Light Scatter Properties Research Articles

Synthetic abnormal mast cell particles successfully mimic neoplastic mast cells by flow cytometry.

Clinical flow cytometry laboratories require quality control materials for assay development, validation, and performance monitoring, including new reagent lot qualification. However, finding suitable controls for populations with uncommonly expressed antigens or for rare populations, such as mast cells, can be difficult. To that end, we evaluated synthetic abnormal mast cell particles (SAMCP), developed together with, and manufactured by, Slingshot Biosciences. The SAMCP's were designed to phenotypically mimic abnormal neoplastic mast cells: they were customized to have the same light scatter and autofluorescence properties of mast cells, along with surface antigen levels of CD45, CD33, CD117, CD2, CD25, and CD30 consistent with that seen in mast cell disease. We evaluated several performance characteristics of these particles using ARUP's high sensitivity clinical mast cell assay, including limit of detection, off-target activity and FMO controls, precision, scatter properties of the particles utilizing several different cytometer platforms, and particle antigen stability. The phenotype of the SAMCP mimicked abnormal mast cells, and they could be distinguished from normal native mast cells. FMO controls demonstrated specificity of each of the markers, and no off-target binding was detected. The limit of detection of the particles spiked into normal bone marrow was found to be ≤0.003% in a limiting dilution assay. The mast cell particles were found to perform similarly on Becton Dickinson Lyric, Cytek Aurora, and Beckman Coulter Navios and CytoFLEX platforms. Within run and between run precision were less than 10% CV. SAMCP were stable up to 13 days with minimal loss of antigen fluorescence intensity. The SAMCP's were able to successfully mimic neoplastic mast cells based on the results of our high sensitivity mast cell flow cytometry panel. These synthetic cell particles represent an exciting and innovative technology, which can fulfill vital needs in clinical flow cytometry such as serving as standardized control materials for assay development and performance monitoring.

Metabolic exhaustion and casein kinase 1α drive deguelin-induced premature red blood cell death

1. Deguelin (DGN), a retinoid isolated from many plants, exhibits a potent anticancer activity against a wide spectrum of tumour cells. There is a dearth of evidence, however, regarding the toxicity of DGN to red blood cells (RBCs). This is relevant given the prevalent chemotherapy-associated anaemia observed in cancer patients. 2. RBCs were exposed to 1-100 μM of DGN for 24 h at 37 °C. Haemolysis and related markers were photometrically measured while flow cytometry was employed to detect phosphatidylserine exposure through Annexin-V-FITC binding and light scatter properties. Additionally, cytosolic Ca2+ and reactive oxygen species were quantified using Fluo4/AM and H2DCFDA, respectively. DGN was also tested against specific signalling inhibitors in addition to vitamin C and ATP. 3. DGN caused a significant increase in Annexin-V-positive cells which was accompanied by cell shrinkage without Ca2+ elevation or oxidative stress. DGN also elicited dose-responsive haemolysis which was ameliorated by preventing KCl efflux and in the presence of sucrose, D4476, and ATP. In whole blood, DGN significantly reduced the reticulocyte count and increased platelet distribution width and large cell count. 4. DGN triggers premature RBC eryptosis and haemolysis through casein kinase 1α and ATP depletion, and exhibits a specific toxicity towards reticulocytes and platelets.

Does the Inclusion of CD34+ B-cell Progenitors (Hematogones) in Stem Cell Enumeration of Apheresis Product Using ISHAGE Protocol Affect the Final Harvest dose Adequacy and the Outcome of Transplantation? A Single Institution Experience from Southern India.

