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Flow-cytometric immune phenotyping is influenced by cryopreservation and inter-laboratory variability limiting comparability in multicenter studies. We assessed a system of optimized, pre-mixed dry-antibody panel tubes requiring small amounts of whole blood for validity, reliability and challenges in a Canadian multicenter study (POSITIVE) with long-distance sample shipping, using standardized protocols. Thirty-seven children awaiting solid-organ transplant were enrolled for parallel immune-phenotyping with both validated, optimized in-house panels and the dry-antibody system. Samples were collected before, 3 and 12 months post-transplant. Quality-assurance measures and congruence of phenotypes were compared using Bland-Altman comparisons, linear regression and group comparisons. Samples showed excellent lymphocyte viability (mean 94.8 %) and recovery when processed within 30 h. Comparing staining methods, significant correlations (Spearman correlation coefficient >0.6, p < 0.05), mean difference <5 % and variation 2SD <25 % were found for natural-killer, T and B cells, including many immunologically important cell subsets (CD8+, naïve, memory CD4+ T; switched-memory, transitional B). Some subgroups (plasmablasts, CD1d+CD5hi B cells) showed weak correlations, limiting interpretation reliability. The dry-antibody system provides a reliable method for standardized analysis of many immune phenotypes after long-distance shipping when processed within 30 h, rendering the system attractive for pediatric studies due to small blood amounts required and highly standardized processing and analysis., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Seema Mital reports a relationship with Bristol Myers Squibb that includes: consulting or advisory. Seema Mital reports a relationship with Tenaya Therapeutics that includes: consulting or advisory. Lavinia Ionescu reports a relationship with Beckman Coulter Canada that includes: employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Innate and adaptive immune responses at mucosal surfaces play a role in protection against most infectious diseases. However, the relative importance either of mucosal versus systemic, or of cellular versus humoral immunity in protection against such infections remains unclear. We aimed to determine the relative percentages and reproducibility of detection of five major T lymphocyte phenotypes in stimulated whole mouth fluid (SWMF); to compare matched mucosal and blood phenotypes; to evaluate the consistency of phenotypes in SWMF over time; and to determine any associations with age or gender. Peripheral blood and SWMF samples were collected from 194 participants and sequential concomitant samples were collected from 27 of those and from 12 subjects living with HIV. CD3, CD4, CD8, Th1 and Th2 T lymphocyte phenotypes were determined by FACS. All the five T lymphocyte phenotypes were detected consistently by FACS in PBMC and SWMF with experimental replicates (N = 10; PBMC CV: 3-30%; SWMF CV: 12-36%). In longitudinal samples detection rates were reproducible in both fluids but variations were higher in SWMF (CV: 23-79.6%) than PBMC (CV: 9.7-75%). Statistically significant correlations of the percentages of all the T lymphocyte phenotypes except CD8 was seen between the two fluids. In PBMCs a negative correlation with age was found with CD3, CD4 and CD8 phenotypes, whilst a positive correlation was found in both SWMF and PBMC with the Th2 phenotype. CD3, CD4 and CD8 phenotypes in SWMF and PBMCs from an HIV-positive cohort were not significantly correlated in contrast with the HIV-negative controls. Our study provides a robust FACS protocol for the detection of the five major T lymphocyte phenotypes in SWMF which should prove useful for research with other mucosal fluids., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Advances in single-cell level analytical techniques, especially cytometric approaches, have led to profound innovation in biomedical research, particularly in the field of clinical immunology. This has resulted in an expansion of high-dimensional data, posing great challenges for comprehensive and unbiased analysis. Conventional manual analysis is thus becoming untenable to handle these challenges. Furthermore, most newly developed computational methods lack flexibility and interoperability, hampering their accessibility and usability. Here, we adapted Seurat, an R package originally developed for single-cell RNA sequencing (scRNA-seq) analysis, for high-dimensional flow cytometric data analysis. Based on a 20-marker antibody panel and analyses of T-cell profiles in both adult blood and cord blood (CB), we showcased the robust capacity of Seurat in flow cytometric data analysis, which was further validated by Spectre, another high-dimensional cytometric data analysis package, and conventional manual analysis. Importantly, we identified a unique CD8 + T-cell population defined as CD8 + CD45RA + CD27 + CD161 + T cell that was predominantly present in CB. We characterised its IFN-γ-producing and potential cytotoxic properties using flow cytometry experiments and scRNA-seq analysis from a published dataset. Collectively, we identified a unique human CB CD8 + CD45RA + CD27 + CD161 + T-cell subset and demonstrated that Seurat, a widely used package for scRNA-seq analysis, possesses great potential to be repurposed for cytometric data analysis. This facilitates an unbiased and thorough interpretation of complicated high-dimensional data using a single analytical pipeline and opens a novel avenue for data-driven investigation in clinical immunology., (© 2024 The Authors. Immunology published by John Wiley & Sons Ltd.)

The use of flow cytometry in mice is constrained by several factors, including the limited availability of mouse-specific antibodies and the need to work with small volumes of peripheral blood. This is particularly challenging for longitudinal studies, as serial blood samples should not exceed 10% of the total blood volume in mice. To address this, we have developed two novel flow cytometry panels designed to extensively analyze immune cell populations in mice during longitudinal studies, using only 50 µL of peripheral blood per panel. Additionally, a third panel has been designed to conduct a more detailed analysis of cytotoxic and inhibitory markers at the end point. These panels have been validated on a lipopolysaccharide (LPS)-induced lung inflammation model. Two experiments were conducted to 1) validate the panels' sensitivity to immune challenges ( n =12) and 2) to assess intrinsic variability of measurements ( n =5). In both experiments, we collected 50 µL of peripheral blood for each cytometry panel from the maxillary venous sinus. All antibodies were titrated to identify the optimal concentration that maximized the signal from the positive population while minimizing the signal from the negative population. Samples were processed within 1 hour of collection using a MACSQuant Analyzer 16 cytometer. Our results demonstrate that these immunological panels are sensitive enough to detect changes in peripheral blood after LPS induction. Moreover, our findings help determine the sample size needed based on the immune population variability. In conclusion, the panels we have designed enable a comprehensive analysis of the murine immune system with a low blood volume requirement, enabling the measure of both absolute values and relative percentages effectively. This approach provides a robust platform for longitudinal studies in mice and can be used to uncover significant insights into immune responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Barco-Tejada, López-Esteban, Mulero, Pion, Correa-Rocha, Desco and Cussó.)

