Cytometry Part B: Clinical Cytometry最新文献

Antibody titration is essential for optimizing platelet flow cytometry, a technique widely used to evaluate platelet phenotype, activation status, and function. This manuscript outlines practical approaches for platelet antibody titration in whole blood, with tailored strategies for constitutive and inducible markers. It emphasizes the use of appropriate controls, consideration of marker coexpression, and selection of subsaturating antibody concentrations to maximize signal resolution while minimizing background and artifactual activation. Quantitative metrics such as the stain index and separation index are introduced as tools for evaluating staining performance. The discussion also addresses key technical variables, including combinatorial titration, spillover spreading, lot variability, and antibody-induced activation. Titration under final assay conditions is recommended to ensure reproducibility and biological relevance. These strategies provide a foundation for developing robust, high-resolution platelet assays that support both research and clinical applications, particularly as flow cytometry evolves toward greater automation and standardization.

Platelet refractoriness is caused by antibodies against human leukocyte antigens or human platelet antigens. However, readily applicable assays that assist in selecting immunologically compatible platelet units remain limited. We established a flow cytometric platelet crossmatching assay and assessed its clinical utility by interpreting the results in conjunction with post-transfusion corrected count increments (CCI). Platelets were incubated with serum that may contain anti-platelet antibodies. Using flow cytometry, CD41-expressing platelets were gated, and the median fluorescence intensity (MFI) of fluorescein isothiocyanate (FITC)-conjugated anti-human IgG was measured. The MFI ratio was calculated as test sample MFI divided by baseline negative control MFI. The cutoff value and limit of detection (LoD) were estimated. Platelet crossmatching was performed using residual segments of transfused platelet units and patient serum, and the results were retrospectively interpreted in conjunction with CCIs and clinical findings. The MFI ratios were clearly distinguishable among the three groups: high-positive controls, low-positive controls, and known negative samples (p < 0.001). The cutoff value was calculated to be 1.35, and the LoD was 1.53. In total, eight platelet transfusion events in five patients were analyzed. Four cases were interpreted as non-immune refractoriness, and the remaining four showed adequate post-transfusion platelet increments. All corresponding platelet crossmatching results were negative, which was considered appropriate given that the assay is designed to reflect immune refractoriness. The flow cytometric platelet crossmatching assay was established and demonstrated to be applicable. The assay can help predict transfusion outcomes in alloimmunized patients and contribute to the selection of compatible blood units.

Multiparametric flow cytometry is a highly valuable method for the assessment of measurable residual disease (MRD) in multiple myeloma patients. The aim of the study was to evaluate a one-tube MRD panel on a DxFlex flow cytometer including all EuroFlow recommended immunophenotypic markers (i.e., cytoplasmic light chain kappa and lambda, CD19, CD27, CD38, CD45, CD56, CD81, CD117, CD138) extended by CD200 to a total of 11 fluorochromes in one tube. Bone marrow aspirates from clinical routine underwent an ammonium chloride-based bulk lysis, followed by staining the surface antibodies and, after permeabilization, kappa, and lambda light-chain intracellular staining. We acquired 1 × 10⁷ cells per sample with a DxFlex flow cytometer. We determined the limit of detection (LOD) and lower limit of quantification (LLOQ) as recommended by the International Myeloma Working Group. For the clinical evaluation, 68 samples from 53 patients with multiple myeloma under or after treatment were analyzed with the one-tube MRD panel, and the results were compared with our routine plasma cell panel (RPCP). Six of the 68 samples were additionally sent to another laboratory to confirm our results with the contemporary EuroFlow next-generation flow (NGF) assay. For our novel one-tube MRD panel we determined a LOD of 0.00016% (1.6 × 10^-6) and a LLOQ of 0.00059% (5.9 × 10^-6). Out of 68 specimens, 55 (80.9%) showed concordant results between the MRD- and the RPC-panel. Thirteen (19.1%) specimens showed a distinct population of abnormal plasma cells with the MRD panel not detectable with the RPC panel. The six samples simultaneously measured with our novel one-tube MRD panel and the EuroFlow NGF assay showed concordant results. The novel one-tube MRD panel meets the quality specifications of the International Myeloma Working Group. Our clinical evaluation found higher sensitivity for the one-tube MRD panel when compared to our RPC panel and concordant results with the contemporary EuroFlow NGF assay. Therefore, our novel one-tube MRD panel is well-suited for detecting MRD in multiple myeloma patients.

The International Harmonisation Protocol (IHP) for Reticulocyte Percentage (%Retic) in CLSI-H44-A2 is a microscopic procedure performed on a new-methylene-blue-stained blood film (NMB-IHP). However, IHP-compliant Measurement Procedures (MPs) based on flow cytometry (FCM) are recommended for practical and accurately assigned reference values. Thiazole orange (TO) for nucleic acid staining and CD235a immunostaining for erythrocyte gating are currently widespread for MP using FCM (TO/CD235a-MP); however they have not been validated using NMB-IHP. We recently developed MP using FCM with anti-CD45/CD41a/CD61 antibodies, that are utilized for excluding platelets from the un-nucleated blood cell fraction to select erythrocytes (TO/CD41a/CD61-MP). The aim of this study is to validate TO/CD41a/CD61-MP for measurement of reticulocytes as an IHP-compliant MP. First, TO/CD235a-MP was validated as an IHP-compliant MP by comparing it with NMB-IHP. Then TO/CD41a/CD61-MP was compared with TO/CD235a-MP. The practical utility of TO/CD41a/CD61-MP was evaluated using XN-2000™(Sysmex) and Celltac G + ™(Nihon Kohden) hematology analyzers to assess the accuracy of different nucleic acid staining methods. We first confirmed that TO/CD235a-MP was consistent with NMB-IHP, then evaluated TO/CD41a/CD61-MP using TO/CD235a-MP. Regression analysis demonstrated consistency between TO/CD41a/CD61-MP and TO/CD235a-MP, thereby establishing TO/CD41a/CD61-MP as IHP-compliant MP. In the accuracy assessment, the correlation coefficients to TO/CD41a/CD61-MP were observed to be 0.97 for both hematology analyzers with %Retic ranging from 0.0 to 8.2. TO/CD41a/CD61-MP serves effectively as an IHP-compliant MP. This study provides the first empirical validation of FCM-based MPs.