Rare acute leukemia (AL) components or subtypes such as blastic plasmacytoid dendritic cell neoplasm (BPDCN) or early T-cell precursor acute Lymphoblastic Leukemia (ETP-ALL) can be difficult to detect by routine flow cytometry due to their immunophenotypes overlapping with other poorly differentiated AL. We hypothesized that using standardized EuroFlow™ Consortium approach could better diagnose such entities among cases that previously classified as acute myeloid leukemia (AML)-M0, AML with minimal differentiation, AML with myelodysplasia-related changes without further lineage differentiation, and AL of ambiguous lineage. In order to confirm this hypothesis and assess whether these AL subtypes such as BPDCN and ETP-ALL had previously gone undetected, we reanalyzed 49 banked cryopreserved sample cases using standardized EuroFlow™ Consortium panels. We also performed target sequencing to capture the mutational commonalities between these AL subtypes. Reanalysis led to revised or refined diagnoses for 23 cases (47%). Of these, five diagnoses were modified, uncovering 3 ETP-ALL and 2 typical BPDCN cases. In 12 AML cases, a variable proportion of immature plasmacytoid dendritic cell and/or monocytic component was newly identified. In one AML case, we have identified a megakaryoblastic differentiation. Finally, in five acute lymphoblastic leukemia (ALL) cases, we were able to more precisely determine the maturation stage. The application of standardized EuroFlow flow cytometry immunophenotyping improves the diagnostic accuracy of ALs and could impact treatment decisions.

The FDA-approved ClearLLab 10C Reagents Panel (Beckman Coulter) simplified the diagnosis of leukemias and lymphomas by flow cytometry. However, the requirement of using 3 × 10 cells/mL cannot be met for paucicellular samples. Therefore, we tested whether this 10-color panel can be reliably employed to analyze specimens with low cell concentrations. Serial dilutions of 16 samples (5 normal, 11 abnormal), yielding concentrations ranging from 3.0 × 10 to 0.0469 × 10 cells/mL (64-fold difference), were stained using the B-cell and T-cell panels of the ClearLLab 10C system, and mean fluorescence intensity (MFI) was measured for each antibody. For each cell dilution, the deviation from the value obtained with the FDA-approved concentration of 3.0 × 10 cells/mL was calculated. The agreement between the highest and lowest cell concentration data was evaluated by the Bland and Altman method, Pearson's and Spearman's correlation analyses, and linear regression. In all patients, the antigen expression pattern was similar at all cell concentrations tested, and the mean deviation of the MFI from the value obtained using 3.0 × 10 cells/mL never exceeded 10% for any of the antibodies. The Bland-Altman method demonstrated the similarity between results obtained with the FDA-approved cell concentration and a 64-fold diluted cell suspension, and a high positive correlation was found between MFI acquired under these two conditions. The tests utilizing the lowest density of cells yielded the same patterns of antigen expression in all patients as those performed with the FDA-approved concentration, documenting a 100% concordance between these two protocols. The ClearLLab 10C panel can reliably determine the expression of markers of leukemias and lymphomas in paucicellular samples containing as little as 0.0469 × 10 cells/mL (64-fold lower than the FDA-approved concentration). This finding markedly expands the applicability of the ClearLLab 10C platform in a clinical setting.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common enzymopathy that affects red blood cells (RBCs) and renders them susceptible to oxidative stress. G6PD deficiency can cause hemolytic anemia, especially after exposure to certain drugs or infections. The diagnosis of G6PD deficiency is usually based on spectrophotometric measurement of enzyme activity, but this method has limitations in heterozygous females and in patients with other hematological disorders. In this study, we evaluated the use of flow cytometry as an alternative method for detecting G6PD deficiency in 514 samples (265 females and 249 males) from a clinical laboratory. We compared the results of flow cytometry with those of spectrophotometry and molecular analysis, and assessed the performance of flow cytometry in different subgroups of patients. We found that flow cytometry was able to identify G6PD deficiency in most cases, with high sensitivity and specificity. Flow cytometry also allowed the quantification of the percentage of G6PD-deficient RBCs, which varied among heterozygous females due to X-chromosome inactivation. Moreover, flow cytometry detected several cases of G6PD deficiency that were missed by spectrophotometry, especially in heterozygous females with normal or subnormal enzyme activity. However, flow cytometry also showed some false negative results, mainly in patients with sickle cell disease. Therefore, flow cytometry is a reliable and efficient tool for screening G6PD deficiency, but some precautions should be taken in interpreting the results in patients with other hematological conditions.