Measurable residual disease (MRD) surveillance in acute myeloid leukemia (AML) may identify patients destined for relapse and thus provide the option of pre-emptive therapy to improve their outcome. Whilst flow cytometric MRD (Flow-MRD) can be applied to high-risk AML/ myelodysplasia patients, its diagnostic performance for detecting impending relapse is unknown. We evaluated this in a cohort comprising 136 true positives (bone marrows preceding relapse by a median of 2.45 months) and 155 true negatives (bone marrows during sustained remission). At an optimal Flow-MRD threshold of 0.040%, clinical sensitivity and specificity for relapse was 74% and 87% respectively (51% and 98% for Flow-MRD ≥ 0.1%) by 'different-from-normal' analysis. Median relapse kinetics were 0.78 log 10 /month but significantly higher at 0.92 log 10 /month for FLT3-mutated AML. Computational (unsupervised) Flow-MRD (C-Flow-MRD) generated optimal MRD thresholds of 0.036% and 0.082% with equivalent clinical sensitivity to standard analysis. C-Flow-MRD-identified aberrancies in HLADRlow or CD34+CD38low (LSC-type) subpopulations contributed the greatest clinical accuracy (56% sensitivity, 90% specificity) and notably, by longitudinal profiling expanded rapidly within blasts in > 40% of 86 paired MRD and relapse samples. In conclusion, flow MRD surveillance can detect MRD relapse in high risk AML and its evaluation may be enhanced by computational analysis., (© 2024. The Author(s).)

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only in the brain, but also in the skull. A key step toward analyzing changes in immune cell populations in both the brain and skull bone marrow after brain damage (e.g., stroke) is to obtain sufficient numbers of high-quality immune cells for downstream analyses. Here, two optimized protocols are provided for isolating immune cells from the brain and skull bone marrow. The advantages of both protocols are reflected in their simplicity, speed, and efficacy in yielding a large quantity of viable immune cells. These cells may be suitable for a range of downstream applications, such as cell sorting, flow cytometry, and transcriptomic analysis. To demonstrate the effectiveness of the protocols, immunophenotyping experiments were performed on stroke brains and normal brain skull bone marrow using flow cytometry analysis, and the results aligned with findings from published studies.

As the dimensionality, throughput and complexity of cytometry data increases, so does the demand for user-friendly, interactive analysis tools that leverage high-performance machine learning frameworks. Here we introduce FlowAtlas: an interactive web application that enables dimensionality reduction of cytometry data without down-sampling and that is compatible with datasets stained with non-identical panels. FlowAtlas bridges the user-friendly environment of FlowJo and computational tools in Julia developed by the scientific machine learning community, eliminating the need for coding and bioinformatics expertise. New population discovery and detection of rare populations in FlowAtlas is intuitive and rapid. We demonstrate the capabilities of FlowAtlas using a human multi-tissue, multi-donor immune cell dataset, highlighting key immunological findings. FlowAtlas is available at https://github.com/gszep/FlowAtlas.jl.git., Competing Interests: JJ reports receiving consulting fees and grant support from Sanofi Genzyme and Enhanc3DGenomics, and consulting fees from Roche. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funders did not play a part in study design, data collection, decision to publish, or preparation of the manuscript. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Coppard, Szep, Georgieva, Howlett, Jarvis, Rainbow, Suchanek, Needham, Mousa, Menon, Feyertag, Mahbubani, Saeb-Parsy and Jones.)

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.)

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.)

Obesity-induced chronic low-grade inflammation, also known as metaflammation, results from alterations of the immune response in metabolic organs and contributes to the development of fatty liver diseases and type 2 diabetes. The diversity of tissue-resident leukocytes involved in these metabolic dysfunctions warrants an in-depth immunophenotyping in order to elucidate disease etiology. Here, we present a 30-color, full spectrum flow cytometry panel, designed to (i) identify the major innate and adaptive immune cell subsets in murine liver and white adipose tissues and (ii) discriminate various tissue-specific myeloid subsets known to contribute to the development of metabolic dysfunctions. This panel notably allows for distinguishing embryonically-derived liver-resident Kupffer cells from newly recruited monocyte-derived macrophages and KCs. Furthermore, several adipose tissue macrophage (ATM) subsets, including perivascular macrophages, lipid-associated macrophages, and pro-inflammatory CD11c + ATMs, can also be identified. Finally, the panel includes cell-surface markers that have been associated with metabolic activation of different macrophage and dendritic cell subsets. Altogether, our spectral flow cytometry panel allows for an extensive immunophenotyping of murine metabolic tissues, with a particular focus on metabolically-relevant myeloid cell subsets, and can easily be adjusted to include various new markers if needed., (© 2024 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.)

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.)

To achieve high-sensitivity cell measurements (<1 in 10 5  cells) by flow cytometry (FCM), the minimum number of acquired cells must be considered and conventional immunophenotyping protocols fall short of these numbers. The bulk lysis (BL) assay is a standardized erythrocyte lysing approach that allows the analysis of the millions of cells required for high-sensitivity measurable residual disease (MRD) detection. However, this approach has been associated with significant cell loss, along with potential over or underestimates of rare cells when using this method. The aim of this study was to evaluate bulk lysis protocols and compare them with minimal sample perturbation (MSP) protocols, which are reported to better preserve the native cellular state and avoid significant cell loss due to washing steps. To achieve this purpose, we first generated an MRD model by spiking fresh peripheral blood with K562 cells, stably expressing EGFP, at known percentages of EGFP positive cells to leukocytes. Samples were then prepared with BL and MSP protocols and analyzed using FCM. For all percentages of K562 cells established and evaluated, a significant decrease of this population was detected in BL samples compared with MSP samples, even at low K562 cell percentages. Significant decreases for non-necrotic cells were also observed in BL samples relative to MSP samples. In conclusion, the evaluation of the potential effects of BL protocols in obtaining the final count is of great interest, especially for over- or under-estimation of target cells, as in the case of measurable residual disease. Since conventional flow cytometry or minimal sample perturbation assays fall short in obtaining the minimum numbers required to reach high sensitivity measurements, significant efforts may be needed to improve bulk lysis solution reagents., (© 2024 International Society for Advancement of Cytometry.)

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.)

