that high-parameter cytometry methods that enable simultaneous analysis of all bone marrow cell types could enhance the diagnostic utility of immunophenotypic analysis in MDS., (© 2020 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.)

Detection of minimal/measurable residual disease (MRD) in acute myeloid leukemia (AML) is important for guiding patient-specific clinical management. Natural killer (NK) cells can express various markers not typically associated with NK lineage, potentially confounding the detection of MRD by flow cytometry. We have observed CD33 expression on NK cells when evaluating for AML MRD in routine clinical practice in multiple patient samples. To characterize CD33 expression on NK cells, 40 peripheral blood or bone marrow samples with NK cells present at >5% of lymphocytes were selected for further assessment of NK cell phenotype and CD33 expression. Seven of the 40 samples (17.5%) were found to have CD33 expression on at least 5% of the NK cells. The CD33-positive NK cell population accounted for an average of 11.4% of NK cells (median 11.9%, range 8.0-15.3%) and 2.2% of total white cells (median 1.1%, range 0.1-10.1%). This NK cell subset expressed bright CD2, bright CD56, and dim CD16. On average, CD33 expression on NK cells was dimmer than on monocytes (mean median fluorescence intensity ratio 0.4; range 0.1-1.0). This study characterizes expression of CD33 on NK cells. Recognition of this pattern of antigen expression is critical in evaluating samples for MRD in patients with myeloid neoplasms, particularly AML., (© 2019 International Clinical Cytometry Society.)

High-dimensional mass cytometry data potentially enable a comprehensive characterization of immune cells. In order to positively affect clinical trials and translational clinical research, this advanced technology needs to demonstrate a high reproducibility of results across multiple sites for both peripheral blood mononuclear cells (PBMC) and whole blood preparations. A dry 30-marker broad immunophenotyping panel and customized automated analysis software were recently engineered and are commercially available as the Fluidigm® Maxpar® Direct™ Immune Profiling Assay™. In this study, seven sites received whole blood and six sites received PBMC samples from single donors over a 2-week interval. Each site labeled replicate samples and acquired data on Helios™ instruments using an assay-specific acquisition template. All acquired sample files were then automatically analyzed by Maxpar Pathsetter™ software. A cleanup step eliminated debris, dead cells, aggregates, and normalization beads. The second step automatically enumerated 37 immune cell populations and performed label intensity assessments on all 30 markers. The inter-site reproducibility of the 37 quantified cell populations had consistent population frequencies, with an average %CV of 14.4% for whole blood and 17.7% for PBMC. The dry reagent coupled with automated data analysis is not only convenient but also provides a high degree of reproducibility within and among multiple test sites resulting in a comprehensive yet practical solution for deep immune phenotyping., (© 2019 International Clinical Cytometry Society.)

Background: Daratumumab (DARA) is a humanized Immunoglobulin G(IgG)1-kappa monoclonal antibody against CD38 antigen that is shown to improve outcomes in relapsed/refractory plasma cell myeloma (PCM) patients. Since CD38 is expressed by different hematopoietic elements, DARA has the potential to interfere with flow cytometric assessment of bone marrow specimens., Methods: Flow cytometric analysis of bone marrow samples from 10 PCM on DARA and 5 control samples was performed using two different antibody panels., Results: Bone marrow samples from PCM patients on DARA exhibited a population of CD19+ CD10+ B-lymphoid cells with kappa light chain restriction. Further morphological and immunophenotypic studies suggested that this population represents marrow hematogones. Marrow hematogones from control samples showed normal immunophenotypic profiles., Conclusion: DARA on the surface of hematogones interferes with flow cytometric clonality study leading to artifactual kappa light chain restriction, which can result in false interpretation of a concurrent clonal B-cell proliferation. In the era of rapidly growing list of therapeutic monoclonal antibodies, flow cytometry pathologists should be aware of potential interferences to avoid misdiagnosis. © 2019 International Clinical Cytometry Society., (© 2019 International Clinical Cytometry Society.)

Background: T-cell/histiocyte rich large B-cell lymphoma (THRLBCL) is B-cell lymphoma in which rare neoplastic cells are embedded in a reactive infiltrate. We describe the first characterization of the neoplastic cells by flow cytometry (FC)., Methods: Using FC, we immunophenotyped the neoplastic cells of 11 cases of THRLBCL and 11 cases of DLBCL, NOS (controls). Neoplastic THRLBCL cells were also purified by flow cytometric cell sorting (FCCS)., Results: A neoplastic THRLBCL population was detected by FC in 9 of 11 cases (82%). Neoplastic THRLBCL cells demonstrated an aberrant germinal center B-cell immunophenotype (bright CD20, bright CD40; positive for Bcl-6 and CD75; weakly positive for CD32; negative for IgH). With regard to adhesion molecules, CD54 was overexpressed, CD58 expression varied between cases, and CD50 expression was intermediate. Evaluation of immunomodulatory receptors demonstrated that PD-L2 was weakly expressed and PD-L1 was variably expressed. Finally, FCCS of two cases showed large multi-lobated cells with morphology consistent with neoplastic cells of THRLBCL., Conclusions: The immunophenotype identified and the morphology of the FCCS purified cells confirms the FC defined populations are neoplastic cells from THRLBCL. While the cohort is small, neoplastic THRLBCL cells lack surface immunoglobulins. CD40, CD50, and CD54 were overexpressed in THRLBCL relative to DLBCL, NOS, perhaps contributing to the predominance of T cells in THRLBCL. Expression of CD32, PD-L1, and PD-L2 may be useful in distinguishing THRLBCL and NLPHL. Finally, the FC assays will be useful for purifying neoplastic cells of THRLBCL and for diagnostic immunophenotyping of THRLBCL. © 2019 International Clinical Cytometry Society., (© 2019 International Clinical Cytometry Society.)