Hematogones have similar antigenic and light scatter properties when compared to CD34+ hematopoietic stem cells (HSC) but they form a separate cluster with dimmer CD45 expression. These should be excluded while enumerating HSC, as their inclusion can overestimate and hence affect the final dose of HSC. However, their exact impact on the outcome of HSC transplant (HSCT) is not entirely known and hence this study was undertaken to address these issues, if any. This was a retrospective study in which patients undergoing HSCT were included, and flow cytometric enumeration was done on the apheresis product using single platform ISHAGE protocol. The gating of all plots was reviewed and carefully studied for hematogone population which would have otherwise been included in the original gating. Totally 78 patients underwent HSCT during the study period. On re-analysis, it was found that 10/78 (12.8%) cases had a separate hematogone population which was included in the HSC in the original analysis. Out of these 10 cases, 7/51 and 3/27 were in autologous and allogenic subgroup respectively. However, all the ten cases ultimately had adequate final stem cell dose and had successful engraftment. The inclusion of hematogones in CD34+ HSC enumeration of apheresis products did not yield any impact on neither the final dose nor the outcome of transplant in this study. However, it is recommended to exclude them from the final count when they are > 10% of the final HSC lest it overestimate the final harvest dose and outcome of HSCT.

Flow Cytometric Analysis and Sorting of Plant Chromosomes.

Flow cytometry offers a unique way of analyzing and manipulating plant chromosomes. During a rapid movement in a liquid stream, large populations can be classified in a short time according to their fluorescence and light scatter properties. Chromosomes whose optical properties differ from other chromosomes in a karyotype can be purified by flow sorting and used in a range of applications in cytogenetics, molecular biology, genomics, and proteomics. As the samples for flow cytometry must be liquid suspensions of single particles, intact chromosomes must be released from mitotic cells. This protocol describes a procedure for preparation of suspensions of mitotic metaphase chromosomes from meristem root tips and their flow cytometric analysis and sorting for various downstream applications.

Study of DNA ploidy in newly diagnosed multiple myeloma by flow cytometry and its correlation with disease prognosis and outcome

Objectives: Multiparameter flow cytometry is increasingly being used in determining DNA content in several hematological malignancies where abnormal DNA ploidy is a useful prognostic marker. This study was done to analyze the DNA ploidy of plasma cells in multiple myeloma (MM) by flow cytometry and explore its role as a prognostic factor. Material and Methods: Propidium-iodide staining technique and gating based on light scatter properties were used. DNA index (DI) of the tumor sample was calculated as the ratio of the DNA content of the G0-G1 population of the myeloma cells with the normal control cells (lymphocytes) present in the same sample. Based on DI, ploidy was categorized as diploidy, hypodiploidy, and hyperdiploidy and the results were correlated with the clinical outcome. Results: Among 32 patients, none had hypodiploid DNA content, 8 (25%) patients had hyperdiploid DNA, and 24 (75%) patients had diploid DNA. There was no significant association between DI and international staging system staging (P = 0.68), Eastern Cooperative Oncology Group Performance Status (P = 0.59), and post-induction remission status (P = 0.10). The median overall survival (OS) in the study patients was 20 (CI 11.4–28.8) months and the median progression-free survival was not reached. There was no difference in the OS among patients with diploid MM and hyperdiploid MM (P = 0.84). Conclusion: Although hyperdiploid MM has been reported to have a better prognosis than diploid MM, we did not find any significant difference possibly due to the small sample size. Nevertheless, flow cytometry is a useful tool in DNA ploidy analysis and its role as a prognostic factor in various hematologic malignancies including MM can be further explored.

Induction of hemolysis and eryptosis by occupational pollutant nickel chloride is mediated through calcium influx and p38 MAP kinase signaling.