Acute myeloid leukemia (AML) is a heterogenous blood cancer with a dismal prognosis. It emanates from leukemic stem cells (LSCs) arising from the genetic transformation of hematopoietic stem cells (HSCs). LSCs hold prognostic value, but their molecular and immunophenotypic heterogeneity poses challenges: there is no single marker for identifying all LSCs across AML samples. We hypothesized that imaging flow cytometry (IFC) paired with artificial intelligence-driven image analysis could visually distinguish LSCs from HSCs based solely on morphology. Initially, a seven-color IFC panel was employed to immunophenotypically identify LSCs and HSCs in bone marrow samples from five AML patients and ten healthy donors, respectively. Next, we developed convolutional neural network (CNN) models for HSC-LSC discrimination using brightfield (BF), side scatter (SSC), and DNA images. Classification using only BF images achieved 86.96% accuracy, indicating significant morphological differences. Accuracy increased to 93.42% when combining BF with DNA images, highlighting differences in nuclear morphology, although DNA images alone were inadequate for accurate HSC-LSC discrimination. Model development using SSC images revealed minor granularity differences. Performance metrics varied substantially between AML patients, indicating considerable morphologic variations among LSCs. Overall, we demonstrate proof-of-concept results for accurate CNN-based HSC-LSC differentiation, instigating the development of a novel technique within AML monitoring.

In recent years, the significance of detecting minimal/measurable residual disease (MRD) in chronic lymphocytic leukemia (CLL) has increased due to the availability of highly effective therapeutic agents. Flow cytometry provides notable cost-effectiveness and immediacy, with an expected sensitivity level of approximately 10 -4 . The critical aspect of MRD detection via flow cytometry lies in accurately defining the region containing tumor cells. However, a subset of CLL, known as CLL with atypical immunophenotype, exhibits a distinct cell surface marker expression pattern that can make MRD detection challenging, because these markers often resemble those of normal B cells. To enhance the sensitivity of MRD detection in such atypical cases of CLL, we have capitalized on the observation that cell surface immunoglobulin (sIg) light chains tend to be expressed at a higher level in this subtype. For every four two-dimensional plots of cell surface markers, we used a plot to evaluate the expression of sIg kappa/lambda light chains and identified regions where the kappa/lambda ratio of sIg light chains deviated from a designated threshold within the putative CLL cell region. Using this method, we could detect atypical CLL cells at a level of 10 -4 . We propose this method as an effective MRD assay., (© 2024. The Author(s).)

Objectives: Flow cytometric immunophenotyping (FCI) is a fast and sensitive method for characterizing hematolymphoid neoplasms. It is not widely used in the workup of systemic mastocytosis (SM), in part because of the technical challenges and in part because the utility of FCI in assessing mast cells is not well understood. The objectives of this study were to assess the diagnostic utility of FCI in establishing a diagnosis of SM and distinguishing SM from nonneoplastic mast cells and to examine the immunophenotypic findings among SM subtypes., Methods: We performed FCI on bone marrow samples suspicious for SM using a panel consisting of CD2, CD25, CD30, CD45, CD117, and HLA-DR., Results: The cohort included 88 SM cases: 67 without an associated hematologic neoplasm (AHN) (PureSM) and 21 with an AHN (SM-AHN). We also assessed 40 normal/reactive controls. Overall, FCI was adequate for interpretation in 87 of 88 (99%) cases and detected at least 1 immunophenotypic aberrancy in 100% of SM cases. CD2, CD25, and CD30 were positive in 78%, 98%, and 90% of SM cases vs 0%, 13%, and 13% of cases with normal/reactive mast cells (P < .0001 for all). Two or 3 abnormalities were observed in 92% of SM cases but not in normal/reactive mast cells. Among SM cases, SM-AHN showed statistically significant less CD2 (38% vs 91%, P < .0001) and less co-expression of all 3 aberrant markers (CD2, CD25, and CD30 positive in 38% vs 86% of cases; P < .0001) than PureSM. Immunohistochemical analysis showed consistently weaker or focal expression of CD2, CD25, and CD30 than FCI, with CD2 and CD30 being falsely negative in 40% and 50% cases, respectively. A KIT D816V mutation was detected in 67% of PureSM cases and 76% of SM-AHN cases., Conclusions: Flow cytometric immunophenotyping is a quick, sensitive, high-yield tool for evaluating the immunophenotype of mast cells. An abnormal FCI finding should prompt careful histologic evaluation and sensitive KIT D816V mutation testing to address the possibility of SM. CD2, CD25, and CD30 are important markers for the detection of immunophenotypic aberrancy of mast cells, and their frequencies of aberrancy differ across SM subtypes., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society for Clinical Pathology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Context.—: In 2018 the College of American Pathologists Diagnostic Immunology and Flow Cytometry Committee designed and implemented a new plasma cell neoplasia flow cytometry proficiency testing program-PCNEO-to allow clinical flow cytometry laboratories to monitor and assess their performance compared with a peer group., Objective.—: To report the results from the first 4 years of the PCNEO program., Design.—: Program participants were sent 2 sets of challenges per year, each including 1 wet challenge and 2 dry challenges, with associated clinical and laboratory findings. The wet challenges were composed of myeloma cell line specimens (with or without dilution in preserved whole blood) for flow cytometric analysis. The dry (paper) challenges were composed of clinical case summaries and images of flow cytometric test results from various flow cytometry laboratories of committee members., Results.—: A total of 116 to 145 laboratories from 17 countries enrolled in the proficiency testing program. For the wet challenges, almost all participants (97%-100%; cumulative, 98.2%) correctly identified the presence of neoplastic plasma cell populations based on flow cytometric analysis of undiluted myeloma cell lines. Slightly fewer participants (89.0%-97.4%; cumulative, 95.2%) correctly identified the presence of neoplastic plasma cell populations based on flow cytometric analysis of diluted myeloma cell lines (10% or 50% dilutions into peripheral blood) intended to better represent a typical clinical sample. There was generally agreement among 80% or more of participants for positive or negative staining for CD38, CD138, CD19, CD20, and surface and cytoplasmic κ and λ light chains. Similarly, 84% to 100% of participants were able to correctly identify the presence of neoplastic plasma cell populations in paper challenges, including the presence of small, neoplastic plasma cell populations (0.01%-5.0% clonal plasma cells) and the presence of nonneoplastic plasma cell populations (correctly identified by 91%-96% of participants)., Conclusions.—: Participant performance in the new proficiency testing program was excellent overall, with the vast majority of participants able to perform flow cytometric analysis and identify neoplastic plasma cell populations and to identify small plasma cell clones or expanded populations of reactive plasma cells in dry challenge flow cytometry results. This program will allow laboratories to verify the accuracy of their testing program and test interpretations for the assessment of patients suspected of having a plasma cell neoplasm., Competing Interests: All authors are current or past members of the College of American Pathologists Diagnostic Immunology and Flow Cytometry Committee, except for Bashleben, who is an employee of the College of American Pathologists., (© 2024 College of American Pathologists.)

