Cytometry Part B: Clinical Cytometry最新文献

Quantifying malarial parasite density is crucial for diagnosis and treatment in endemic areas. While Malaria-derived particles (MDPs) have been linked to malaria pathology, a direct quantification method for routine laboratory use remains unestablished. To address this, our study optimized a flow cytometry approach to enumerate MDPs per microliter of blood. Specimens were incubated with propidium iodide and red blood cell (RBC) lysis solution. The number of MDPs was quantified using a CytoFlex flow cytometer, size-standard beads, and counting beads. Electron microscopy was used to study the ultrastructures of the malarial parasites in the lysed RBC specimens. A significant increase in MDP levels was detected in blood samples from P. falciparum and P. vivax infections, but fewer than 1 particle/μL of MDPs were detected in the controls. The number of MDPs correlated with the percentage of infected red blood cells (iRBCs) obtained by manual counting (R² = 0.94). The dilution assay demonstrated a strong correlation between the measured and expected values of the MDPs. An electron microscopic study demonstrated that different stages of malarial parasites exist in lysed RBCs in the form of membrane-bound spherical cells. A positive association was established between parasite density and MDPs across both P. falciparum (R² = 0.94) and P. vivax (R² = 0.91) infections. We demonstrated the potential use of flow cytometry for determining the MDP concentration. The developed approach is reliable and straightforward for the diagnosis and treatment of patients with malarial parasite infection in routine laboratory settings.

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.