Nickel (Ni) is an abundant environmental hazard and an occupational pollutant. Exposure to Ni compounds is prevalent in electroplating workers and in the printing industry, among others. The toxicity of Ni manifests as dermatological, gastrointestinal, respiratory, allergic, and cardiovascular symptoms. In particular, hyperbilirubinemia and reticulocytosis have been detected in intoxicated subjects; an observation possibly implicating selective red blood cell (RBC) toxicity. Herein, the interaction of nickel chloride (NiCl2) with human RBCs and associated molecular mechanisms are described. Cells from healthy donors were incubated for 24 h at 37°C in the presence or absence of 0.5‒10 mM of NiCl2, and cytotoxicity was determined through hemoglobin leakage by colorimetry under different experimental conditions. Eryptotic markers were also identified by flow cytofluorometry using Annexin-V-FITC tagging for phosphatidylserine (PS) exposure, light scatter properties for cellular dimensions, Fluo4/AM labeling for intracellular calcium, and H2DCFDA staining for reactive oxygen species (ROS). Additionally, small molecule inhibitors were used to probe the signaling pathways involved. It was found that NiCl2 at 10 mM caused profound intracellular calcium overload and significant calcium-dependent hemolysis. Also, NiCl2 reduced forward scatter and increased side scatter, Annexin- positive cells, and ROS levels. Importantly, NiCl2-induced hemolysis was significantly attenuated by the exclusion of extracellular calcium, and in the presence of p38 MAP kinase (MAPK) inhibitor SB203580. It is concluded that NiCl2 induces p38 MAPK-dependent hemolysis, and stimulates the canonical features of premature eryptosis. This report presents the first description of the molecular mechanisms underlying the hemolytic and eryptotic potential of NiCl2 and, thus, may explain changes in hematological parameters observed in poisoning victims. Int J Occup Med Environ Health. 2022;35(1):1-11.

Multiparameter flow cytometry in plasma cell disorders: when in doubt, go with the flow.

Multiparameter flow cytometry in plasma cell disorders: when in doubt, go with the flow.

Flow Cytometric Analysis of Microbial Diversity in Patients with Aggressive Lymphoma Disease Undergoing Chemoimmunotherapy

IntroductionMicrobial dysbiosis is associated with increased infectious complications and poorer treatment outcomes in patients with hematologic malignancies (Galloway-Pena et al., 2016, Peled et al., 2020). While the effective reconstitution of microbial diversity by autologous fecal microbiota transfer appears within clinical reach (Malard et al, 2021), diagnosis of dysbiosis typically relies on comparatively slow and laborious molecular techniques. We validated a fast and reliable method to assess and track microbial diversity based on flow cytometric analysis (FCM) of single cell phenotypical traits in stool samples of patients undergoing therapy for aggressive lymphoma disease.MethodsStool samples of 12 patients with B-cell neoplasms and 1 patient with a T-cell neoplasm were collected at the time of diagnosis, directly before each cycle of chemoimmunotherapy administration (CHOP backbone +/- anti-CD20 therapy) and every 3 months during follow-up (2-11 samples per patient totaling n = 77 samples). Microbiome analyses were performed by sequencing the V3/V4 16S rRNA region and by FCM of light scatter properties and DNA content (DAPI staining). Phenotypic and taxonomic alpha diversity (inverse Simpson index, D2) was determined from the flow cytometric profiles and sequencing data, respectively, using the PhenoFlow R package (Props et al., 2016). Longitudinal trends in alpha diversity were evaluated with the SplinectomeR R package (Shields-Cutler et al., 2018).ResultsCorrelation analysis and ordinary least squares regression confirmed a statistically significant association between FCM-based phenotypic and sequencing-based taxonomic alpha diversity in “real-world” patient fecal samples (Pearson's correlation r p = 0.56, r 2 = 0.32, p < 0.001) (Figure 1). Despite the distinct and complex data types generated by the two methods, these results demonstrate that phenotypic diversity measurements obtained from FCM can serve as proxies for taxonomic diversity measurements obtained from 16S rRNA sequencing.Next, we analyzed the dynamics of microbial alpha diversity in our cohort over the course of therapy and follow-up using a spline-based approach optimized for longitudinal data. We demonstrated a statistically significant non-linear trend of alpha diversity with a decrease during the period of chemotherapy administration followed by reconstitution during the immunotherapy and follow-up period (Figure 2) using both FCM and 16S rRNA sequencing (p = 0.001). Notably, the samples collected during the treatment phase were obtained 2-3 weeks after the last administration of chemoimmunotherapy, depicting a progressive and sustained dysbiosis in patients during the months of chemotherapy.ConclusionsWe demonstrated a clear correlation between microbial alpha diversity as determined by 16S rRNA sequencing and by our flow cytometric-based approach. Using FCM analysis, we found a marked decline in diversity during chemotherapy administration. These results are in line with the dysbiosis pattern observed by 16S rRNA sequencing and with previous studies (Rashidi et al., 2019). Our findings thus demonstrate that FCM could provide a fast, first-line point-of-care monitoring of microbial diversity dynamics. [Display omitted] DisclosuresJenq: Merck: Consultancy; Karius: Consultancy; Microbiome DX: Consultancy; Seres: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Kaleido: Membership on an entity's Board of Directors or advisory committees; MaaT Pharma: Membership on an entity's Board of Directors or advisory committees; Prolacta: Membership on an entity's Board of Directors or advisory committees; LISCure: Membership on an entity's Board of Directors or advisory committees. Bullinger: Daiichi Sankyo: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Sanofi: Honoraria; Amgen: Honoraria; Gilead: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Hexal: Consultancy; Novartis: Consultancy, Honoraria; Bayer: Research Funding; Pfizer: Consultancy, Honoraria; Seattle Genetics: Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Menarini: Consultancy; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Astellas: Honoraria. Na: Octapharma: Honoraria, Research Funding; Shire/Takeda: Honoraria, Research Funding; Bristol Myers Squibb: Research Funding.