Background: Neoplasms derived from plasmacytoid dendritic cells (PDCs) are currently divided into two broad categories: mature PDC proliferations associated with myeloid neoplasms (MPDMN) and blastic plasmacytoid dendritic cell neoplasm (BPDCN); only BPDCN is recognized in the WHO 2016 classification of hematopoietic neoplasms. We present seven patients with high grade myeloid neoplasms (MNs), mostly acute leukemias, having a spectrum of PDC differentiation and not fitting with MPDMN or BPDCN., Methods: We analyzed seven MN cases having increased myeloblasts and prominent CD56-negative PDC proliferations comprising 5-26% of bone marrow or blood cellularity as measured by flow cytometry. The cases included five acute myeloid leukemia (three FAB M4 subtype, two unclassified), one mixed phenotype acute leukemia, and one case of unclassified MN., Results: Six cases demonstrated immunophenotypic evidence of PDC differentiation from leukemic blasts, based on variable expression of CD34, CD45, CD123, and CD304 by the leukemic cells. Four cases had circulating PDC populations in blood. None of the cases met clinical or pathologic criteria for BPDCN. Morphologic review was available for four acute leukemia cases and demonstrated either nodular or interstitial infiltrates of PDCs. All cases had an aggressive clinical course, and three cases had FLT3 ITD mutation., Conclusions: These cases demonstrate that high grade MNs, in particular AML, can exhibit PDC differentiation, with or without monocytic differentiation, in a manner distinct from MPDMN or BPDCN. The existence of MNs with immature PDC proliferations suggests that there is a broader spectrum of PDC-associated neoplasms than currently recognized. © 2019 International Clinical Cytometry Society., (© 2019 International Clinical Cytometry Society.)

Background: Extranodal natural killer (NK)/T-cell lymphoma, nasal type (ENKTL-N) is an aggressive lymphoma typically diagnosed by examining small biopsy specimens. Flow cytometry is very valuable for the diagnosis and classification of several kinds of hematolymphoid neoplasms but has not been widely used for diagnosing ENKTL-N., Methods: We systematically investigated the flow cytometry characteristics of 26 solid tissue biopsy specimens of ENKTL-N at initial diagnosis and compared the results with those from reactive NK-cells in the nasal/nasopharyngeal region and peripheral blood., Results: Our study revealed seven flow cytometry (FCM)-based characteristics for distinguishing between the neoplastic cells and reactive NK-cells, including (1) the proportion of NK-cells among total lymphocytes >10%; (2) forward scatter >10 5 ; (3) mean fluorescence intensity of CD56 > 5,000; (4) aberrant antigen expression or loss; (5) skewed killer cell immunoglobulin-like receptor repertoire; (6) homogenously positive for CD38; and (7) positive for CD30 or CD336. FCM-based immunophenotyping is a potentially feasible and convenient approach for discriminating cellular lineages, evaluating the activation status of NK-cells, and selecting potential therapy targets of ENKTL-N., Conclusions: Flow cytometry is very valuable for facilitating routine diagnosis, confirming clonality, predicting the cellular lineage, and guiding individual treatment for ENKTL-N. © 2019 International Clinical Cytometry Society., (© 2019 International Clinical Cytometry Society.)

