Classifying flow cytometry data using Bayesian analysis helps to distinguish ALS patients from healthy controls.

Introduction: Given its wide availability and cost-effectiveness, multidimensional flow cytometry (mFC) became a core method in the field of immunology allowing for the analysis of a broad range of individual cells providing insights into cell subset composition, cellular behavior, and cell-to-cell interactions. Formerly, the analysis of mFC data solely relied on manual gating strategies. With the advent of novel computational approaches, (semi-)automated gating strategies and analysis tools complemented manual approaches. Methods: Using Bayesian network analysis, we developed a mathematical model for the dependencies of different obtained mFC markers. The algorithm creates a Bayesian network that is a HC tree when including raw, ungated mFC data of a randomly selected healthy control cohort (HC). The HC tree is used to classify whether the observed marker distribution (either patients with amyotrophic lateral sclerosis (ALS) or HC) is predicted. The relative number of cells where the probability q is equal to zero is calculated reflecting the similarity in the marker distribution between a randomly chosen mFC file (ALS or HC) and the HC tree. Results: Including peripheral blood mFC data from 68 ALS and 35 HC, the algorithm could correctly identify 64/68 ALS cases. Tuning of parameters revealed that the combination of 7 markers, 200 bins, and 20 patients achieved the highest AUC on a significance level of p < 0.0001. The markers CD4 and CD38 showed the highest zero probability. We successfully validated our approach by including a second, independent ALS and HC cohort (55 ALS and 30 HC). In this case, all ALS were correctly identified and side scatter and CD20 yielded the highest zero probability. Finally, both datasets were analyzed by the commercially available algorithm 'Citrus', which indicated superior ability of Bayesian network analysis when including raw, ungated mFC data. Discussion: Bayesian network analysis might present a novel approach for classifying mFC data, which does not rely on reduction techniques, thus, allowing to retain information on the entire dataset. Future studies will have to assess the performance when discriminating clinically relevant differential diagnoses to evaluate the complementary diagnostic benefit of Bayesian network analysis to the clinical routine workup.

Medical Devices; Hematology and Pathology Devices; Classification of the Flow Cytometric Test System for Hematopoietic Neoplasms. Final order.

The Food and Drug Administration (FDA or we) is classifying the flow cytometric test system for hematopoietic neoplasms into class II (special controls). The special controls that apply to the device type are identified in this order and will be part of the codified language for the flow cytometric test system for hematopoietic neoplasms' classification. We are taking this action because we have determined that classifying the device into class II (special controls) will provide a reasonable assurance of safety and effectiveness of the device. We believe this action will also enhance patients' access to beneficial innovative devices, in part by reducing regulatory burdens.

A classification tree approach for improving the utilization of flow cytometry testing of blood specimens for B-cell non-Hodgkin lymphoproliferative disorders.

We sought to improve the diagnostic efficiency of flow cytometry investigation on blood by developing data-driven ordering guidelines. Our goal was to improve flow cytometry utilization by decreasing negative testing, therefore reducing healthcare costs. We investigated several laboratory tests performed alongside flow cytometry to identify biomarkers useful in excluding non-leukemic bloods. Test results and patient demographic features were subjected to receiver-operator characteristic (ROC) curve, logistic regression and classification tree analyses to find significant predictors and develop decision rules. Our data show that, in the absence of a compelling clinical indication, flow cytometry testing is largely non-informative on bloods from patients less than 50 years of age having an absolute lymphocyte count (ALC) below 5.0 × 10(9)/L. For patients over age 50 having an ALC below this value, a ferritin value above 450 µg/L is counter-indicative of B-cell clonality. Using these guidelines, 26% of cases were correctly predicted as negative with greater than 97% accuracy.

Colourful death: six-parameter classification of cell death by flow cytometry--dead cells tell tales.

The response of the immune system against dying and dead cells strongly depends on the cell death phenotype. Beside other forms of cell death, two clearly distinct populations, early apoptotic and secondary necrotic cells, have been shown to induce anti-inflammation/tolerance and inflammation/immune priming, respectively. Cytofluorometry is a powerful technique to detect morphological and phenotypical changes occurring during cell death. Here, we describe a new technique using AnnexinA5, propidiumiodide, DiIC1(5) and Hoechst 33342 to sub-classify populations of apoptotic and/or necrotic cells. The method allows the fast and reliable identification of several different phases and pathways of cell death by analysing the following cell death associated changes in a single tube: cellular granularity and shrinkage, phosphatidylserine exposure, ion selectivity of the plasma membrane, mitochondrial membrane potential, and DNA content. The clear characterisation of cell death is of major importance for instance in immunization studies, in experimental therapeutic settings, and in the exploration of cell-death associated diseases. It also enables the analysis of immunological properties of distinct populations of dying cells and the pathways involved in this process.

Description of flow cytometry examinations related to human cell differentiation molecules in clinical immunology.

