Cytometry Part B: Clinical Cytometry最新文献

A reduced proportion of peripheral class-switched memory B cells (CSM-B cells) is presumed to indicate ineffective germinal activity. The extent that this finding corresponds to a plausible germinal center failure pathophysiology in patients not diagnosed with CVID or hyper IgM syndrome is not known. We asked if patients with low CSM-B cells are more likely to demonstrate failure to produce serum IgA and IgG than counterparts with nonreduced class-switched memory B cell levels, regardless of diagnosis. Patients with low CSM-B cell levels regardless of diagnosis were retrospectively compared with their counterparts without CSM-B cell reductions for demographics and serum immunoglobulin levels. Patients were further divided based on whether CSM-B cell levels remained low over time or fluctuated, and these groups were compared for serum immunoglobulin levels and diagnoses. Of 305 patients, those with CSM-B cell (n = 50) reductions were more likely to have low serum IgA and IgG than those without reductions. Of the 78 patients in whom CSM-B cells were measured repeatedly over time, 21 patients had low CSM-B cell levels, but this finding was persistent in only 10. Patients with persistent CSM-B cell reductions universally had severe serum IgA and IgG deficiencies and patients with transient CSM-B cell reduction often did not. These two groups contained divergent diagnoses and likely represent separate pathophysiologic groups. Quantifying CSM-B cells as a percentage of CD27+ B cells repeatedly over time is a robust approach to identifying patients with a plausible germinal center failure endotype.

The clinical and immunophenotypic attributes of reactive γδ T-cell expansions are less well characterized than their malignant counterparts, which can pose diagnostic challenges. This study aims to investigate the characteristics and long-term clinical outcomes of reactive γδ T-cell expansions. A retrospective review was performed to identify patients with expanded γδ T-cell population (>15% of T-cells) by flow cytometry in peripheral blood and/or bone marrow specimens over a 17-year period. The cases were divided into reactive and malignant categories and their clinical and immunophenotypic findings were compared. Clinical follow-up was performed. 97 patients were identified including 19 malignant and 78 reactive cases with a variety of underlying conditions. The median absolute γδ T-cell count and median percentage of γδ T-cells per total T-cells were significantly lower in reactive vs. malignant cases (p = 0.0001 and p < 0.00001, respectively). Reactive cases showed more frequent brighter surface CD3 expression (87.1% vs. 42.1%; p < 0.0001), no discrete loss of CD7 (0% vs. 36.9%; p < 0.0001), less frequent lack of CD5 (25.7% vs. 42.4%; p < 0.0001), and no homogeneous CD56 expression (0% vs. 31.6%; p > 0.0001) as compared with malignant cases. Upon long-term follow-up, none of the reactive cases showed clinical evidence of malignant evolution. Reactive expansions of γδ T-cells can be seen in a variety of conditions including hematologic neoplasms, autoimmune and post-transplant states, and infections. Such cases have significantly lower γδ T-cell counts and percentages and no discrete loss of CD7. Lack of CD5 on its own is not an indication of immunophenotypic aberrancy in γδ T-cells. Upon long-term clinical follow-up, such reactive expansions show no evidence of evolution to γδ T-cell malignancies.

BH3 profiling can assess global mitochondrial priming and dependence of leukemic cells on specific BH3 anti-apoptotic proteins such as BCL-2. In acute myeloid leukemia (AML), proof-of-concept prognostic studies have been performed on archived samples variably accounting for molecular genetics. We undertook a single-center feasibility study of a simplified flow-based assay to determine the absolute mitochondrial priming and BCL-2 dependence in consecutive AML patients. When possible, results on the leukemic fraction were normalized to the cognate lymphocyte population (relative priming and BCL-2 dependence). Samples from 97 (89.8%) of the 108 referred patients were successfully processed. Relative priming and BCL-2 dependence could be determined in 62 (67.4%) and 67 (62.0%) samples, respectively. Absolute mitochondrial priming was lower in patients having previously failed intensive chemotherapy compared to chemotherapy-naïve patients (p = 0.01), but its prognostic impact was limited. Conversely, relative BCL-2 independence tended to predict worse EFS (HR = 2.51, p = 0.07) and OS (HR = 2.79, p = 0.10) independently of adverse genetic risk. Our results show that simplified BH3 profiling can be prospectively assessed in AML patients but that its prognostic use may require internal normalization. Future studies should compare its relevance with other functional assays such as ex vivo drug testing or BH3 protein expression.

Flow cytometry is a high-throughput, high-dimensional technique that generates large sets of single-cell data. Prior to analyzing this data, it is common to exclude any events that contain two or more cells, multiplets, to ensure downstream analysis and quantification is of single-cell events, singlets, only. The process of singlet discrimination is critical yet fundamentally subjective and time-consuming; it is performed manually by the user, where the proper exclusion of multiplets depends on the user's expertise and often varies from experiment to experiment. To address this problem, we have developed an algorithm to automatically discriminate singlets from other unwanted events such as multiplets and debris. Using parameters derived from imaging, the algorithm first identifies high-density clusters of events using a density-based clustering algorithm, and then classifies the clusters based on their properties. Multiplets are discarded in the first step, while singlets are distinguished from debris in the second step. The algorithm can use different strategies on imaging feature selection-based user's preferences and imaging features available. In addition, the relative importance of singlets precision vs. sensitivity can be further tweaked via a density coefficient adjustment. Twenty-two datasets from various sites and of various cell types acquired on the BD FACSDiscover™ S8 Cell Sorter with CellView™ Image Technology were used to develop and validate the algorithm across multiple imaging feature sets. A consistent singlets precision >97% with a solid >88% sensitivity has been demonstrated with a LightLoss feature set and the default density coefficient. This work yields a high-precision, high-sensitivity algorithm capable of objective and automated singlet discrimination across multiple cell types using various imaging-derived parameters. A free FlowJo™ Software plugin implementation is available for simple and reproducible singlet discrimination for use at the beginning of any user's workflow.