Automated analysis of flow cytometry data can improve objectivity and reduce analysis time but has generally required work by software and algorithm experts. Here, we investigated the performance of BD ElastiGate™ Software (hereafter ElastiGate), which allows users to automate gating by selecting gated training files, then uses elastic image registration to gate new files. Three assays of increasing complexity were examined: TBNK, stem cell enumeration (SCE), and lymphoid screening tube (LST). For TBNK analysis, 60 peripheral blood (PB) samples from normal, HIV+, and controls were tested with ground truth analysis by an existing automated method. For SCE, 128 samples including bone marrow (BM), cord blood (CB), and apheresis were tested with analysis by multiple manual analysts. For LST, 80 PB and 28 BM samples were tested with manual analysis. For ElastiGate, a minimal number of training files was selected. Results were compared by Bland-Altman or F1 score analysis. For TBNK, ElastiGate using three training files (1 control, 1 normal, 1 HIV+) showed mean %bias across all reported populations between -1.48% and 7.13% (average 2.08%). For SCE, ElastiGate using three BM and two CB training files showed median F1 scores >0.93 in comparison to >0.94 and >0.92 for two other manual analysts. For LST, ElastiGate using four training files for each of PB and BM showed median F1 scores >0.945 for 13 of 14 PB populations and 10 of 14 BM populations, with generally similar or better performance for normal samples compared to abnormal; populations with lower scores were often associated with lower agreement between manual analysts. Based on analysis of three assays with four sample types of increasing complexity, ElastiGate with minimal training files may perform as an automated gating assistant. The results reported here are for research use only, not for use in diagnostic or therapeutic procedures.

Cervical cancer (CC) is the fourth most common malignant tumor in women worldwide. Detecting different biomarkers together on single cells by novel method mass cytometry could contribute to more precise screening. Liquid-based cytology (LBC) cervical samples were collected (N = 53) from women categorized as normal and precancerous lesions. Human papillomavirus was genotyped by polymerase chain reaction, while simultaneous examination of the expression of 29 proteins was done by mass cytometry (CyTOF). Differences in cluster abundances were assessed with Spearman's rank correlation as well as high dimensional data analysis (t-SNE, FlowSOM). Cytokeratin (ITGA6, Ck5, Ck10/13, Ck14, Ck7) expression patterns allowed determining the presence of different cells in the cervical epithelium. FlowSOM analysis enabled to phenotype cervical cells in five different metaclusters and find new markers that could be important in CC screening. The markers Ck18, Ck18, and CD63 (Metacluster 3) showed significantly increasing associated with severity of the precancerous lesions (Spearman rank correlation rho 0.304, p = 0.0271), while CD71, KLF4, LRIG1, E-cadherin, Nanog and p53 (Metacluster 1) decreased with severity of the precancerous lesions (Spearman rank correlation rho -0.401, p = 0.0029). Other metaclusters did not show significant correlation, but metacluster 2 (Ck17, MCM, MMP7, CD29, E-cadherin, Nanog, p53) showed higher abundance in low- and high-grade intraepithelial lesion cases. CyTOF appears feasible and should be considered when examining novel biomarkers on cervical LBC samples. This study enabled us to characterize different cells in the cervical epithelium and find markers and populations that could distinguish precancerous lesions.

The gut-liver axis includes the bidirectional communication between the gut and the liver, and thus covers signals from liver-to-gut and from gut-to-liver. Disruptions of the gut-liver axis have been associated with the progression of chronic liver diseases, including alcohol-related and metabolic dysfunction-associated steatotic liver disease and cholangiopathies. Immune cells and their expression of pattern recognition receptors, activation markers or immune checkpoints might play an active role in the communication between gut and liver. Here, we present a 26-color full spectrum flow cytometry panel for human cells to decipher the role of circulating immune cells in gut-liver communication during the progression of chronic liver diseases in a non-invasive manner, which has been optimized to be used on patient-derived whole blood samples, the most abundantly available clinical material. Our panel focuses on changes in pattern recognition receptors, including toll-like receptors (TLRs) or Dectin-1, and also includes other immunomodulatory molecules such as bile acid receptors and checkpoint molecules. Moreover, this panel can be utilized to follow the progression of chronic liver diseases and could be used as a tool to evaluate the efficiency of therapeutic targets directed against microbial mediators or modulating immune cell activation.