B-cell progenitors (hematogones) are markedly decreased or completely absent in the bone marrows of myelodysplastic syndromes (MDS), and the finding is considered as an important feature in MDS flow cytometry immunophenotyping. We studied CD34+ hematogones as a proportion of total CD34+ cells in 160 treatment naïve low grade primary MDS patients, consecutively collected over a two-year period. While confirming that the median CD34 + hematogones was significantly decreased (1.08%, 0%, to 67.86%), we observed variably preserved CD34 + hematogones in some MDS patients. Using a 5% cutoff, a total of 46 (29%) MDS patients had ≥5% CD34 + hematogones, significantly overrepresented by MDS with ring sideroblasts (RS) (MDS-RS) (18/40, 45%, vs. 28/120, 23%, P = 0.015). While we did not observe unique features among MDS-RS, mutations were noticeably absent in a significant number of MDS without RS (37% vs. 14%, P = 0.013), including TP53 mutations (0% vs.16.5%, P = 0.021) if ≥5% CD34 + hematogones were present. Although the follow up was short (18.5 months, 0 to 151.0), a better overall survival was observed in MDS with ≥5% CD34 + hematogones, either as a group (P = 0.008) or among patients with no RS (P = 0.028). In multivariate analysis, reduced hematogones remained to be significant hazard (P = 0.015). In summary, preserved CD34 + hematogones (≥5%) are seen in over a quarter of primary low-grade MDS and these cases are overrepresented by MDS with RS or MDS with no detectable mutations. The finding is new, and this phenomenon may in part attribute to preservation of B-cell differentiation of CD34+ progenitors, and it is associated with a better prognosis in low grade MDS patients. © 2019 International Clinical Cytometry Society., (© 2019 International Clinical Cytometry Society.)

Background: In patients with acute myeloid leukemia (AML), CD56 expression has been associated with adverse clinical outcome. We reported on a phenotype associated with very poor prognosis (RAM) in children enrolled in the Children's Oncology Group trial AAML0531 (Brodersen et al. Leukemia 30 (2016) 2077-2080). RAM is also characterized in part by high-intensity expression of the CD56 antigen. Herein, we investigate underlying biological and clinical differences among CD56-positive AMLs for patients in AAML0531., Methods: For 769 newly diagnosed pediatric patients with de novo AML enrolled in AAML0531, bone marrow specimens were submitted for flow cytometric analysis. For each patient, an immunophenotypic expression profile (IEP) was defined by mean fluorescent intensities of assayed surface antigens. Unsupervised hierarchical clustering analysis (HCA) was completed to group patients with similar immunophenotypes. Clusters were then evaluated for CD56 expression. Principal component analysis (PCA) was subsequently applied to determine whether CD56-positive patient groups were nonoverlapping., Results: HCA of IEPs revealed three unique phenotypic clusters of patients with CD56-positive AML, and PCA showed that these three cohorts are distinct. Cohort 1 (N = 77) showed a prevalence of t(8;21) patients (72%), Cohort 2 (N = 52) a prevalence of 11q23 patients (69%), and Cohort 3 (RAM) (N = 16) a prevalence of patients with co-occurrence of the CBFA2T3-GLIS2 fusion transcript (63%). The 5-year event-free survival (EFS) for Cohorts 1, 2, and 3 were 69, 39, and 19%, respectively., Conclusions: When leukemia is considered by its multidimensional immunophenotype and not by the expression of a single antigen, correlations are seen between genotype and there are significant differences in patient outcomes. © 2019 International Clinical Cytometry Society., (© 2019 International Clinical Cytometry Society.)

Background: Despite bone marrow (BM) immunophenotyping by flow cytometry has progressively been recognized as an important tool for the diagnosis of myelodysplastic syndromes (MDS), the sparse knowledge about normal erythroid maturation and the lack of markers for erythroid characterization is a major shortcoming., Methods: Here, we analyzed the expression of CD43 and CD49d, two markers included in the diagnostic panel for B-cell chronic lymphoproliferative disorders (B-CLPD), in the CD34 + compartment of normal BM and along the normal and dysplastic erythroid maturation. For this, 13 normal BM aspirates and 18 BM aspirates from MDS patients were studied by flow cytometry., Results: Normal BM presented a higher expression of CD43 and CD49d among CD34 + erythroid precursors, compared to CD34 + cells committed to the remaining hematopoietic cell lineages. CD43 expression progressively decreased along the normal erythroid maturation, whereas CD49d levels increased from Stage I to Stage II, were maintained in Stages II and III, and then decreased until the last stage of maturation. In MDS, the expression of CD43 and CD49d followed a similar pattern, but with decreased expression levels for both markers, observed in all erythroid maturation stages (P < 0.05)., Conclusions: Our results point to the usefulness of CD43 and CD49d, two markers commonly present in B-CLPD diagnosis panels, in the identification of dysplastic phenotypic features in the erythroid lineage. This allows a feasible and inexpensive way to identify patients who would benefit from a more extensive study to evaluate the presence of MDS, during the processing of suspected B-CLPD samples. © 2019 International Clinical Cytometry Society., (© 2019 International Clinical Cytometry Society.)

