Cytometry Part B: Clinical Cytometry最新文献

Accurate quantification of chimeric antigen receptor (CAR) T cells is essential for monitoring post-infusion CART expansion and persistence and for real-time clinical decision-making. Multiparameter flow cytometry (MFC) enables rapid, live-cell detection with absolute quantification and concurrent immunophenotypic characterization. This review focuses on the practical and technical aspects of flow cytometry-based CAR T-cell monitoring, including selection of CAR detection reagents (target-specific, construct-specific, and target-agnostic strategies), assay optimization, purpose-driven panel design, and matrix-appropriate validation for peripheral blood and other clinically relevant specimens. We also address assay considerations unique to gene-edited allogeneic CAR T-cell products, including the use of surrogate immunophenotypic approaches when construct-specific reagents are unavailable. Finally, we discuss the role of MFC in identifying CAR T-cell clonal expansions and in evaluating suspected secondary hematolymphoid neoplasms in the post-CAR T setting.

This article details the development of a tissue-based receptor occupancy (RO) assay using colonic pinch biopsies to enable the evaluation of vixarelimab target coverage in a Phase 1c study for inflammatory bowel disease (IBD). Vixarelimab, a monoclonal antibody targeting Oncostatin M Receptor Beta subunit (OSMRβ), has demonstrated clinical efficacy in treating inflammatory conditions such as prurigo nodularis and chronic pruritus. It was more recently studied in patients with ulcerative colitis (UC). Understanding the drug's distribution and retention in gut tissues is essential for assessing its therapeutic potential for IBD. While RO assays are powerful tools for evaluating drug target engagement, there are gaps in best practices for their development, especially when applied to sample types not readily available as single-cell suspensions. This article addresses challenges associated with tissue processing, cell recovery, and accurate interpretation of RO results, offering insights into overcoming limitations in sample availability and variability in drug-target interactions during assay development.

Systemic mastocytosis (SM) is a clonal mast cell (MC) disorder characterized by aberrant immunophenotypes, including expression of CD25, CD2, and occasionally CD30. CD123, the α-subunit of the interleukin-3 receptor, is a therapeutic target in hematologic malignancies and has been reported to be expressed on neoplastic MCs by immunohistochemistry (IHC) with prognostic implications. This study aims to characterize CD123 expression in SM by flow cytometry. We retrospectively analyzed 142 bone marrow samples from 79 SM patients (81 diagnostic samples) and 25 controls with normal MC immunophenotype. Flow cytometry was performed using a clinically validated 9-color mast cell tube which included CD123. Data collected included SM subtype, clinical and laboratory features, MC burden, and marker expression. Statistical analyses were performed in R. CD123 was expressed on MCs in 91% of SM cases (ISM 92%, SM-AHN 94%, SSM 100%, ASM 100%, MCL 50%). Median percentage of MCs positive for CD123 was 53.9% (IQR 8.1-83.4). Compared to prior IHC data (overall 64% positivity), flow cytometry demonstrated more cases with CD123 expression by MCs. No significant correlations were observed between CD123 expression and serum tryptase, KIT D816V allele burden, or MC burden. CD123 is frequently expressed on neoplastic MCs in SM by flow cytometry, across all subtypes. These findings support further investigation of CD123 as a therapeutic target and warrant correlation with IHC and clinical outcomes in larger cohorts.