Human cell differentiation molecules, also known as "CD", are cell-surface molecules reacting with different monoclonal antibodies. The fraction of cells belonging to each immunophenotype is measured by flow cytometry. This brief communication proposes the application into clinical immunological laboratories, of the syntax recommended by the International Union of Pure and Applied Chemistry and the International Federation of Clinical Chemistry and Laboratory Medicine to describe biological properties. Following this syntax, all properties related to human cell differentiation molecules examined by flow cytometry, but not limited to, may be described in the same way. In addition, the application of the IUPAC-IFCC recommended syntax in clinical immunology may facilitate unequivocal written or electronic communication between healthcare professionals world-wide.

[Early diagnosis of bladder carcinoma]. / Wczesna diagnostyka raka pecherza moczowego.

Asymptomatic erythrocyturia is an early symptom of urinary tracts and kidney diseases, including bladder carcinoma. The aim of the research was to compare the diagnostic validity of the cytological urine analysis and the DNA flow cytometry in detecting cancer cells in urine and bladder washings, taken from patients with asymptomatic erythrocyturia, as an early symptom of the bladder carcinoma in situ. The research was conducted on a group of 48 patients (32 male, 16 female, aged 28-55) with asymptomatic erythrocyturia, caused, in 16 cases, by bladder carcinoma in situ, in 18 cases, by bladder carcinoma in situ with urinary tracts infection, and in 14 cases, by the infection alone. Flow cytomery showed a higher sensitivity and a higher negative prediction value in detecting cancer cells in bladder washings. Flow cytometry analysis of DNA and phase S is used for detecting early disturbances in the cell cycle which result in aneuploidia, which is impossible to detect in cytological analysis. However peculiarity and positive prediction value were the same (100%) in both methods. On the basis of the research it has been proved that asymptomatic erythrocyturia classifies patients for further, in-depth diagnostic examination for the presence of bladder carcinoma in situ. Furthermore, morning urine and bladder washings analysis, which are non-intrusive tests, are an outstanding diagnostic material for screening for this disease. Detecting aneuploidia with flow cytometry can be an early-detection screening test for bladder carcinoma, while the cytological tests should still be used for confirming the diagnosis.

Primera experiencia en aislamiento de celulas endoteliales humans en Bolivia / First experience in the isolation of human endothelian cells in Bolivia

INTRODUCCIÓN: las células endoteliales pueden proveer información valiosa con respecto a muchos procesos patológicos como la aterosclerosis, inflamación, neoplasia y angiogénesis. Este trabajo describe la primera experiencia boliviana en el aislamiento y cultivo de células endoteliales humanas derivadas de la vena umbilical (HUVEC). MÉTODOS: Un segmento largo de cordón umbilical se utilizó para el aislamiento y fue tratado por digestión con colagenasa. Las células endoteliales fueron desprendidas de la íntima y posteriormente cultivadas en medio de cultivo M199 y suero fetal bovino. Técnicas morfológicas, inmunohistoquímicas y de citometría de flujo fueron utilizadas para identificar estas células. RESULTADOS: Se examinaron las células endoteliales obtenidas por análisis morfológico e inmunohistoquímico. El marcaje con CD34 fue positivo para más del 90% de las células obtenidas por digestión con colagenasa analizado por citometría de flujo. CONCLUSIÓN Se logró aislar y cultivar HUVEC de manera exitosa. Una gran variedad de experimentos y aplicaciones en ciencias biomédicas pueden ser potencialmente factibles.

INTRODUCTION: endothelial cell study yields valuable information concerning many pathologic processes such as atherosclerosis, inflammation, neoplasia and angiogenesis. This paper describes the first Bolivian experience in isolation and culture of human umbilical vein endothelial cells (HUVEC). METHODS: a long segment of the umbilical cord was processed by collagenase digestion. Endothelial cells were detached from intima and further cultured using M199 culture media. Morphologic, immunochemistry and flow cytometry approaches were used to identify these cells. RESULTS: morphologic and immunochemistry analysis of the pool of obtained cells were positive for HUVEC. CD34 staining was positive in more than 90% of the cells obtained by collagenase digestion as assessed by flow cytometry. CONCLUSION: HUVEC were successfully isolated for the first time in Bolivia. A great deal of further experiments and applications in biomedical sciences is possible

High-speed cell sorting: fundamentals and recent advances.

Cell sorters have undergone dramatic technological improvements in recent years. Driven by the increased ability to differentiate between cell types, modern advances have yielded a new generation of cytometers, known as high-speed cell sorters. These instruments are capable of higher throughput than traditional sorters and can distinguish subtler differences between particles by measuring and processing more optical parameters in parallel. These advances have expanded their use to facilitate genomic and proteomic discovery, and as vehicles for many emerging cell-based therapies. High-speed cell sorting is becoming established as an essential research tool across a broad range of scientific fields and is poised to play a pivotal role in the latest therapeutic modalities.

[A historical review of flow cytometers and characteristics of commercial instruments].

Instruments utilized in flow cytometry are called flow cytometers and they can be classified into two kinds, namely, cell analyzers and cell sorters. Important technological developments related to the flow cytometer and various kinds of laboratory instruments are reviewed historically. Commercial cell sorters and cell analyzers produced by several companies are compared and the characteristics of each are listed.