HLA-B27 is a major histocompatibility complex (MHC) class I antigen which exhibits strong association (90%) with ankylosing spondylitis. HLA-B27 detection in patients by flow cytometry is a widely used clinical test, performed on many different flow cytometer models. We sought to develop and validate a test conversion protocol for the HLA-B27 test performed on the BD FACSCanto to BD's newer FACSLyric flow cytometers. The development and validation experiments were performed using anti-HLA-B27*FITC/CD3*PE antibody-stained whole blood patient specimens. The anti-HLA-B27*FITC logarithmic median fluorescence (LMF) results on the BD FACSCanto were converted to median fluorescence intensity (MFI) values on the BD FACSLyric. Clustering of the HLA-B27 positive and negative values, using a 3rd order polynomial equation, resulted in a conversion of the BD FACSCanto cutoff values, negative (<150 LMF) and positive (≥160 LMF), to negative (<4530 MFI) and positive (≥6950 MFI) on the BD FACSLyric. Accuracy was assessed by comparing the flow results obtained on the BD FACSCanto and BD FACSLyric to a molecular PCR based assay. Additional validation parameters (compensation verification, intra- and inter-assay precision, and instrument comparison) were performed per the recommendations outlined in the Clinical and Laboratory Standards Institute (CLSI) H62 guidelines for validation of flow cytometry assays.

High-grade B-cell lymphomas (HGBCL) represent a heterogeneous group of very rare mature B-cell lymphomas. The 4th revised edition of the WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues (WHO-HAEM) previously defined two categories of HGBCL: the so-called double-hit (DHL) and triple-hit (THL) lymphomas, which were related to forms harboring MYC and BCL2 and/or BCL6 rearrangements, and HGBCL, NOS (not otherwise specified), corresponding to entities with intermediate characteristics between diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL), without rearrangement of the MYC and BCL2, and/or BCL6 genes. In the 5th edition of the WHO-HAEM, DHL with MYC and BCL2 rearrangements or THL were reassigned as DLBCL/HGBCL with MYC and BCL2 rearrangements (DLBCL/HGBL-MYC/BCL2), whereas the category HGBCL, NOS remains unchanged. Characterized by an aggressive clinical presentation and a poor prognosis, HGBCL is often diagnosed at an advanced, widespread stage, leading to potential disseminated forms with a leukemic presentation, or spreading to the bone marrow (BM) or other biological fluids. Flow cytometric immunophenotypic study of these disseminated cells can provide a rapid method to identify HGBCL. However, due to the scarcity of cases, only limited data about the immunophenotypic features of HGBCL by multiparametric flow cytometry are available. In addition, identification of HGBCL cells by this technique may be challenging due to clinical, pathological, and biological features that can overlap with other distinct lymphoid malignancies, including Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), and even B acute lymphoblastic leukemia (B-ALL). In this study, we aimed to characterize the detailed immunophenotypic portrait of HGBCL, evaluating by multiparametric flow cytometry (MFC) the expression of 26 markers on biological samples obtained from a cohort of 10 newly-diagnosed cases and comparing their level of expression with normal peripheral blood (PB) B lymphocytes (n = 10 samples), tumoral cells from patients diagnosed with B-ALL (n = 30), BL (n = 13), or DLBCL (n = 22). We then proposed a new and simple approach to rapidly distinguish disseminated forms of HGBCL, BL, and DLBCL, using the combination of MFC data for CD38, BCL2, and CD39, the three most discriminative markers explored in this study. We finally confirmed the utility of the scoring system previously proposed by Khanlari to distinguish HGBCL cells from B lymphoblasts of B-ALL. In conclusion, we described a distinct immunophenotypic portrait of HGBCL cells and proposed a strategy to differentiate these cells from other aggressive B lymphoma entities in biological samples.