Introduction: Lymphocyte subset enumeration by flow cytometry is important for the therapeutic monitoring of a range of conditions. However, current bead-based methodologies do not produce metrologically traceable results. Here we compare an established bead-based methodology with a volumetric-based system traceable to an internationally recognised reference method., Method: A total of 118 samples received for lymphocyte subset analysis were tested using an established bead-based technique (BD Multitest™ 6-colour TBNK assay using Trucount™ tubes on a BD FACSLyric flow cytometer), followed by a volumetric method on the Sysmex XF-1600 flow cytometer using Exbio Kombitest 6-colour TBNK reagent. All samples were tested in accordance with the manufacturer's instructions., Results: Absolute count values from both methodologies for CD3+, CD3 + CD4+, CD3 + CD8+, CD19+ and CD3-CD16+/CD56+ lymphocyte populations were compared using linear regression (R 2 for all parameters >0.95) and Bland-Altman analysis. There was no significant bias (where p < 0.05) for absolute CD3 + CD4+ lymphocytes in the defined therapeutic range of 0-250 cells/μL (mean bias: 0.27 cells/μL). Although positive biases were seen for CD3 + CD4+ lymphocytes (over the entire range tested: 14-1798 cells/μL) and CD3-CD16+/CD56+ lymphocytes (mean bias: 10.83 cells/μL and 6.79 cells/μL, respectively). Negative biases were seen for CD3 + CD8+ and CD19+ lymphocytes (mean bias: -29.17 cells/μL and - 18.76 cells/μL, respectively)., Conclusion: A high degree of correlation was found for results from both methodologies and observed bias was within the limits of clinical acceptability for all populations. This shows that the metrologically traceable lymphocyte subset absolute counts produced by the Sysmex XF-1600 are robust within clinically required limits., (© 2023 The Authors. International Journal of Laboratory Hematology published by John Wiley & Sons Ltd.)

We report the development of an optimized 50-color spectral flow cytometry panel designed for the in-depth analysis of the immune system in human blood and tissues, with the goal of maximizing the amount of information that can be collected using currently available flow cytometry platforms. We established and tested this panel using peripheral blood mononuclear cells (PBMCs), but included CD45 to enable its future use for the analysis of human tissue samples. The panel contains lineage markers for all major immune cell subsets, and an extensive set of phenotyping markers focused on the activation and differentiation status of the T cell and dendritic cell (DC) compartment. We outline the biological insight that can be gained from the simultaneous measurement of such a large number of proteins and propose that this approach provides a unique opportunity for the comprehensive exploration of the immune status in human samples with a limited number of cells. Of note, we tested the panel to be compatible with cell sorting for further downstream applications. Furthermore, to facilitate the wide-spread implementation of such a panel across different cohorts and samples, we established a trimmed-down 45-color version which can be used with different spectral cytometry platforms. Finally, to generate this panel, we utilized not only existing panel design guidelines, but also developed new metrics to systematically identify the optimal combination of 50 fluorochromes and evaluate fluorochrome-specific resolution in the context of a 50-color unmixing matrix., (© 2024 The Author(s). Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.)

Background: The diagnosis of coeliac disease (CD) in adults is based on clinical, serological and histological criteria. The inappropriate performance of intestinal biopsies, non-specificity of mild histological lesions and initiation of a gluten-free diet (GFD) before biopsy may hamper the diagnosis. In these situations, determining the intraepithelial lymphogram of the duodenum by flow cytometry (IEL-FC) can be helpful., Objectives: To describe the clinical scenarios in which the IEL-FC is used and its impact on the diagnosis of CD., Methods: All adult patients with suspected CD at three tertiary centres for whom the duodenal histology and IEL-FC were available were identified. Catassi and Fasano's diagnostic criteria and changes to a CD diagnosis after the IEL-FCs were collected., Results: A total of 348 patients were included. The following indications for an IEL-FC formed part of the initial study for CD (38%): negative conventional work-up (32%), already on a GFD before duodenal biopsies (29%) and refractoriness to a GFD (2%). The IEL-FC facilitated a definitive diagnosis in 93% of patients with an uncertain diagnosis who had had a conventional work-up for CD or who were on a GFD before histology., Conclusions: The IEL-FC facilitates the confirmation or rejection of a diagnosis of CD in clinical scenarios in which a conventional work-up may be insufficient.

POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein [M-protein], and skin changes) is a rare systemic disorder characterized by various symptoms caused by underlying plasma cell (PC) dyscrasia. Detection of monoclonal PCs is mandatory for the diagnosis of POEMS syndrome; however, the usefulness of EuroFlow-based next-generation flow cytometry (EuroFlow-NGF) in POEMS syndrome for detecting monoclonal PCs in bone marrow (BM) and the gating strategy suitable for flow cytometry study of POEMS syndrome remain unknown. We employed EuroFlow-NGF-based single-tube eight-color multiparameter flow cytometry (MM-flow) and established a new gating strategy (POEMS-flow) to detect the monoclonal PCs in POEMS syndrome, gating CD38 broadly from dim to bright and CD45 narrowly from negative to dim compared to MM-flow. MM-flow detected monoclonal PCs in 9/25 (36.0%) cases, including 2/2 immunofixation electrophoresis (IFE)-negative cases (100%). However, POEMS-flow detected monoclonal PCs in 18/25 cases (72.0%), including 2/2 IFE-negative cases (100%). POEMS-flow detected monoclonal PCs with immunophenotypes of CD19 - in 17/18 (94.4%). In six cases where post-treatment samples were available, the size of the clones was significantly reduced after the treatment (P = 0.031). POEMS-flow can enhance the identification rate of monoclonal PCs in POEMS syndrome and become a valuable tool for the diagnosis of POEMS syndrome., (© 2024. The Author(s).)