Mixed phenotype acute leukemias (MPALs) represent a rare subgroup of acute leukemias with a poor prognosis. Proper diagnosis and classification of MPAL is extremely important for patients' outcome. Morphology and flow cytometry recognize two types of MPAL: the "bilineal" MPAL with the coexistence of two blast populations of different lineage and truly "biphenotypic" MPAL coexpressing markers of more than one lineage in a homogenous blast population, respectively. The WHO 2008 classification further delineated three categories: associated with t(9;22)/BCR-ABL1 fusion gene, associated with KMT2A (mixed lineage leukemia) rearrangements, and nonotherwise specified. These categories remained unchanged in the WHO2016 update. Molecular studies have further underlined the heterogeneity of MPAL. In this review, rules for the correct assignment of acute leukemia to the MPAL category are discussed, including both flow cytometry and immunohistochemistry on bone marrow or other tissues biopsies. Comparison of the immunophenotypic classification proposals is provided outlining the explorations mandatory for definitive diagnosis. An extensive review of published data summarizes the reported cytogenetic and molecular anomalies. New developments in the understanding of the early stages of hematopoiesis provide clues to the possible etiopathology of these diseases. Finally, current treatment recommendations are summarized and referenced for clinical use, pointing out that allogeneic hematopoietic stem cell transplantation at an early stage should be considered (at least in adult patients). © 2019 International Clinical Cytometry Society., (© 2019 International Clinical Cytometry Society.)

Background: Clinical diagnostic laboratories are subject to numerous regulations imposed by government agencies. Laboratory developed tests for flow cytometry panels are essentially restricted to the use of analyte-specific reagents (ASR) antibodies. With the advances in clinical flow cytometry systems, there is a trend toward the utilization of blue/red/violet laser flow systems and 8 to 10-color panels. Currently, the selection of commercially available ASR antibodies for the violet laser is very limited. The market is dominated by Brilliant Violet 421 (BV421) manufactured by BD Biosciences and Pacific Blue (PB) manufactured by Beckman Coulter. In this study, we compare BV421 and PB conjugated ASR antibodies., Methods: Whole blood was stained and acquired on a Gallios flow cytometer system. For single color staining, the stain index (SI) was calculated. For the two panels, the compensation matrix was calculated and the performance of the antibody cocktails analyzed in FCS Express., Results: The results show that five out of six tested BV421 conjugated antibodies have significantly higher SI than their PB counterparts. Furthermore, BV421 antibodies require less compensation for spillover than PB. Finally, BV421 conjugated antibodies give better separation between negative and positive populations in the context of an 8 and 10 color panel without affecting the intensity of the other dyes., Conclusions: Overall, using BV421 conjugated antibodies results in better separation between populations compared to PB conjugated antibodies without negatively affecting other fluorochromes in our panels. We conclude that the BV421 conjugated ASR antibodies are currently the better available option for clinical flow panels. © 2017 International Clinical Cytometry Society., (© 2017 International Clinical Cytometry Society.)

Background: Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare acquired hematopoietic stem cell disorder characterized by an inability to make Glyco-Phosphatidyl-Inositol (GPI)-linked cell surface structures. Fluorescent proaerolysin (FLAER-Alexa488) is increasingly used to detect GPI-deficient WBCs by flow cytometry. However, FLAER is not available in all countries and is expensive to obtain in others. An earlier study to compare FLAER-based and non-FLAER assays confirmed very good agreement between the two tubes suggesting a cost effective simultaneous evaluation of PNH neutrophils and monocytes is possible without FLAER., Methods: We have used a single tube approach with a 7-color assay comprising FLAER-CD157-CD15-CD64-CD24-CD14-CD45. Conjugates were carefully selected and validated so that stained samples could be analyzed on either 10-color Navios or 8-color FACSCanto II platforms. The 6-color (minus CD14) and 5-color (minus CD24 and CD14) versions were also developed and compared with our predicate clinical lab 5-color assay comprising FLAER-CD157PE-CD64ECD-CD15PC5-CD45PC7., Results/conclusions: CD15-gated PNH neutrophil clone size was quantified using either FLAER and CD157, FLAER and CD24, or CD157 and CD24. CD64-gated PNH monocyte clone size was quantified using either FLAER and CD157, FLAER and CD14, or CD157 and CD14. Analysis of >40 PNH samples showed that the FLAER-based plots derive virtually identical data to the non-FLAER plot for neutrophils (R 2  = 1) and monocytes (R 2  = 0.9999) and that closely similar data can be acquired using both Canto II and Navios platforms with 7-, 6-, and 5-color versions of the assay. Assessment of non-PNH samples confirmed extremely low background rate of PNH phenotypes (neutrophils and monocytes) with all three approaches. © 2018 International Clinical Cytometry Society., (© 2018 International Clinical Cytometry Society.)