Multicolor flow cytometry (MFC) is crucial in detecting occult or minimal bone marrow (BM) involvement by non-Hodgkin lymphomas (NHL), which may not be detected using trephine biopsy or imaging studies. Detection of low-level BM involvement can be challenging without definite immunophenotypic aberrancies. We studied the utility of CD305 in MFC detection of minimal BM involvement by B-NHL, especially in the absence of aberrancies by commonly used markers. The study included 1084 consecutive BM samples submitted for the staging of B-NHLs (excluding CLL) over two years. Samples were studied for morphological, immunophenotypic, and histopathological assessment. MFC studies were performed using 10-13 color MFC, including CD305-antibody (clone, DX26). Minimal BM involvement was defined with a cutoff of ≤10% lymphoma cells in viable cells on MFC assessment. Of 1084, 148 samples revealed overt morphological involvement by B-NHL and were excluded from analysis. BM samples of 172/936 patients were morphologically negative but revealed involvement using MFC independently. Corresponding trephine biopsy involvement was detected in only 79/172 (45.9%) patients. On MFC, 23/172 samples showed BM involvement with >10% lymphoma cells, and 149/172 (86.6%) samples revealed minimal involvement. In 54/149 (36.24%) samples, lymphoma cells were detected only with aberrant loss of CD305 expression. In 78 of the remaining 95 samples (82.1%), it provided an immunophenotypic aberrancy addition to other markers and supported the results. CD305 is a highly useful marker in the flow cytometric assessment of minimal BM involvement by B-NHL. MFC is a superior modality to trephine biopsy in detecting low-level BM involvement.

Hematologic neoplasms with germline predisposition have been increasingly recognized as a distinct category of tumors over the last few years. As such, this category was added to the World Health Organization (WHO) 4th edition as well as maintained in the WHO 5th edition and International Consensus Classification (ICC) 2022 classification systems. In practice, these tumors require a high index of suspicion and confirmation by molecular testing. Flow cytometry is a cost-effective diagnostic tool that is routinely performed on peripheral blood and bone marrow samples. In this review, we sought to summarize the current body of research correlating flow cytometric immunophenotype to assess its utility in diagnosis of and clinical decision making in germline hematologic neoplasms. We also illustrate these findings using cases mostly from our own institution. We review some of the more commonly mutated genes, including CEBPA, DDX41, RUNX1, ANKRD26, GATA2, Fanconi anemia, Noonan syndrome, and Down syndrome. We highlight that flow cytometry may have a role in the diagnosis (GATA2, Down syndrome) and screening (CEBPA) of some germline predisposition syndromes, although appears to show nonspecific findings in others (DDX41, RUNX1). In many of the others, such as ANKRD26, Fanconi anemia, and Noonan syndrome, further studies are needed to better understand whether specific flow cytometric patterns are observed. Ultimately, we conclude that further studies such as large case series and organized data pipelines are needed in most germline settings to better understand the flow cytometric immunophenotype of these neoplasms.

The Clinical and Laboratory Standards Institute (CLSI) H62-Validation of Assays Performed by Flow Cytometry guideline, released in 2021, provides recommendations for platform workflow and quality system essentials, instrument setup and standardization, assay development and optimization and fit-for-purpose analytical method validation. In addition, CLSI H62 includes some recommendations for the validation strategies after a validated flow cytometric method has been modified. This manuscript builds on those recommendations and discusses the impact of different types of assay modifications on assay performance. Recommendations regarding which validation parameters to evaluate depending on the type of modification are provided. The impact of assay modification on the assay's intended use is discussed. When recommending minor deviations from the CLSI H62 process for a laboratory-initiated assay revision (e.g., specimen numbers for sensitivity, specificity, or precision studies), a rationale based on expert opinion is provided with the understanding that not every laboratory, assay type, and circumstance can be comprehensively addressed in this paper. These recommendations are meant as a practical recommendation and are not intended to be restrictive, prescriptive, or understood as necessarily sufficient to meet every specific requirement from regulatory bodies (e.g., FDA or New York State Department of Health).

The most important reason for dismal outcomes in acute myeloid leukemia (AML) is the development of relapse. Leukemia stem cells (LSCs) are hypothesized to initiate relapse, and high CD34+CD38- LSC load is associated with poor prognosis. In 10% of AML patients, CD34 is not or is low expressed on the leukemic cells (<1%), and CD34+CD38- LSCs are absent. These patients are classified as CD34-negative. We aimed to determine whether the primitive marker CD133 can detect LSCs in CD34-negative AML. We retrospectively quantified 148 CD34-negative patients for proportions of CD34-CD133+ and CD133+CD38- cell fractions in the diagnostic samples of CD34-negative patients in the HOVON102 and HOVON132 trials. No prognostic difference was found between patients with high or low proportions of CD34-CD133+, which is found to be aberrantly expressed in AML. A high level of CD133+CD38- cells was not associated with poor overall survival, and expression in AML was similar to normal bone marrow. To conclude, CD133 is useful as an additional primitive marker for the detection of leukemic blast cells in CD34-negative AML. However, CD133+CD38 alone is not suitable for the detection of LSCs at diagnosis.