Basophils are effector cells in type I hypersensitivity reactions. Upon cross-linking of surface-bound (allergen-specific) IgE, basophils release a variety of mediators. Determining the activation of basophils ex vivo with anti-IgE (a basophil activation test or BAT) can be achieved using flow cytometry. However, this requires an experienced laboratory, hampering penetration of the BAT into general allergen diagnostics. Automation of the BAT could make this requirement redundant. Automation of the BAT enables rapid analysis in a field study setting of the effect of prolonged endurance exercise that can prime circulating basophils. A modified near-patient BAT (activation by anti-IgE and fMLF) was developed to identify basophils (SSC^Low and CD193^bright) and their activation status (based on expression of CD63, CD11b and CD62L) using blood samples taken from 7 healthy anonymous volunteers of the hospital's donor service. Due to logistic reasons, the modified BAT (mBAT) was developed at room temperature. Second, blood samples from 18 healthy volunteers were taken during a multiple day walking event to determine the effect of exercise on basophil activation in situ. Eosinophil (SSC^high and CD193^bright) and neutrophil (SSC^high and CD193^dim) activation were also measured as comparators. Anti-IgE- and fMLF-induced activation of basophils in whole blood of both groups showed reproducibility and optimal activation at 1 μg/mL and 0.1 μM, respectively. The field study showed that exercise led to activation of basophils in situ. The fully automated, 24/7 mBAT is feasible for near-patient analysis of priming and/or activation of innate immune cells. This can bring basophil activation testing by mBAT to peripheral care institutes as it does not depend on a specialized laboratory and/or personnel. This study indicates that prolonged exercise leads to activation of basophils in vivo, which might be used to study exercise intolerance in asthmatics.

Antigen expression on residual blast cells in acute megakaryoblastic leukemia (AMKL, classified as AML-M7 by FAB criteria) may change after treatment, potentially affecting both immunophenotypic characterization and minimal/measurable residual disease (MRD) monitoring. This study aimed to characterize post-therapy immunophenotypic alterations in AMKL and to determine whether specific patterns of antigenic change exist between samples obtained at initial presentation (IP group) and those obtained at MRD-positive status after therapy (MRD group). This retrospective descriptive study included 110 patients diagnosed with AMKL at Hebei Yanda Lu Daopei Hospital between January 1, 2009 and December 31, 2024 (male:female = 57:53; 103 pediatric and 7 adult cases). Immunophenotypes at initial diagnosis and after treatment were analyzed by flow cytometry. The chi-square test was used to compare antigen expression between the IP and MRD groups. Flow cytometric immunophenotypes differed by at least three antigens (including CD33, CD61, and CD42b) between initial presentation and post-therapy samples. Compared with the IP group, the MRD group showed a significantly higher frequency of loss of megakaryocytic markers, including CD61 (11/109, 10.1% vs. 30/109, 27.5%; p < 0.05) and CD42b (6/106, 5.7% vs. 22/101, 21.8%; p < 0.05). Partial loss of CD13 expression was also more frequent in the MRD group (18/99, 18.2% vs. 2/83, 2.4%; p < 0.05). No significant differences were observed in the expression of progenitor-associated markers (CD34, CD117), myeloid markers (CD33, CD11b), or other antigens (HLA-DR, CD7, CD56, CD42a) between the two groups (p > 0.05). Lineage-specific markers MPO and CD22, the monocytic marker CD14, and lymphoid markers CD10 and CD5 were negative in both groups. In contrast, aberrant expression of cCD3 (2/89, 2.2%) and CD19 (3/85, 3.5%) was observed in a small subset of IP cases. Overall, 100 of 110 patients (90.9%) showed changes in at least one antigen after therapy. By lineage category, alterations were most frequent in megakaryocytic markers (CD61, CD42b, CD41a, CD42a; 64/110, 58.2%), followed by myeloid antigens (HLA-DR, CD33, CD13, CD11b; 54/108, 50.0%), progenitor-associated antigens (CD34, CD117; 53/110, 48.2%), and lymphoid antigens (CD7, CD56; 24/107, 22.4%). In addition, CD110 was consistently expressed in all 26 AMKL cases tested, whereas only 18% (9/50) of non-AMKL AML cases were CD110-positive (p < 0.05). Significant immunophenotypic differences, particularly involving CD61, CD42b, and CD13, exist between IP and MRD samples in AMKL. Antigenic shifts affecting megakaryocytic, myeloid, progenitor-associated, and lymphoid markers are common after chemotherapy. For MRD assessment, the use of more specific megakaryocytic markers such as CD110, together with comprehensive multiparameter flow cytometry panels, may improve detection accuracy.