Background: The data on the clinical utility of the quantitative assessment of immunophenotypes in distinguishing mature CD5-positive B-cell neoplasms is limited. The study aim was to assess the diagnostic value of the quantitative assessment of a panel of 18 markers and to identify the most informative ones., Methods: The immunophenotype of the neoplastic population was determined in diagnostic specimens from 188 patients. BD FACSCanto II flow cytometer and FACSDiva software were used to analyze the positivity/negativity and mean fluorescence intensity (MFI) of the surface expression of 18 markers. Advanced data mining methods were used to define the key differential diagnostic features of CLL/SLL (chronic lymphocytic leukemia/small lymphocytic lymphoma), MCL (mantle cell lymphoma), and CD5+ MZL (marginal zone lymphoma)., Results: The most informative markers for the distinction of CLL/SLL, MCL, CD5+ MZL, including atypical cases, were the MFI values of CD79b, CD20, CD23, CD43, CD38, CD11c, FMC7, CD200, kappa light chain, and their combinations. CD23 and CD200 were the most discriminant between CLL/SLL and MCL and CD23 plus CD79b between CLL/SLL and CD5+ MZL. The quantitative analysis of the most informative markers failed to accurately distinguish MCL and CD5+ MZL. The study highlights the data mining methods for the analysis and selection of the most informative immunophenotypic markers and for the design of a predictive model (diagnostic classifier), minimizing the subjectivity of expert-based assessment., Conclusions: Our data confirmed that the quantification of the expression of informative markers increases the diagnostic value of immunophenotyping in mature CD5+ B-cell neoplasms. © 2017 International Clinical Cytometry Society., (© 2017 International Clinical Cytometry Society.)

Background: Measurement of lymphocyte subpopulations is a crucial parameter in the diagnosis and monitoring of therapy in a wide variety of clinical conditions. We compared different flow cytometry-based methods to determine lymphocyte subsets counts of routine samples by volumetric AQUIOS CL (Beckman Coulter) and bead-based FACS CANTO II (BD Biosciences) cytometers. We evaluated the possible decrease of the labor intensive technical work using the fully automated AQUIOS CL system in the pre and post analytical steps in comparison with our routine flow cytometer FACS CANTO II, toward the reduction of laboratory analytical turnaround time (TAT)., Methods: The analytical performance of AQUIOS CL flow cytometer compared with FACS CANTO II was evaluated testing 224 routine samples, attending the Immunology and Allergology Laboratory Unit, S. Giovanni di Dio Hospital, (Florence, Italy) between September and October 2015., Results: Bland-Altman plot of the differences between the two methods showed an excellent agreement for absolute and percentage counts. Furthermore, the study showed that automated AQUIOS CL system is simple to be used during all analytical steps with a reduction of TAT., Conclusion: In routine conditions, AQUIOS CL flow cytometer could be a suitable tool for subpopulations subset analysis. © 2017 International Clinical Cytometry Society., (© 2017 International Clinical Cytometry Society.)

Background: In many studies it would be advantageous if blood samples could be collected and analyzed using flow cytometry at a later stage. Ideally, sample collection should involve little hands-on time, allow for long-term storage, and minimally influence the samples., Methods: Here we establish a flow cytometry antibody panel that can be used to determine granulocytes, monocytes, and lymphocyte subset concentrations in fresh and frozen whole blood using TruCount technology., Results: The panel can be used on fresh whole-blood samples as well as whole-blood samples that have been frozen after mixing with 10% DMSO. Concentrations in frozen and fresh sample is highly correlated both when frozen within 4 h and the day after collection (r ≥ 0.98), and the estimated concentration in frozen samples was between 91 and 94% of that in fresh samples for all cell types., Conclusion: Using this method whole-blood samples can be frozen using a simple preparation method, and stored long-term before accurate determination of cell concentration. This allows for standardized analysis of the samples at a reference laboratory in multi-center studies. © 2016 International Clinical Cytometry Society., (© 2016 International Clinical Cytometry Society.)

Background: Multiparametric flow cytometry is a useful tool for diagnosis of plasma cell (PC) dyscrasias and assessment of minimal residual disease in plasma cell myeloma (PCM). However, the immunophenotypic differences between the clonal PCs of PCM and those of monoclonal gammopathy of undetermined significance (MGUS) as well as the correlation of these flow cytometric markers with pertinent laboratory parameters have not been evaluated., Methods: We retrospectively identified all newly diagnosed treatment-naive PCM and MGUS patients between 09/2014 and 06/2015 who underwent 10-color flow-cytometric evaluation: CD45, CD38, CD138, cKappa, cLambda, CD19, CD27, CD28, CD56, CD117. FACSDiva analysis was used to identify antigenic aberrancies and associations with pertinent laboratory parameters were evaluated., Results: All cases demonstrated at least two aberrancies. There was a trend toward a greater number of aberrancies in PCM, with 68% showing >/= 4 aberrancies compared with 44% in MGUS (P = 0.11). The only marker more frequently aberrant in one disease class was CD19, aberrant in 68% of PCM and 25% of MGUS (P < 0.01). In PCM, significant associations were found for CD56 non-aberrancy (P = 0.05) and the presence of amyloid and CD27 aberrancy and normal serum albumin (P = 0.05). In MGUS, CD117 expression was associated with normal hemoglobin (P = 0.03)., Conclusions: The PCs of PCM show a trend toward more antigenic aberrancy than those of MGUS. There is significant association between the antigenic profiles of PCM/MGUS and clinical parameters including amyloidosis, albumin level, and hemoglobin. © 2018 International Clinical Cytometry Society., (© 2018 International Clinical Cytometry Society.)