Background: The goal was to improve the clinical cognition of Ph-positive mixed phenotype acute leukemia and avoid misdiagnosis or delayed diagnosis., Methods: The clinical manifestations and laboratory results (bone marrow cell morphology, multiparameter flow cytometry, and cytogenetics) of a case of Ph-positive mixed phenotype acute leukemia were analyzed, and related literature was reviewed., Results: Blood routine: WBC 386.35 x 109/L, HGB 117.00 g/L, PLT 31 x 109/L; 80% of the original cells can be seen by artificial classification. Morphological examination of bone marrow cells showed that the proliferation of nucleated cells was obviously active, and the original cells accounted for 76%. The size of the original cells was somewhat uniform, most of the cells had less mass, were stained light grayish blue, the cytoplasm particles were not obvious, the nuclei were mostly round or quasi-round, some of them showed distortion and nuclear notch, and the chromatin was coarse. Some of the cells were rich in mass, small azurin granules were seen, the nuclei were regular, most of them were round, the chromatin was fine, the myeloperoxidase and esterase staining were negative, the eosinophils accounted for 2.5%, and the basophils accounted for 0.5%. Flow cytometry immunotyping: Two groups of abnormal cells were seen in the bone marrow. 1. A group included 12.32% of nuclear cells and showed abnormal myeloid primitive cell phenotype. Main expression: CD117, CD34, CD38, HLA-DR, CD33, CD64, CD123, weak expression: CD13, CD19. 2. The other group included 45.61% of the nuclear cells and had a B-lymphoblastic phenotype. Main expression: CD34, CD38, HLA-DR, CD123, CD19, CD10, CD9, cCD79a, TDT, weak expression of CD13, CD22. Mixed phenotype acute leukemia (M/B) immunophenotype was considered. Chromosome: 46,XY,t(9; 22)(q34;q11.2) [20]. BCR-ABL (P210) fusion gene was positive., Conclusions: Mixed phenotype acute leukemia (MPAL) is a rare type of malignant hematologic disease. Its diagnosis is based on the comprehensive evaluation of bone marrow cell morphology, immunophenotype, molecular and cytogenetic features.

Assessing minimal residual disease (MRD) in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) is essential for adjusting therapeutic strategies and predicting relapse. Quantitative polymerase chain reaction (qPCR) is the gold standard for MRD. Alternatively, flow cytometry is a quicker and cost-effective method that typically uses leukaemia-associated immunophenotype (LAIP) or different-from-normal (DFN) approaches for MRD assessment. This study describes an optimized 12-colour flow cytometry antibody panel designed for BCP-ALL diagnosis and MRD monitoring in a single tube. This method robustly differentiated hematogones and BCP-ALL cells using two specific markers: CD43 and CD81. These and other markers (e.g. CD73, CD66c and CD49f) enhanced the specificity of BCP-ALL cell detection. This innovative approach, based on a dual DFN/LAIP strategy with a principal component analysis method, can be used for all patients and enables MRD analysis even in the absence of a diagnostic sample. The robustness of our method for MRD monitoring was confirmed by the strong correlation (r = 0.87) with the qPCR results. Moreover, it simplifies and accelerates the preanalytical process through the use of a stain/lysis/wash method within a single tube (<2 h). Our flow cytometry-based methodology improves the BCP-ALL diagnosis efficiency and MRD management, offering a complementary method with considerable benefits for clinical laboratories., (© 2024 British Society for Haematology and John Wiley & Sons Ltd.)

Background: For many years it has been postulated that the immune system controls the progress of multiple myeloma (MM). However, the phenotypes of T cells in MM remain to be elucidated. In this study, we compared the phenotypes of T cells, which were obtained from the peripheral blood, in MM patients with those in healthy donors (HD). The expression of CCR7, CD57, CD28, HLA-DR, CD38, CD45RA, and CD45RO were assessed on T cells from MM patients and HDs using multicolor flow cytometry (MFC)., Methods: For this study, 17 newly diagnosed MM patients were selected, and 20 healthy people were selected as a control group. MFC was used to detect the markers on T cells., Results: We detected significant increases in the expression levels of HLA-DR, CD38, and CD57on CD8+ T cells, significant decreases in the expression levels of CD28 and CD45RA on CD8+ T cells, and a decrease of CD4+ effec-tor T cells in MM patients, compared to the HD group., Conclusions: Our study shows that the accumulation of peripheral CD8+CD57+T cells, CD8+CD38high T cells, and CD8+HLA-DR+CD38high T cells is reflective of an ongoing antitumor T cell response and a progressive immune dysfunction in MM. During chemotherapy, the recovery of immune function can be monitored by detecting the proportion of activated molecules of T lymphocytes.

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.)

Cross reactivities are known for human leukocyte antigen inside HLA-B7 related Cross-Reactive Group (B7CREG). Some CE-IVD flow-cytometry kits use double monoclonal antibodies (mAb) to distinguish HLA-B27 and HLA-B7 but practice reveals more complexes results. This study explores the performances of this test. Analysis of 466 consecutive cases using HLA-B27 IOTest™ kit on a Navios™ cytometer from Beckman-Coulter, partially compared to their genotypes. Expected haplotypes HLA-B27-/HLA-B7- (undoubtedly HLA-B27 negative) and HLA-B27+/HLA-B7- (undoubtedly HLA-B27+) were clearly identified according to the manufacturer's instructions. On the opposite, patients strongly labeled with anti-HLA-B7 showed three different phenotypes regarding anti-HLA-B27 labeling: (1) most of the cases were poorly labeled in accordance with cross reactivity inside B7CREG (HLA-B27-/HLA-B7+ haplotype); (2) rare cases had strong B7 and B27 labeling corresponding to HLA-B27+/HLA-B7+ haplotype; (3) even less cases had strong labeling by anti-HLA-B7 but non for anti-HLA-B27, all expressing HLA-B44 and no B7CREG molecules. Surprisingly, more cases were not labeled with anti-HLA-B7 antibody but partially labeled with anti-HLA-B27 suggesting another cross reactivity out of B7CREG. mAb HLA typing suggests new, cross reactivities of anti-HLA-B27 antibody to more molecules out of B7CREG and of anti-HLA-B7 antibody but not anti-HLA-B27 to HLA-B44 molecule also out of B7CREG. HLA-B27 could surely be excluded in most samples labeled with HLA-B27, below a "grey zone" on intermediate intensity. More comparison is needed in future studies., (© 2024 International Society for Advancement of Cytometry.)