A Machine Learning Approach to the Classification of Acute Leukemias and Distinction From Nonneoplastic Cytopenias Using Flow Cytometry Data.

Flow cytometry (FC) is critical for the diagnosis and monitoring of hematologic malignancies. Machine learning (ML) methods rapidly classify multidimensional data and should dramatically improve the efficiency of FC data analysis. We aimed to build a model to classify acute leukemias, including acute promyelocytic leukemia (APL), and distinguish them from nonneoplastic cytopenias. We also sought to illustrate a method to identify key FC parameters that contribute to the model's performance. Using data from 531 patients who underwent evaluation for cytopenias and/or acute leukemia, we developed an ML model to rapidly distinguish among APL, acute myeloid leukemia/not APL, acute lymphoblastic leukemia, and nonneoplastic cytopenias. Unsupervised learning using gaussian mixture model and Fisher kernel methods were applied to FC listmode data, followed by supervised support vector machine classification. High accuracy (ACC, 94.2%; area under the curve [AUC], 99.5%) was achieved based on the 37-parameter FC panel. Using only 3 parameters, however, yielded similar performance (ACC, 91.7%; AUC, 98.3%) and highlighted the significant contribution of light scatter properties. Our findings underscore the potential for ML to automatically identify and prioritize FC specimens that have critical results, including APL and other acute leukemias.

Stimulation of sCD14-ST (Presepsin) Secretion By Peripheral Blood Mononuclear Cells after Candida Albicans Lysate and Lipopolysaccharide Exposure

