Cytometry Part B: Clinical Cytometry最新文献

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.

Melanoma is an aggressive skin tumor whose incidence is rising sharply, and for which the determination of new prognostic factors is a major challenge. In oncology, circulating tumor cells (CTCs) are at the heart of much research, as they represent a source of tumor material obtained non-invasively by liquid biopsy. With this in mind, this prospective, longitudinal study looked at the detection of CTCs in melanoma patients using the flow cytometry technique, and constitutes a proof-of-principle study, as molecular biology is the most widely used technique today to detect CTCs. The labeling strategy showed high sensitivity and specificity for melanoma cells. All 35 patients in the cohort presented at least one CTC at inclusion, demonstrating that the cells circulate regardless of the stage of the disease. However, a significant increase in the number of CTCs was observed in metastatic stages compared with non-metastatic stages. With regard to the main prognostic factors for melanoma, no significant association was found between the number of CTCs and Breslow thickness or the presence of ulceration. This study must be continued in order to increase the size of the sample, with a more consistent longitudinal follow-up, in order to gain a better understanding of the prognostic significance of CTCs.

Characterization of the tumor immune microenvironment (TIME) of pituitary neuroendocrine tumors (PitNETs) is crucial for understanding the behavior of different types of PitNETs and identification of possible causes of their aggressiveness, rapid growth, and resistance to therapy. High-dimensional flow cytometry (FC) is a promising technology for studying TIME but poses unique technical challenges, especially when applied to solid tissues and PitNETs, in particular. This paper evaluates the potential of FC for analyzing TIME in PitNETs by addressing methodological difficulties across all stages of the workflow and proposing solutions. We developed a protocol for preparing single-cell suspensions from PitNET tissues for FC. This involved optimization of enzymatic digestion and comparison of it with mechanical tissue dissociation assessing cell yield, viability, and target antigen expression. We designed four multicolor FC panels to analyze major lymphocyte and myeloid cell subsets including determination of subpopulations of T, B, NK cells and their activation and cytotoxic potential, neutrophils, monocytes, CD68 + CD64 + CD11b^low macrophages of M2 and M1 subtypes, and two types of myeloid suppressor cells - PMN-MDSC and M-MDSC. Principles of multicolor panel design, spreading error, and importance of voltage balance for proper flow cytometer setting are discussed. The panels were validated and demonstrated the feasibility of their simultaneous use on pituitary tumor surgical tissue for comprehensive TIME characterization. We compared lymphocyte frequencies in blood, PitNETs, and three sequential PitNET eluates to find out the contamination level of PitNET samples with blood leukocytes. To address technical challenges, we propose a strategy of logical data gating that removes spurious signals from aggregates, dead cells, and subcellular debris that can interfere with analysis. Our results indicate that despite all technical difficulties, multiparametric FC can effectively characterize different types of PitNETs. This enhanced understanding of the immune infiltrate provides valuable insights into PitNET biology and advances clinical diagnostics.