Background: Current flow-cytometric plasma cell (PC) gating is based on CD138, CD38, and CD45 expression. CD138 is known for variable expression and loss during storage and processing. Introduction of anti-CD38 and anti-CD138 monoclonal-antibody therapies has limited the use of these markers during follow-up. Hence, additional reliable PC-gating markers are required. Recently, CD229 has been claimed as an alternative PC-gating marker. However, these studies are limited to a small cohort of samples. We evaluated the utility of CD229 as a new PC-gating marker in routine laboratory practice., Methods: Expression of CD229 was studied in 310 bone marrow (BM) samples (251 plasma-cell disorders and 59 controls) and compared with CD138 and CD38 expression. We also evaluated the effect of additional processing for cytoplasmic immunoglobulin-light-chains (CyIgL) staining on the quantitation of PC., Results: Expression of CD229 was consistently stronger on PC than other hematopoietic-cells (p < 0.001). PC-percentages using CD229 in combination with CD38 or CD138 and CD45 revealed high correlation with a reference gating-strategy using CD138, CD38 and CD45 (r = 0.98, r = 0.99 r = 0.99 respectively) and r = 0.92 using CD229 and CD45 without CD38 or CD138. In contrast, CD138 expression showed significant variability (CV-MFI, 97.5) and loss from PC in 53% of samples. Quantitation of PC was found to be lower in 69.3% and higher in 30.7% samples processed for CyIgL-staining as compared to surface-staining., Conclusions: CD229 is a reliable new alternative PC-gating marker in routine laboratory practice. Quantitation of PC based on CD138 expression or from samples processed for CyIgL-staining should be used with caution. © 2018 International Clinical Cytometry Society., (© 2018 International Clinical Cytometry Society.)

Background: Flow cytometric intracellular myeloperoxidase (MPO) staining of leukemic blasts is a useful tool in diagnosis of leukemia subtype. Interpretation of high MPO-positivity can be a diagnostic challenge in B-lineage acute lymphoblastic leukemia (B-ALL). While very few such cases have been reported, high MPO positive B-ALL cases without additional myeloid antigen positivity are suspect and require further investigation., Methods: Three pediatric cases of B-ALL with strong MPO staining (clone 8E6; Invitrogen) at diagnosis and three others with negative MPO staining were studied by flow cytometry and immunohistochemistry. In-vitro drug cytotoxicity, oxidative stress generation, and immunophenotyping using other MPO clones were performed to further investigate MPO presence., Results: Expectedly, normal myeloid cells in all six samples were positive and mature lymphocytes negative for MPO staining. However, MPO monoclonal antibody (mAb) obtained from clones other than Invitrogen and other myeloid-specific mAbs gave negative results suggesting false positivity of the initial MPO staining. Immunohistochemistry for MPO was also negative on all six cases tested. Furthermore, in-vitro vincristine cytotoxicity was greater in leukemic cells from MPO false-positive cases compared with MPO-negative B-ALL samples, demonstrating indirect lack of MPO activity. Moreover, drug treatment did not lead to generation of reactive oxidative species, also reflective of lack of significant MPO presence., Conclusions: The cause of false-positive MPO staining remains unknown in these three cases; a cross reactivity could be the culprit. Caution should be given to similar phenomena and detailed investigation may contribute to the understanding of altered protein expression in such outlier cases. © 2017 International Clinical Cytometry Society., (© 2017 International Clinical Cytometry Society.)

Background: Primary mediastinal large B cell lymphoma (PMLBCL) is a B cell non-Hodgkin lymphoma (B-NHL) that shows morphologic, immunophenotypic, and genetic similarities to classical Hodgkin lymphoma (CHL). We evaluated the neoplastic and reactive infiltrate of PMLBCL by flow cytometry (FC)., Methods: A total of 24 cases of PMLBCL were retrospectively characterized using FC combinations for B-NHL, T-NHL, and CHL., Results: The CHL assay identified the neoplastic population (NP) in all cases, while the B-NHL assay identified the NP in 18 of 24 cases. In four cases, the neoplastic population was retrospectively identified in the B-NHL tube, given the CHL tube-derived immunophenotype. Neoplastic cells of PMLBCL displayed a B cell immunophenotype (CD19/CD20 + ), with CD40 consistently and brightly expressed. Most cases lacked immunoglobulin light chains, CD10, and CD15; CD30 was frequently expressed. All cases showed expression of CD71 and CD95. The reactive infiltrate in PMLBCL showed increases in T cells relative to reactive tissues. Lower CD4/CD8 ratios in PMLBCL relative to CHL and reactive tissues were also observed. Although not seen as commonly as with CHL, 41% of cases showed a reactive CD3/CD4/CD7 bright /CD45 bright+ T cell population. Reactive populations seen in PMLBCL were similar to that seen in diffuse large B cell lymphoma, not otherwise specified., Conclusions: FC can detect the NP of PMLBCL and a FC assay for CHL performed better than a B-NHL assay in this regard. The neoplastic cells showed a B cell immunophenotype with over-expression of CD40. The reactive infiltrate in PMLBCL shows unique features including a prominent CD8 + T cell infiltrate. © 2017 International Clinical Cytometry Society., (© 2017 International Clinical Cytometry Society.)

