Cytometry Part B: Clinical Cytometry最新文献

Central nervous system (CNS) involvement in multiple myeloma (MM) is a rare but severe complication with a poor prognosis. The identification of malignant plasma cells in cerebrospinal fluid (CSF) is essential for early diagnosis and intervention. However, the sensitivity of traditional diagnostic methods like cytology is low, especially in samples with low-cell counts. This study aimed to develop a multidimensional radar dot-plot analysis using Kaluza software to enhance the sensitivity and specificity of flow cytometry for detecting abnormal plasma cells in CSF. One hundred and twenty-five CSF samples were sent for flow cytometric testing to investigate the central nervous system involvement of MM. Finally, 89 samples from 40 patients were included in our study. Multicolor flow cytometry was performed using an 8-color labeling method, and radar dot-plot analysis was developed using diagnostic bone marrow samples to distinguish normal plasma cells, abnormal plasma cells, and cellular debris. The sensitivity of the novel method was evaluated by diluting myeloma bone marrow cells in pooled CSF samples to simulate low cell counts. Of the 125 CSF specimens, 16 samples from 4 patients showed abnormal plasma cells using both conventional and multidimensional flow cytometry analysis. Discordant results were found in 32 samples (25%), where conventional analysis suggested the presence of abnormal cells, but these were ruled out by multidimensional analysis. Sensitivity testing showed that the multidimensional dot-plot method outperforms conventional two-dimensional dot-plot analysis, as the radar dot plot can be used to identify abnormal cells in samples diluted to 5 WBC/μL, where the cell count of abnormal plasma cells is < 1 cell/μL. Our results showed that the new radar dot-plot analysis can increase the sensitivity and specificity of flow cytometry in MM for the detection of CNS involvement, even in low-cell-count CSF samples, regardless of whether the sample was obtained in a tube containing special reagent or not (TransFix/EDTA CSF Sample Storage tubes). This approach improves diagnostic accuracy, reduces the number of false positive cases caused by antibodies adhering to cell debris, and provides a reliable tool for assessing neurological complications in MM. Further validation is needed in a larger number of cases and testing of the method on different antibody panels.

T-lineage acute lymphoblastic leukemia (T-ALL) is an aggressive neoplasm of immature T cells. Flow cytometry plays a critical role in the diagnosis and management of the disease. It is used to establish the abnormal immature T-cell phenotype and to distinguish the early T-cell precursor (ETP)-ALL from more mature types at diagnosis. The evaluation of mediastinal disease is often complicated by the difficulty of the phenotypic distinction between the normal thymic precursors and the abnormal T lymphoblasts. Follow-up measurements of minimal/measurable residual disease (MRD) are critical for therapy decision-making and prognostication. In the MRD setting, flow cytometry requires a high degree of analytical expertise and assessment of numerous antigens. To address the diagnostic and monitoring challenges, we developed a single-tube 21-antigen assessment with simplified analysis. The assay distinguishes between normal thymic precursors and T lymphoblasts in tissue samples, enables evaluation of ETP versus non-ETP phenotypes, and allows for MRD assessment below 0.01% robust to antigenic changes.

Monitoring subpopulations of CD4⁺ T cells in blood, especially regulatory CD4⁺CD25⁺Foxp3⁺CD127^loT cells, has the potential to identify tolerance to transplants and defects that cause autoimmunity. CD45RA is expressed by naïve/resting CD4⁺, not by activated cells. Staining CD45RA with CD25 or Foxp3 identifies five populations of CD4⁺ T cells, three Treg, and two CD4⁺Foxp3⁻T cells. CD25 is induced on activation of effector cells and is constitutively expressed by Treg. Examining Foxp3⁺ cells in CD4⁺CD25⁺CD127^lo, identified three Treg populations. It is not known how stable these populations of CD4⁺T cells are within individuals and between individuals. Repeated estimations over 3 years in 10 HV showed the proportion of cells in the three Treg populations was stable, whereas the two Foxp3⁻ populations varied. In a larger cohort of 110 samples, females had higher numbers of CD4⁺ cells than males. As a percentage of lymphocytes, there was no sex difference in the proportion of cells in the five populations. With age, there were fewer total Treg, with fewer resting Treg but an increase in activated Treg. Activation of both naïve CD4⁺ T cells and Treg induces expression of chemokine receptors associated with Th1, Th17, and Th2 responses that promote their infiltration into sites of inflammation. Activated Treg expressed CCR4 and, in addition, expressed CXCR3 (Th1), CCR6 (Th17), or neither CXCR3 nor CCR6 (Th2). Some Treg expressed both CCR6 and CXCR3. HLA-DR and CD39 were expressed by activated Treg, and many cells expressed both. There was low PD-1 expression. The stability of the major Treg populations suggested it could be feasible to establish normal ranges for the three Treg populations. Staining for chemokine receptors and Treg effector molecules in activated Treg populations may detect changes in immune homeostasis and tolerance.

The free carcinoma cells (FCC) in peritoneal lavage fluid are an independent adverse prognostic factor in patients with gastric carcinoma. Detection of FCC in the pre-operative peritoneal lavage fluid is critical, as patients with FCC do not have a survival advantage with curative cytoreductive (CCR) surgery. Cytology is currently used to assess FCC, but its sensitivity is poor and there is a need for better sensitive techniques. We attempted to study the efficiency of intra-operative flow cytometry (FCM) in detecting FCC in peritoneal lavage fluid of gastric carcinoma patients. In this prospective study, 32 peritoneal lavage fluids were analyzed intra-operatively by cytology and FCM. The median time taken for sample processing was 47 min. The concordance was achieved in 84% (27/32) of samples. FCM detected FCC in 17 peritoneal lavage fluids, of which only 12 were reported positive by cytology. Five cases that had a FCC burden of less than 0.01% were reported negative by cytology. FCC with programmed death-ligand 1 (PD-L1) expression of greater than 50% was noted in 12 cases. Intra-operative FCM improves the detection of FCC in peritoneal lavage fluid compared to cytology. Due to higher sensitivity, flow cytometry offers a promising adjuvant to cytology and helps in deciding on judicious radical CCR.