Distinguishing T-cell from NK-cell neoplasms can occasionally be challenging, as neoplastic T cells can lose T-cell markers such as surface CD3 (sCD3) and CD5 while acquiring NK-cell markers such as CD16, CD56, and CD94. In this study, we present a series of 7 mature T-cell lymphoma/leukemia cases with NK-like immunophenotypes to clarify diagnostic approaches for lineage determination. The cohort included 3 cases of peripheral T-cell lymphoma, not otherwise specified, 2 cases of T-large granular lymphocytic leukemia, and 2 cases of hepatosplenic T-cell lymphoma. In all cases, flow cytometry immunophenotypic analysis showed that the neoplastic cells exhibited an NK-like immunophenotype characterized by expression of CD56 and CD94 in all 7 cases, with 4 cases also positive for CD16. Concurrently, the neoplastic cells in all cases were negative for the T-cell markers, including sCD3, CD4, CD5, TCRαβ, TCRγδ, and TRBC1 by flow cytometry. These immunophenotypic features mimicked NK cells. But in T-cell receptor (TCR) gene rearrangement analysis, all 7 cases had monoclonal TRG rearrangements, and 4 cases also showed concurrent TRB rearrangements. Immunohistochemical analysis showed that all 6 tested cases were positive for BCL11B, and 1 case was positive for TCRαβ. Additionally, 2 of 3 cases assessed using a T-cell-specific CD3 antibody were positive for cytoplasmic CD3 by flow cytometry. In summary, this study illustrates that neoplastic T cells can show an NK-like immunophenotype. Features useful for supporting T-lineage in this context include cytoplasmic CD3 expression assessed using a T-cell-specific CD3 antibody, clonal TCR gene rearrangement, BCL11B expression, and detection of TCRαβ, TCRγδ, or TRBC by immunohistochemistry.

Minimal/measurable residual disease detection is routinely performed as part of post-diagnostic treatment plans for many types of cancer, for which multiparameter flow cytometry is one possible modality frequently used. We propose a machine learning approach for binary prediction of minimal residual disease status with flow cytometry data. Our method involves the projection of cells from the original feature space to a low-dimensional embedding in which cells are clustered by similarity, and the cluster-wise cell proportions are used for prediction as well as regression. This is a weakly supervised approach in that the only annotation required for the training set data is the percentage of neoplastic cells present in each case. We demonstrate the applicability of our method with respect to a cohort of chronic lymphocytic leukemia patient data to obtain high levels of accuracy. We contrast our approach with other proposed machine learning methods for application to minimal residual disease prediction.

Measurable residual disease (MRD) monitoring by multiparameter flow cytometry (MFC) is well established in bone marrow (BM) samples for acute myeloid leukemia (AML), but its use in peripheral blood (PB) remains less developed. We adapted a semi-automated and well validated MFC-MRD assay, originally developed for BM, to PB aiming to evaluate its applicability and concordance with established methods. To transfer the MFC-MRD assay from BM to PB, we incorporated population-specific reference values derived from healthy and chemotherapy-exposed non-AML controls. MRD detection was based on a combined aberrant leukemia associated immunophenotype (aLAIP) and different from normal (DfN) approach. We compared MFC-MRD detection in paired PB and BM samples and evaluated concordance with molecular MRD (Mol-MRD) diagnostics. Additional analyses included assessment of mast cells as markers for BM hemodilution as well as time requirements for analyses. Reference values for aberrant populations in PB were generally lower than in BM, particularly considering immature markers. At follow-up, MFC-MRD positivity was less common in PB than in BM, leading to a concordance rate of 73.7%. Both quantitative and qualitative differences in aberrant antigen expression between PB and BM were observed. Most cases of MRD positivity were characterized by newly emerging aberrant features rather than persistent ones. Concordance between MFC-MRD from PB and Mol-MRD from BM was 64.6%, similar to that of MFC-MRD from BM versus Mol-MRD from BM (58.5%). Most discordant cases (MFC-MRD^pos/Mol-MRD^neg) may reflect clonal hematopoiesis or limited spectrum of molecular panels. Mast cell quantification proved useful in identifying hemodiluted BM samples, which comprised ~15% of cases. Inter-rater agreement for MRD detection was strong (Kα > 0.8). The MFC-MRD gating and evaluation was rapid (~2 min/sample in both PB and BM). The adapted MFC-MRD assay is feasible and robust in PB, offering a fast and reproducible alternative to BM-based MRD monitoring. However, the prognostic relevance of the proposed method needs to be validated in a prospective cohort of patients.