Background: Flow cytometry (FC) is a reliable tool for diagnosing and monitoring of plasma cell myeloma (PCM). Recent studies used FC for quantifying plasma cells (PCs) in peripheral blood (PB) using various panels, and an adverse prognostic effect of circulating PCs (cPCs) has been reported. We investigated the prognostic implication of cPCs quantified using a simple panel in patients with PCM., Methods: Bone marrow (BM) and PB of 85 patients with PCM were analyzed by five-color FC at time of diagnosis. A serial gating strategy for quantification used CD38/CD138 to gate PCs in 100,000-200,000 acquired events, with subsequent gating for CD19, CD56, and CD45, to identify aberrant immunophenotypes., Results: cPCs were observed in 74.1% patients (63/85, median 0.067% leukocytes). Patients were grouped based on a cPC cut-off level of 0.02% derived using the receiver operating characteristic curves. Compared with patients with cPCs < 0.02% (n = 28), those with cPCs ≥ 0.02% (n = 57) showed lower hemoglobin (P = 0.003) and platelets (P = 0.014), but higher calcium, M-protein and BM PCs (P = 0.013, 0.029, and P < 0.001, respectively). Survival analysis of 74 patients showed that cPCs ≥ 0.02% predicted shorter progression-free and overall survival (P = 0.001 and 0.013, respectively), and this negative prognostic impact was retained in multivariate analysis (P = 0.023)., Conclusions: Flow cytometric quantification of cPCs using five surface antigens (CD138, CD38, CD56, CD19, and CD45) is a sensitive and simple method that can be used for assessing PCM prognosis; it would allow clinical laboratories to readily adopt a risk stratification strategy based on cPC levels in PCM patients. © 2017 International Clinical Cytometry Society., (© 2017 International Clinical Cytometry Society.)

Background: Accuracy of bone marrow (BM) blast count in low-risk myelodysplastic syndromes (MDS) still remains a challenge though it is essential for prognosis. We investigated whether the enumeration of CD34+ myeloid cells by flow cytometry immunophenotyping (FCI) could be used as a consistent parameter for clinical MDS studies., Methods: Six clinical centers entered the study and information on their FCI protocols was recorded. Sixty-seven flow cytometry listmodes from BM samples of patients with low-risk MDS with <5% BM blasts were exchanged among participants in two different rounds. Interlaboratory variations on the quantification of CD34+ myeloid cells were calculated and strategies to solve differences were evaluated., Results: An overall "very good" agreement on CD34+ cell count among participants (intraclass correlation coefficient = 0.720) was observed, but agreement was "low" in 22 files. No single parameter could fully explain all discrepancies, but 3 technical issues were identified as relevant: the use of the CD34/CD45/CD117/HLA-DR mAb combination, acquisition of ≥50,000 events and a low percentage of debris/aggregates. The frequency of discordant results increased with the accumulation of pitfalls (none, 16%; 1 pitfall, 40%; 2 pitfalls, 83%; P = 0.006). Finally, the use of a common gating strategy for analysis increased the percentage of files with "very good" agreement to 100%., Conclusions: Prevention of specific technical pitfalls is mandatory to reach a good reproducibility of CD34+ cell count among centers. These recommendations set the basis for laboratory standardization and enable the use of CD34+ cell enumeration as additional information in low-risk MDS patients. © 2017 International Clinical Cytometry Society., (© 2017 International Clinical Cytometry Society.)

Helper innate lymphoid cells (ILCs), the most recently identified population of the ILC family, play a fundamental role in the restoration of tissue integrity, in the protection against infiltrating pathogens as well as in tumor immune-surveillance. ILCs have been divided into three main subsets, ILC1, ILC2, and ILC3, that can be specifically activated by different signals coming either indirectly from pathogens or from other cell populations, including cancer cells. Following activation, ILCs are in turn able to promptly secrete a wide range of soluble mediators that modulate effector cell functions. The discovery and the study of these immune cells is now offering important opportunities for innovative therapies of allergic airway diseases, inflammatory disorders and might be crucial for the discovery of new targets for the therapy of cancer. It is therefore fundamental that the scientific community establishes harmonized guidelines to obtain a consensus in the identification and phenotypical and functional characterization of ILCs. © 2018 International Clinical Cytometry Society., (© 2018 International Clinical Cytometry Society.)