Background:Сluster of differentiation 14 (CD14) is a specific high-affinity receptor for lipopolysaccharides (LPSs) on monocytes, macrophages and granulocytes. Soluble CD14 subtype (sCD14-ST, Presepsin) is a shortened soluble N-terminal fragment of CD14. Levels of sCD14-ST have a strong correlation with severe bacterial infection which can be used as diagnostic tool. However, CD14 recognize not only LPS, but also a variety of ligands associated also with Gram-positive bacteria, fungi, and viruses. Here we report data about impact of C.albicans antigens on monocytes and presepsin level in vitro .Donors and Methods:Peripheral Blood Mononuclear Cells (PBMCs) from 19 healthy volunteers (12 women,7 men, age 20-38, median 28 years) were isolated on media gradient Fiqoll-Histopaque (p=1.077 g/ml) by standard protocol. Fraction of monocytes in PMBC was defined by light scatter properties on flow cytometry (BD FACS Canto II, USA). PBMCs with 0.5 x 106 of monocytes in RPMI-1640 (with 10% of autologous serum) were incubated in 5% CO2, 370C with LPS (GIEM, Moscow, Russia) at the final concentration - 1 µcg/ml (“LPS” tube); with Candida albicans lysates (Sanum-Kehlbeck GmbH & Co, Germany) at the final concentration 200 ng/ml (“ C.albicans ” tube) and control tube with RPMI-1640 (with 10% of autologous serum) and without PMBC (“Serum” tube). Twenty-four hours and 48 hours after stimulation a 150 µL-samples were collected and centrifuged. Presepsin concentration were measured using a chemiluminescent enzyme immunoassay method, performed on PATHFAST analyzer (Mitsubishi Chemical Medience Corporation, Tokyo, Japan). Mann-Whitney U test was used for nonparametric data analysis between two groups. Wilcoxon signed-rank test was used to compare repeated measures. A p-value less than 0.05 was considered as significant.Results:Median (with ranges) of sCD14-ST concentration 24 hours after beginning of stimulation was 59,8 pg/ml (29,7-140), 84 pg/ml (38,8-133) and 34,6 pg/ml (18,5-81,8) for “LPS”, “ C.albicans ” and “Serum” tube respectively. 48 hours after beginning of stimulation sCD14-ST concentration increased to 85,8 pg/ml (45-146), 114 pg/ml (62,3-198) and 40,8 pg/ml (25,3-74,8) also for “LPS”, “ C.albicans ” and “Serum” tube respectively. According to our data (see Figure 1), the level of sCD14-ST in supernatant after 24 and 48 hours in « C.albicans » tube was significantly higher than in «LPS» (p <0.05) and in serum tube (p<0.0001).Conclusion:Here we report data about impact of C.albicans antigens on monocytes and sCD14-ST level in vitro . Obtained data show us that not only bacterial infection can lead to increased level of sCD14-ST (Presepsin) and lead us to preposition that extra-high level of sCD14-ST (Presepsin) can be found in patients with severe fungal infections. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Issue Highlights — May 2021

Issue Highlights — May 2021

Chromosome analysis and sorting.

Flow cytometric analysis and sorting of plant mitotic chromosomes has been mastered by only a few laboratories worldwide. Yet, it has been contributing significantly to progress in plant genetics, including the production of genome assemblies and the cloning of important genes. The dissection of complex genomes by flow sorting into the individual chromosomes that represent small parts of the genome reduces DNA sample complexity and streamlines projects relying on molecular and genomic techniques. Whereas flow cytometric analysis, that is, chromosome classification according to fluorescence and light scatter properties, is an integral part of any chromosome sorting project, it has rarely been used on its own due to lower resolution and sensitivity as compared to other cytogenetic methods. To perform chromosome analysis and sorting, commercially available electrostatic droplet sorters are suitable. However, in order to resolve and purify chromosomes of interest the instrument must offer high resolution of optical signals as well as stability during long runs. The challenge is thus not the instrumentation, but the adequate sample preparation. The sample must be a suspension of intact mitotic metaphase chromosomes and the protocol, which includes the induction of cell cycle synchrony, accumulation of dividing cells at metaphase, and release of undamaged chromosomes, is time consuming and laborious and needs to be performed very carefully. Moreover, in addition to fluorescent staining chromosomal DNA, the protocol may include specific labelling of DNA repeats to facilitate discrimination of particular chromosomes. This review introduces the applications of chromosome sorting in plants, and discusses in detail sample preparation, chromosome analysis and sorting to achieve the highest purity in flow‐sorted fractions, and their suitability for downstream applications.