Background Aims: The successful development of CD19-targeted chimeric antigen receptor (CAR) T-cell therapies has led to an exponential increase in the number of patients recieving treatment and the advancement of novel CAR T products. Therefore, there is a strong need to develop streamlined platforms that allow rapid, cost-effective, and accurate measurement of the key characteristics of CAR T cells during manufacturing (i.e., cell number, cell size, viability, and basic phenotype)., Methods: In this study, we compared the novel benchtop cell analyzer Moxi GO II (ORFLO Technologies), which enables simultaneous evaluation of all the aforementioned parameters, with current gold standards in the field: the Multisizer Coulter Counter (cell counter) and the BD LSRFortessa (flow cytometer)., Results: Our results demonstrated that the Moxi GO II can accurately measure cell number and cell size (i.e., cell volume) while simultaneously assessing simple two-color flow cytometry parameters, such as CAR T-cell viability and CD4 or CAR expression., Conclusions: These measurements are comparable with those of gold standard instruments, demonstrating that the Moxi GO II is a promising platform for quickly monitoring CAR T-cell growth and phenotype in research-grade and clinical samples., Competing Interests: Declaration of Competing Interest MR holds patents related to CAR T cells that are licensed to Novartis, Tmunity (Kite) and viTToria biotherapeutics. MR has served as a consultant for nanoString, BMS, GSK, GLG, GuidePoint, Sana, Bayer and AbClon. MR receives research funding from AbClon, Beckman Coulter, Oxford Nano Imaging, Curiox and viTToria biotherapeutics. MR is the scientific founder of viTToria biotherapeutics. The Ruella laboratory was provided a Moxi GO II device and supplies to perform this study. GD is an employee of ORFLO Technologies. All other authors declare no competing interests., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Introduction: Exploring monocytes' roles within the tumor microenvironment is crucial for crafting targeted cancer treatments., Methods: This study unveils a novel methodology utilizing four 20-color flow cytometry panels for comprehensive peripheral immune system phenotyping, specifically targeting classical, intermediate, and non-classical monocyte subsets., Results: By applying advanced dimensionality reduction techniques like t-distributed stochastic neighbor embedding (tSNE) and FlowSom analysis, we performed an extensive profiling of monocytes, assessing 50 unique cell surface markers related to a wide range of immunological functions, including activation, differentiation, and immune checkpoint regulation., Discussion: This in-depth approach significantly refines the identification of monocyte subsets, directly supporting the development of personalized immunotherapies and enhancing diagnostic precision. Our pioneering panel for monocyte phenotyping marks a substantial leap in understanding monocyte biology, with profound implications for the accuracy of disease diagnostics and the success of checkpoint-inhibitor therapies. Key findings include revealing distinct marker expression patterns linked to tumor progression and providing new avenues for targeted therapeutic interventions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Xiao, Geng, Wang, Zeng, Lai, Gong and Chen.)

Introduction: B cells play a pivotal role in adaptive immunity which has been extensively characterised primarily via flow cytometry-based gating strategies. This study addresses the discrepancies between flow cytometry-defined B cell subsets and their high-confidence molecular signatures using single-cell multi-omics approaches., Methods: By analysing multi-omics single-cell data from healthy individuals and patients across diseases, we characterised the level and nature of cellular contamination within standard flow cytometric-based gating, resolved some of the ambiguities in the literature surrounding unconventional B cell subsets, and demonstrated the variable effects of flow cytometric-based gating cellular heterogeneity across diseases., Results: We showed that flow cytometric-defined B cell populations are heterogenous, and the composition varies significantly between disease states thus affecting the implications of functional studies performed on these populations. Importantly, this paper draws caution on findings about B cell selection and function of flow cytometric-sorted populations, and their roles in disease. As a solution, we developed a simple tool to identify additional markers that can be used to increase the purity of flow-cytometric gated immune cell populations based on multi-omics data ( AlliGateR ). Here, we demonstrate that additional non-linear CD20, CD21 and CD24 gating can increase the purity of both naïve and memory populations., Discussion: These findings underscore the need to reconsider B cell subset definitions within the literature and propose leveraging single-cell multi-omics data for refined characterisation. We show that single-cell multi-omics technologies represent a powerful tool to bridge the gap between surface marker-based annotations and the intricate molecular characteristics of B cell subsets., Competing Interests: RB-R is a co-founder of Alchemab Therapeutics Ltd and consultant for Alchemab Therapeutics Ltd, Roche, Enara Bio, UCB and GSK. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Pernes, Alsayah, Tucci and Bashford-Rogers.)

Technological advancements in fluorescence flow cytometry and an ever-expanding understanding of the complexity of the immune system, have led to the development of large flow cytometry panels, reaching up to 40 markers at the single-cell level. Full spectrum flow cytometry, that measures the full emission range of all the fluorophores present in the panel instead of only the emission peaks is now routinely used in many laboratories internationally, and the demand for this technology is rapidly increasing. With the capacity to use larger and more complex staining panels, optimized protocols are required for the best panel design, panel validation and high-dimensional data analysis outcomes. In addition, for ex vivo experiments, tissue preparation methods for single-cell analysis should also be optimized to ensure that samples are of the highest quality and are truly representative of tissues in situ. Here we provide optimized step-by-step protocols for full spectrum flow cytometry panel design, tissue digestion and panel optimization to facilitate the analysis of challenging tissue types., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Multiparameter flow cytometry is a common tool for assessing responses of T, B, and other cells to pathogens or vaccines. Such responses are likely to be important for predicting the efficacy of an HIV vaccine, despite the elusive findings in HIV vaccine trials to date. Fortunately, flow cytometry has evolved to be capable of readily measuring 30-40 parameters, providing the ability to dissect detailed phenotypes and functions that may be correlated with disease protection. Nevertheless, technical hurdles remain, and standardization of assays is still largely lacking. Here an optimized protocol for antigen-specific T cell monitoring is presented, with specific variations for particular markers. It covers the analysis of multiple cytokines, cell surface proteins, and other functional markers such as CD107, CD154, CD137, etc. References are given to published panels of 8-28 colors., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The use of flow cytometry for immunophenotyping is contingent on the ability to accurately assign biological relevance to the detected signal. This process has historically been challenging when defining IgE expressing B cells or IgE expressing antibody-secreting cells due to widespread expression of receptors for IgE on various leukocyte subsets, including human B cells. Here we describe our implementation of intracellular staining for human IgE following a blocking step to negate the challenge of surface-bound IgE. We also describe our experience with a human B cell culture system that can be used to robustly validate this approach before application to primary human samples. Orthogonal confirmatory techniques remain essential; these are not described in detail, but several possible strategies are suggested., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Mass cytometry permits the high dimensional analysis of cellular systems at single-cell resolution with high throughput in various areas of biomedical research. Here, we provide a state-of-the-art protocol for the analysis of human peripheral blood mononuclear cells (PBMC) by mass cytometry. We focus on the implementation of measures promoting the harmonization of large and complex studies to aid robustness and reproducibility of immune phenotyping data., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Background: The alteration of cerebrospinal fluid (CSF) in hematologic neoplasms is a poor prognostic marker. The characteristics of CSF are usually analyzed by flow cytometry or cytology. However, paucicellular CSF samples (≤5 cells/dL) can sometimes be considered unsuitable for analysis due to the low number of events., Objective: To evaluate the proportion of samples reported as suitable for analysis obtained by cytometry (FCM) and cytology in paucicellular CSF samples., Material and Methods: 169 samples ofpaucicellular CSF corresponding to 115 patients with hematologic neoplasms were selected. The samples were obtained by lumbar puncture in tubes conditioned with EDTA and Transfix®. We characterized the immunophenotype ofCSF samples with an 8-color panel, and 55 samples (32%) were in a small sample tube (SST). In all cases, monocytes were identified by CD14 labeling and T lymphocytes by CD3 labeling. The acquisition was carried out in a FACSCantoII® cytometer, and the analysis was performed using Infinicyt® software., Results: The proportion of samples suitable for analysis was higher in FCM compared to cytology (98% vs 61%, p < 0.000). We identified the presence of T lymphocytes and/or monocytes in most samples (98% and 90%, respectively). In the SST samples, the number of events recorded in low-volume samples (< 1 mL) was lower than in samples with higher volume (140 vs 556, p < 0.001), with a median of identification of 3 cell populations., Conclusion: FCM allows the analysis of a higher proportion ofpaucicellular CSF samples than cytology in hematologic neoplasms study.