The advent of high-dimensional imaging offers new opportunities to molecularly characterize diagnostic cells in disorders that have previously relied on histopathological definitions. One example case is found in tuberous sclerosis complex (TSC), a developmental disorder characterized by systemic growth of benign tumors. Within resected brain tissues from patients with TSC, detection of abnormally enlarged balloon cells (BCs) is pathognomonic for this disorder. Though BCs can be identified by an expert neuropathologist, little is known about the specificity and broad applicability of protein markers for these cells, complicating classification of proposed BCs identified in experimental models of this disorder. Here, we report the development of a customized machine learning pipeline (BAlloon IDENtifier; BAIDEN) that was trained to prospectively identify BCs in tissue sections using a histological stain compatible with high-dimensional cytometry. This approach was coupled to a custom 36-antibody panel and imaging mass cytometry (IMC) to explore the expression of multiple previously proposed BC marker proteins and develop a descriptor of BC features conserved across multiple tissue samples from patients with TSC. Here, we present a modular workflow encompassing BAIDEN, a custom antibody panel, a control sample microarray, and analysis pipelines-both open-source and in-house-and apply this workflow to understand the abundance, structure, and signaling activity of BCs as an example case of how high-dimensional imaging can be applied within human tissues.

The publication of Clinical and Laboratory Standards Institute's guideline H62 has provided the flow cytometry community with much-needed guidance on development and validation of flow cytometric assays (CLSI, 2021). It has also paved the way for additional exploration of certain topics requiring additional guidance. Flow cytometric analysis of rare matrices, or unique and/or less frequently encountered specimen types, is one such topic and is the focus of this manuscript. This document is the result of a collaboration subject matter experts from a diverse range of backgrounds and seeks to provide best practice consensus guidance regarding these types of specimens. Herein, we define rare matrix samples in the setting of flow cytometric analysis, address validation implications and challenges with these samples, and describe important considerations of using these samples in both clinical and research settings.

Measurable residual disease (MRD) is detected in approximately a quarter of AML chemotherapy responders, serving as a predictor for relapse and shorter survival. Immunological control of residual disease is suggested to prevent relapse, but the mechanisms involved are not fully understood. We present a peripheral blood single cell immune profiling by mass cytometry using a 42-antibody panel with particular emphasis on markers of cellular immune response. Six healthy donors were compared with four AML patients with MRD (MRD) in first complete remission (CR1). Three of four patients demonstrated a favorable genetic risk profile, while the fourth patient had an unfavorable risk profile (complex karyotype, TP53-mutation) and a high level of MRD. Unsupervised clustering using self-organizing maps and dimensional reduction analysis was performed for visualization and analysis of immune cell subsets. CD57 natural killer (NK)-cell subsets were found to be less abundant in patients than in healthy donors. Both T and NK cells demonstrated elevated expression of activity and maturation markers (CD44, granzyme B, and phosho-STAT5 Y694) in patients. Although mass cytometry remains an expensive method with limited scalability, our data suggest the utility for employing a 42-plex profiling for cellular immune surveillance in whole blood, and possibly as a biomarker platform in future clinical trials. The findings encourage further investigations of single cell immune profiling in CR1 AML-patients.

Over the last 15 years activity of diagnostic flow cytometry services have evolved from monitoring of CD4 T cell subsets in HIV-1 infection to screening for primary and secondary immune deficiencies syndromes and assessment of immune constitution following B cell depleting therapy and transplantation. Changes in laboratory activity in high income countries have been driven by initiation of anti-retroviral therapy (ART) in HIV-1 regardless of CD4 T cell counts, increasing recognition of primary immune deficiency syndromes and the wider application of B cell depleting therapy and transplantation in clinical practice. Laboratories should use their experience in standardization and quality assurance of CD4 T cell counting in HIV-1 infection to provide immune monitoring services to patients with primary and secondary immune deficiencies. Assessment of immune reconstitution post B cell depleting agents and transplantation can also draw on the expertise acquired by flow cytometry laboratories for detection of CD34 stem cell and assessment of MRD in hematological malignancies. This guideline provides recommendations for clinical laboratories on providing flow cytometry services in screening for immune deficiencies and its emerging role immune reconstitution after B cell targeting therapies and transplantation.