Non-Acute Myeloid Neoplasms With CD34(-) Blasts: Immunophenotypic And Clinical Analysis Of 5 Cases

Abstract Introduction/Objective Non-acute myeloid neoplasms (NAMNs) comprise clonal hematopoietic disorders, characterized by ≤20% myeloblasts in the peripheral blood (PB) and/or bone marrow (BM). The blasts of NAMN are almost exclusively CD34(+), contrasting with blasts in some acute myeloid leukemias (AML). We detail the clinicopathologic features of NAMN with CD34(-) blasts. Methods We searched 981 NAMN pathology reports for CD34(-) blasts by flow cytometry (FC). 8-color FC data was analyzed using cluster analysis to identify all relevant cell populations with a comprehensive antibody panel. CD34(-) blast populations were defined as reproducible clusters with similar light scatter properties across antibody tubes, after exclusion of other cell populations (e.g. basophils, neutrophils, mast cells). Blast aberrancies were defined as differences from established normal antigen expression patterns. PB and BM morphology was reviewed and chart reviews were performed. Results We identified 5 NAMN patients with CD34(-) blasts (2M, 3F; 57-84y/o) including 3 myelodysplastic syndrome (MDSs) with excess blasts, 1 therapy-related MDS, and 1 chronic myelomonocytic leukemia-1 (CMML). Blasts ranged from 0-1.5% in PB and 2.8-8% in BM. CD34(-) blasts accounted for 0.85-8.2% of BM events by FC, with all showing other aberrancies. The CMML patient had CD34(-) blasts with monocytic differentiation; all other CD34(-) blast populations showed expression of CD13, CD33, and CD117 and aberrant under-expression of CD38. Aberrancies were also present in the CD34(+) blasts in all patients (1-4/patient), accounting for 0.01-0.55% of events; 3/5 pts had under-expression of CD33, CD38, CD45, and/or HLA-DR. No cases had adverse cytogenetics. Patient outcomes included: indolent disease (1); died from disease 3 years post-diagnosis and chemotherapy (1); transformed to AML (2; 1-alive in complete remission; 1-died); and alive following allogeneic stem cell transplant (1). Conclusion Our data confirm that CD34(-) blasts are rarely observed in NAMN, and these patients have variable outcomes. Interestingly, all cases showed a concomitant aberrant CD34(+) blast population.

Abnormal Platelet Phenotypes in Patients with Myelodysplastic Syndromes

Background: Hemorrhage and infection are two main causes of death in patients with myelodysplastic syndromes (MDS), mainly due to thrombocytopenia and neutropenia respectively. However, it is becoming increasingly clear that platelet dysfunction can also affect the process of hemostasis and anti-infection. Several studies have evaluated platelet activation in MDS and found platelet dysfunction, but immune-related function of platelets in patients with MDS has not been investigated yet. The aim of this study was to evaluate activation function and immune-related function of platelets in MDS by testing platelet activation-associated phenotypes and immune-associated phenotypes. Methods: We included 29 MDS patients and 22 healthy subjects, and MDS patients were divided into different subgroups (low-risk group and high-risk group; untreated group and treated group; pre-transfusion group and post-transfusion group) according to IPSS-R score, hypomethylating agents (HMAs) therapy and platelet transfusion history to observe phenotype changes under different factors. Platelet light scatter properties, expression of CD41a, expression of activation-associated phenotypes (CD62p and CD63) and expression of immune-associated phenotypes (CD154 and TLR4) were systematically detected by multi-parameter flow cytometry (MFC). Then correlations between phenotypes and clinical indexes were analyzed. Results: Compared to the healthy controls, decreased expression of platelet-specific antigen CD41a was shown in MDS patients (P&lt;0.05). No difference was found in platelet light scatter properties between MDS patients and healthy subjects (P&gt;0.05). Significantly decreased expression frequency and intensity of activation phenotype CD63 were found in patients with MDS (P&lt;0.05). Low-risk MDS showed lower expression frequency while high-risk MDS showed reduced mean fluorescence intensity (MFI) of CD63. Decreased expression of CD154 and TLR4 was found in MDS patients (P&lt;0.05) which was significantly elevated after HMAs therapy (P&lt;0.05). Particularly, high-risk MDS patients again exhibited reduced MFI of CD154 and TLR4 (P&lt;0.05). Correlation analysis showed that the expression of activation-associated phenotypes was not significantly correlated with the severity of bleeding events, but expression of CD62p was negatively correlated with white blood cell count (WBC) (r=-0.496, P=0.006; r=-0.498, P=0.006), absolute neutrophil count (ANC) (r=-0.512, P=-0.005; r=-0.442, P=0.016), fibrinogen (FIB) (r=-0.415, P=0.025; r=-0.510, P=0.005), and D-dimer (DD) (r=-0.547, P=0.002; r=-0.601, P=0.001), and positively correlated with prothrombin time (PT) (r=0.409, P =0.028; r=0.489, P=0.007). Expression of immune-associated phenotypes was not significantly correlated with the severity of infection events (P &gt; 0.05). Conclusion: MDS patients displayed defective expression of both activation- and immune-associated platelet phenotypes, with differential mechanisms between low-risk and high-risk group regarding phenotype alterations. CD62p showed clinical relevance in regard to WBC count and coagulation indexes, and HMAs treatment significantly improved CD154 and TLR4 expression on platelets. Taken together, the findings confirmed impaired platelet phenotypes in MDS which may assist in the diagnosis and identification of MDS patients. Disclosures No relevant conflicts of interest to declare.