Purpose: Using a comprehensive flow cytometric panel, simultaneously obtained mid-luteal immunophenotypes from peripheral blood and endometrium were compared and values correlated. Is a peripheral blood evaluation of reproductive immunophenotype status meritorious relative to local endometrial evaluation to directly assess the peri-implantation environment?, Methods: Fifty-five patients had a mid-luteal biopsy to assess the local endometrial immunophenotype, while simultaneously providing a peripheral blood sample for analysis. Both samples were immediately assessed using a comprehensive multi-parameter panel, and lymphocyte subpopulations were described and compared., Results: Distinct lymphocyte proportions and percentage differences were noted across the two compartments, confirming the hypothesis that they are distinct environments. The ratio of CD4 + to CD8 + T cells were reversed between the two compartments, as were Th1 and Th2-type CD4 + T cell ratios. Despite these differences, some direct relationships were noted. Positive Pearson correlations were found between the levels of CD57 + expressing natural killer cells, CD3 + NK-T cells and CD4 + Th1 cells in both compartments., Conclusions: Flow cytometric evaluation provides a rapid and objective analysis of lymphocyte subpopulations. Endometrial biopsies have become the gold standard technique to assess the uterine immunophenotype in adverse reproductive outcome, but there may still a place for peripheral blood evaluation in this context. The findings demonstrate significant variations in cellular proportions across the two regions, but some positive correlations are present. Immunological assessment of these specific peripheral blood lymphocyte subtypes may provide insight into patients with potential alterations of the uterine immune environment, without the risks and inconveniences associated with an invasive procedure., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The Ki-67 antigen is a nuclear protein with proven prognostic value in different neoplasms and recognizes the predictive value in breast cancer (BC). No consensus exists on the ideal cutoff point. In this study, Ki-67 expression was evaluated in samples of BC by flow cytometry (FC) and compared with immunohistochemical (IHC) examination. For this, the BC tissue samples were sectioned, macerated, filtered, and marked with anti-Ki-67 FITC and anti-CD45 V500 antibodies. We selected the neoplastic cells according to CD45 expression and size and internal complexity (FSC × SSC) using the Infinicity 1.7 software. Lymphocytes were negative control. We compared the results with IHC analyses carried out in parallel and independently. The expression of Ki-67 was evaluated in both methodologies through Bland-Altman analysis. Among the 44 samples analyzed, only three showed bias higher than the established confidence interval (mean bias 2.1%, p = 0.62), with no significant difference for the perfect mean bias (0%). Therefore, one can state that FC provides results equivalent to IHC analysis and possibly analyzes more cells simultaneously. The results obtained in this study show the absence of observational bias through software analysis in a larger number of tumor cell populations. We can conclude that FC may be a promising alternative method for investigating Ki-67 in solid tumours., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Introduction: Stem cell enumeration by the hematopoietic progenitor cells (HPC) mode is a novel method available from Sysmex XN2000 hematology analyzer. A small amount of blood (190 μL) is required, and the results are available in a few minutes without manual gating or presample treatment. The present study compares stem cell measurements using XN2000 analyzer HPC mode and FC500 flow cytometry analyzer using peripheral blood (PB) specimens and apheresis products., Methods: In this prospective study, CD34-positive cell counts were enumerated using an FC500 flow cytometry analyzer and compared with XN2000 Sysmex analyzer (XN-HPC mode) in the same samples. Results were compared using Bland-Altman plots., Results: A total of 103 samples were used. In the PB samples, the median HPC count and CD34-positive cells were 83.5 × 10 6 /L and 78.0 × 10 6 /L, respectively. The mean Bland-Altman difference was 4.5 × 10 6 /L (Limits: -51.7 to 60.7 × 10 6 /L), with a Pearson's correlation of 0.79. In the apheresis products, the median HPC count and CD34-positive cells were 1468 × 10 6 /L (IQR: 1049 - 1960 × 10 6 /L) and 1327 × 10 6 /L (IQR: 910 - 2001 × 10 6 /L), respectively. The mean Bland-Altman difference was 179.0 × 10 6 /L (Limits: -2022.2 - 2380.2 × 10 6 /L), with a Pearson's correlation of 0.58., Conclusion: The XN-HPC mode has an excellent correlation and minimal disagreement for stem cell enumeration in PB compared with flow cytometry and could replace it. There is high disagreement in apheresis products, and therefore, the XN-HPC mode cannot be recommended., (© 2021 John Wiley & Sons Ltd.)