We performed a retrospective analysis of 88 adult patients with B-ALL diagnosed in our center by a flow-cytometric assessment. Immunophenotypic expression of leukemic cells was explored by simultaneous evaluation of positivity, percentage of expressing cells and median fluorescence intensity (MFI). BCR/ABL1 fusion transcripts were assessed by RT-PCR analysis and were identified in 36 patients (40.9%). CD10 and CD34 were positive in the totality of BCR/ABL1-positive cases. Patients with gene rearrangement had a greater frequency of CD66c, CD13 and CD33 positivity compared with BCR/ABL1-negative cases. Moreover, BCR/ABL1-positive cases exhibited a greater median percentage and MFI values of CD13, CD33, CD66c, CD10, CD34 and CD25 expressions, but a lower median percentage and MFI values of CD38 and CD22 expressions than patients without gene rearrangement. Multivariate logistic regression analysis showed that CD10, CD38 and CD13 expressions were independent predictors for the presence of BCR/ABL1 rearrangement. Predictive probabilities of molecular occurrence based on these markers are proposed. © 2017 International Clinical Cytometry Society., (© 2017 International Clinical Cytometry Society.)

Background: CD19-targeted chimeric-antigen receptor-modified T-cells (CAR-T) are promising in the treatment of refractory B-lymphoblastic leukemia (B-ALL). Minimal residual disease (MRD) detection by multicolor flow cytometry (FCM) is critical to distinguish B-ALL MRD from regenerating, non-neoplastic B-cell populations., Methods: FCM was performed on samples from 9 patients with B-ALL treated with CAR-T., Results: All 9 patients showed response to CAR-T. Additionally, FCM revealed circulating CD10 + B cells, potentially mimicking MRD. Circulating CD10+ B-cells were detected in blood from 3 days to 3 months after CAR-T, comprising 73% (median) of B-cells (52-83%, 95%CI). They expressed CD19, CD10, CD20, bright CD9, CD22, CD24, moderate CD38 and dim CD58, but were CD34 (-), with bright CD45 and polyclonal surface light chain immunoglobulin (sIg) expression. A similar CD10 + B-cell subpopulation was detected by marrow FCM, amidst abundant B-cell precursors., Conclusions: These circulating CD10 + B-cells are compatible with immature B-cells, and are a reflection of B-cell recovery within the marrow. They are immunophenotypically distinguishable from residual B-ALL. Expression of light chain sIg and key surface antigens characterizing regenerating B-cell precursors can distinguish immature B-cells from B-ALL MRD and prevent misdiagnosis. © 2017 International Clinical Cytometry Society., (© 2017 International Clinical Cytometry Society.)

Background: B cell lymphomas' (BCL) current diagnosis is usually based on a combination of morphology, immunophenotype, recurrent cytogenetic aberration and clinical features. However, even with these diagnostic tools, a definitive diagnosis can be difficult to achieve. Therefore, the aim of this study was to assess the profile of CD39, CD43, CD81, and CD95 expressions in diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), and Burkitt lymphoma (BL) cases., Methods: To address this issue, we investigated the expression of CD39, CD43, CD81, and CD95 by eight-color flow cytometry in retrospective cases from 2014 to 2016., Results: The study included 27 adult patients diagnosed with DLBCL, FL, and BL during the study period. Four patients were diagnosed with germinal center B cell-like DLBCL (GCB DLBCL), seven with non-GCB DLBCL, nine with FL, and seven with BL. CD39 seems to be especially relevant to differentiate non-GCB DLBCL from BL and from FL. BL showed stronger expression of CD43 when compared to FL and GCB DLBCL. Moreover, CD43 may help to distinguish non-GCB DLBCL from GCB DLBCL. CD81 expression was much stronger in BL when compared to the other three groups of patients. Lastly, CD95 may also help to distinguish BL from the other subtypes, as BL cells expressed this antigen at low levels., Conclusions: In combination, CD39, CD43, CD81, and CD95 expressions appear to be helpful to distinguish CD10 + BCL, particularly BL. Phenotypic distinction between FL and GCB DLBCL remains challenging and requires further studies. © 2017 International Clinical Cytometry Society., (© 2017 International Clinical Cytometry Society.)

Flow cytometry has been traditionally used to diagnose leukaemia and lymphoma in peripheral blood, bone marrow, body fluids, and tissue samples. The diagnosis of a malignant epithelial tumour is usually made by correlation between histopathologic appearance and the use of immunohistochemical staining. A CE approved BerEP4 antibody (anti-EpCAM, CD326) is available for flow cytometric testing, but has been evaluated predominantly in body fluids in the current literature. In this study, we have evaluated the performance of this antibody in detecting the presence of epithelial cells in tissue samples which have traditionally been reported as CD45 negative cells by flow cytometry. Among the 42 cases studied, 21 (50%) were found to be positive for CD326, thereby suggesting epithelial differentiation. The results had good concordance rates (97.6%) with final histopathological diagnosis. The results clearly show that flow cytometric testing for BerEP4 (CD326) can be a useful method for diagnosing nonhaematological malignancies that are poorly differentiated. As this is a rapid method for identifying epithelial differentiation, it can help the histopathologists tailor and rationalise the immunohistochemical panel, with the potential benefits of improving reporting times and work-flow in the laboratory. © 2017 International Clinical Cytometry Society., (© 2017 International Clinical Cytometry Society.)