Flow cytometric features of incidental indolent T lymphoblastic proliferations

Indolent T lymphoblastic proliferations have been reported rarely in extramedullary and extrathymic tissues. Recent work has identified these indolent T lymphoblast populations to be mostly CD4+/CD8+ (characterized by immunohistochemistry), with only limited immunophenotypic evaluation of these populations by flow cytometry (FC). We retrospectively reviewed our institutional FC archives and identified 12 samples from 10 patients with incidental T lymphoblastic populations. Samples were characterized with respect to expression of T-cell antigens, CD45, TdT, CD1a, and T/NK antigens, and light scatter properties. Overall, the proportion of T lymphoblasts was small (range 0.01-8.8% of white cells; mean, 1.7%). Histologic correlation showed scattered immature T lymphoblasts in samples without overt distortion of underlying architecture. T lymphoblasts were identified most frequently in association with Castleman disease (four) or tissues with Castleman features (four), marginal zone B-cell lymphoma (one), or tissue with reactive/atypical changes (three cases). Although three cases were composed predominantly of CD4+/CD8+ T cells, the majority of cases in our cohort (eight) included a major subset of CD4-/CD8- T lymphoblasts by FC (one case CD8+/CD4-), which has not been described previously. There was no evidence of subsequent progression to T lymphoblastic leukemia. Incidental, indolent T lymphoblastic proliferations may be detected by clinical FC. In contrast to reports, we find that these proliferations in clinical samples may contain not only CD4+/CD8+ immature T cells but also CD4-/CD8- immature T cells, expanding the immunophenotypic spectrum of this entity.

Educational no. 2: flow cytometry

Flow cytometry (FC) plays an important role in the diagnosis and classification of hematological disorders and can analyze a wide range of diagnostic samples. It is used in both diagnostic and follow-up testing, and is increasingly important in the detection of very small residual disease populations, i. e., minimal residual disease (MRD). Modern flow cytometers have the capability to analyze several thousands of cells per second and to identify different cell populations by evaluating the expression of specific antigens (markers) along with light scatter properties of the cells. FC can precisely differentiate between B‑ and T‑cell malignancies, between mature and precursor tumors, and among the latter, determine the myeloid or lymphatic lineage.

Molecular Characterization of Gastric Epithelial Cells Using Flow Cytometry.