A comprehensive study of the cellular components of the immune system demands both deep and broad immunophenotyping of numerous cell subsets in an effective and practical way. Novel full-spectrum technology reveals the complete emission spectrum of each dye maximizing the amount of information that can be obtained on a single sample regarding conventional flow cytometry and provide an expanded knowledge of biological processes. In this chapter, we describe a 37-color protocol that allows to identify more than 45 different cell populations on whole blood samples of SARS-CoV-2-infected patients., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

High-sensitivity multicolour flow cytometry (MFC)-based B-lymphoblastic leukaemia (B-ALL) measurable residual disease (BMRD) assay is increasingly being used in clinical practice. Herein, we describe six consistently present low-level populations immunophenotypically mimicking abnormal B-ALL blasts in 441 BMRD samples from 301 children. These included CD19 + CD123 + plasmacytoid dendritic cells differentiating from lymphoid precursors, CD10 + transitional B cells with CD10 + /CD38dim-to-negative/CD20bright/CD45bright phenotype, CD19 + natural killer (NK) cells, CD73bright/CD10 + mesenchymal stromal/stem cells, CD73bright/CD34 + endothelial cells, and a CD34 + CD38dim-to-negative/CD10 - /CD20bright/CD45bright subset of mature B cells. We provide the proportions, comprehensive immunophenotype, and practical clues for proper identification of these low-level populations. Knowledge regarding the presence and immunophenotype of these mimics is essential for accurate interpretation in high-sensitivity MFC-BMRD analysis., (© 2021 British Society for Haematology and John Wiley & Sons Ltd.)

This protocol provides instructions to improve flow cytometry analysis of marrow/peripheral blood cells by avoiding erythrolytic solutions, density gradients, and washing steps. We describe two basic approaches for identifying cell surface antigens with minimal sample perturbation, which have been successfully used to identify healthy and pathologically rare cells. The greatest advantage of these approaches is that they minimize the unwanted effect caused by sample preparation, allowing for improved study of live cells at the point of analysis. For complete details on the use and execution of this protocol, please refer to Petriz et al. (2018)., Competing Interests: M.D.W. and J.A.B. are employees of Thermo Fisher Scientific, which is in the business of selling flow cytometers and flow cytometry reagents. The rest of the authors declare no competing interests., (© 2021 The Author(s).)

Most shared resource flow cytometry facilities do not permit analysis of radioactive samples. We are investigating low-dose molecular targeted radionuclide therapy (MTRT) as an immunomodulator in combination with in situ tumor vaccines and need to analyze radioactive samples from MTRT-treated mice using flow cytometry. Further, the sudden shutdown of core facilities in response to the COVID-19 pandemic has created an unprecedented work stoppage. In these and other research settings, a robust and reliable means of cryopreservation of immune samples is required. We evaluated different fixation and cryopreservation protocols of disaggregated tumor cells with the aim of identifying a protocol for subsequent flow cytometry of the thawed sample, which most accurately reflects the flow cytometric analysis of the tumor immune microenvironment of a freshly disaggregated and analyzed sample. Cohorts of C57BL/6 mice bearing B78 melanoma tumors were evaluated using dual lymphoid and myeloid immunophenotyping panels involving fixation and cryopreservation at three distinct points during the workflow. Results demonstrate that freezing samples after all staining and fixation are completed most accurately matches the results from noncryopreserved equivalent samples. We observed that cryopreservation of living, unfixed cells introduces a nonuniform alteration to PD1 expression. We confirm the utility of our cryopreservation protocol by comparing tumors treated with in situ tumor vaccines, analyzing both fresh and cryopreserved tumor samples with similar results. Last, we use this cryopreservation protocol with radioactive specimens to demonstrate potentially beneficial effector cell changes to the tumor immune microenvironment following administration of a novel MTRT in a dose- and time-dependent manner., (Copyright © 2021 by The American Association of Immunologists, Inc.)

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.)

Multiple myeloma (MM) is a heterogeneous group of mature B-cell diseases that are typically characterized by the presence and accumulation of abnormal plasma cells (PCs), which results in the excess production of monoclonal immunoglobulin and/or light chain found in the serum and/or urine. Multiparametric flow cytometry (MFC) is an indispensable tool to supplement the diagnosis, classification and monitoring of the disease due to its high patient applicability, excellent sensitivity and encouraging results from various clinical trials. In this regard, minimal or, more appropriately, measurable residual disease (MRD) negativity by MFC has been recognized as a powerful predictor of favourable long-term outcomes. Before flow cytometry can be effectively implemented in the clinical setting for MM MRD testing, sample preparation, panel configuration, analysis and gating strategies must be optimized to ensure accurate results. This manuscript will discuss the current consensus guidelines for flow cytometric processing of samples and reporting of results for MM MRD testing. We also discuss alternative approaches to detect plasma cells in the presence of daratumumab treatment. Finally, there is a lack of information describing the subclonal distribution of myeloma cells based on their protein expression. The advent of high-dimensional analysis may assist in following the evolution of antigen expression patterns on abnormal plasma cells in patients with relapsed/refractory disease. This in turn can help identify clonal subtypes that are more aggressive for potential informed decision. An analysis using t-SNE to identify the emergence of PCs subclones by MFC, along with the analysis of their immunophenotypic profiles are presented as a future perspective., (© 2021 John Wiley & Sons Ltd.)

5-Azacitidine (AZA) therapy is used in high-risk myelodysplastic syndrome (MDS) patients who often show abnormalities in their immunophenotype. We explored the potential impact of AZA on these immunophenotypic abnormalities in serial bone marrow studies performed in 81 patients from five centers. We compared the immunophenotypic features before and after therapy with AZA, established definitions consistent with flow cytometry immunophenotyping (FCI) improvement, and explored its clinical significance. After a median of 6 cycles of AZA, 41% of patients showed a FCI improvement and this finding associated with best possible clinical response (P < 0.001). FCI improvement also correlated with hematological improvement (HI) (53/78 patients; 68%), independently of their eligibility for stem cell transplantation. Among patients who achieved a HI after 6 cycles of AZA, the probability of maintaining this response at 12 cycles of AZA was twice as large (67%) for those patients who also achieved a FCI improvement after 6 cycles of AZA as compared to patients who did not (33%, P < 0.01). These findings support that monitoring of the immunophenotypic abnormalities during therapy with AZA may assist in redefining the quality of response in patients with MDS.

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.)