The ability to analyze individual epithelial cells in the gastric mucosa would provide important insight into gastric disease, including chronic gastritis and progression to gastric cancer. However, the successful isolation of viable gastric epithelial cells (parietal cells, neck cells, chief cells, and foveolar cells) from gastric glands has been limited due to difficulties in tissue processing. Furthermore, analysis and interpretation of gastric epithelial cell flow cytometry data has been difficult due to the varying sizes and light scatter properties of the different epithelial cells, high levels of autofluorescence, and poor cell viability. These studies were designed to develop a reliable method for isolating viable single cells from the corpus of stomachs and to optimize analyses examining epithelial cells from healthy and diseased stomach tissue by flow cytometry. We performed a two stage enzymatic digestion in which collagenase released individual gastric glands from the stromal tissue of the corpus, followed by a Dispase II digestion that dispersed these glands into greater than 1 × 106 viable single cells per gastric corpus. Single cell suspensions were comprised of all major cell lineages found in the normal gastric glands. A method describing light scatter, size exclusion, doublet discrimination, viability staining, and fluorescently-conjugated antibodies and lectins was used to analyze individual epithelial cells and immune cells. This technique was capable of identifying parietal cells and revealed that gastric epithelial cells in the chronically inflamed mucosa significantly upregulated major histocompatibility complexes (MHC) I and II but not CD80 or CD86, which are costimulatory molecules involved in T cell activation. These studies describe a method for isolating viable single cells and a detailed description of flow cytometric analysis of cells from healthy and diseased stomachs. These studies begin to identify effects of chronic inflammation on individual gastric epithelial cells, a critical consideration for the study of gastric cancer.

TALEN-mediated gene targeting in porcine spermatogonia.

Spermatogonia represent a diploid germ cell population that includes spermatogonial stem cells. In this report, we describe new methods for isolation of highly enriched porcine spermatogonia based on light scatter properties, and for targeted mutagenesis in porcine spermatogonia using nucleofection and TALENs. We optimized a nucleofection protocol to deliver TALENs specifically targeting the DMD locus in porcine spermatogonia. We also validated specific sorting of porcine spermatogonia based on light scatter properties. We were able to obtain a highly enriched germ cell population with over 90% of cells being UCH-L1 positive undifferentiated spermatogonia. After gene targeting in porcine spermatogonia, indel (insertion or deletion) mutations as a result of non-homologous end joining (NHEJ) were detected in up to 18% of transfected cells. Our report demonstrates for the first time an approach to obtain a live cell population highly enriched in undifferentiated spermatogonia from immature porcine testes, and that gene targeting can be achieved in porcine spermatogonia which will enable germ line modification.

Human group 2 innate lymphoid cells do not express the IL-5 receptor

This work was part funded by (for CEB) Airway Disease Predicting Outcomes through Patient Specific Computational Modelling (AirPROM) project (funded through FP7 EU grant), Wellcome Senior Fellowship, (for DJC) Medical Research Council (MRC) and Asthma UK (Centre Grant: G1000758), from the NIHR Biomedical Research Centre based at Guy's and St Thomas' NHS Foundation Trust and King's College London, an equipment grant from the Midlands Asthma and Allergy Research Association (MAARA) and (for CEB and DJC) the National Institute for Health Research (NIHR) Leicester Respiratory Biomedical Unit. BMJR was funded by an MRC and Asthma UK PhD studentship. This paper presents independent research funded by the NIHR.

High sensitivity detection and sorting of infectious human immunodeficiency virus (HIV-1) particles by flow virometry

Detection of viruses by flow cytometry is complicated by their small size. Here, we characterized the ability of a standard (FACSAria II) and a sub-micron flow cytometer (A50 Micro) to resolve HIV-1 viruses. The A50 was superior at resolving small particles but did not reliably distinguish HIV-1, extracellular vesicles, and laser noise by light scatter properties alone. However, single fluorescent HIV-1 particles could readily be detected by both cytometers. Fluorescent particles were sorted and retained infectivity, permitting further exploration of the functional consequences of HIV-1 heterogeneity. Finally, flow cytometry had a limit of detection of 80 viruses/ml, nearly equal to PCR assays. These studies demonstrate the power of flow cytometry to detect and sort viral particles and provide a critical toolkit to validate methods to label wild-type HIV-1; quantitatively assess integrity and aggregation of viruses and virus-based therapeutics; and efficiently screen drugs inhibiting viral assembly and release.