Comparison of methods for in-house screening of HLA*B57:01 to prevent abacavir hypersensitivity in HIV-1 care

Abacavir is a nucleoside reverse transcriptase inhibitor used as part of combination antiretroviral therapy in HIV-1-infected patients. Because this drug can cause a hypersensitivity reaction that is correlated with the presence of the HLA-B*57:01 allotype, screening for the presence of HLA-B*57:01 is recommended before abacavir initiation. Different genetic assays have been developed for HLA-B*57:01 screening, each with specific sensitivity, turnaround time and assay costs. Here, a new real-time PCR (qPCR) based analysis is described and compared to sequence specific primer PCR with capillary electrophoresis (SSP PCR CE) on 149 patient-derived samples, using sequence specific oligonucleotide hybridization combined with high resolution SSP PCR as gold standard. In addition to these PCR based methods, a complementary approach was developed using flow cytometry with an HLA-B17 specific monoclonal antibody as a pre-screening assay to diminish the number of samples for genetic testing. All three assays had a maximum sensitivity of >99. However, differences in specificity were recorded, i.e. 84.3%, 97.2% and >99% for flow cytometry, qPCR and SSP PCR CE respectively. Our data indicate that the most specific and sensitive of the compared methods is the SSP PCR CE. Flow cytometry pre-screening can substantially decrease the number of genetic tests for HLA-B*57:01 typing in a clinical setting

Quantification of gliadin levels to the picogram level by flow cytometry

Celiac disease is a widely prevalent enteropathy caused by intolerance to gliadin, one of the gluten proteins. We developed two methods for the analysis of gliadin levels. Both methods use flow cytometry and rat antibodies against a 16-residue peptide of gliadin. The peptide is common to the alpha-, beta-, gamma-, and omega-gliadins

Comparison of Neural Stem Cells Neurogenesis by Using Flow Cytometry versus Manual Counting Method

Finding factors that can increase neurogenesis are of great importance. To find these factors, it seems that neural stem cells culture is an ideal method. To analyze the effect of different factors in a limited period of time and with the least cost, finding an easier and more efficient method than normal manual counting method is needed. The aim of this study was using flow cytometry as an alternative method to evaluate neural stem cells neurogenesis. Neural stem cells from E14 mouse brain have been differentiated in a one- step and a two -step methods. After performing immunohistochemistry for neuronal and astrocytic markers, manual and flow cytometry methods have been compared in determining the percentage of neurons and astrocytes. Then, the percentage of neurons and astrocytes generated in two different differentiation methods has been compared using flow cytometry. Our findings showed that there wasn't any statistical difference between manual and flow cytometry methods in determining the percentage of neurons and astrocytes. Comparing differentiation methods by flow cytometry, showed that the percentage of both neurons and astrocytes were significantly different in theses two methods (p<0.001). Flow cytometry is a simple and reliable method that can replace manual counting method to evaluate neurogenesis of the neural stem cells. This method would be very useful especially when a high content screening of different factors and compounds is needed

Data Quality Assessment of Ungated Flow Cytometry Data in High

Background: The recent development of semi-automated techniques for staining and analyzing flow cytometry samples has presented new challenges. Quality control and quality assessment are critical when developing new high throughput technologies and their associated information services. Our experience suggests that significant bottlenecks remain in the development of high throughput flow cytometry methods for data analysis and display. Especially, data quality control and quality assessment are crucial steps in processing and analyzing high throughput flow cytometry data. Methods: We propose a variety of graphical exploratory data analytic tools for exploring ungated flow cytometry data. We have implemented a number of specialized functions and methods in the Bioconductor package rflowcyt. We demonstrate the use of these approaches by investigating two independent sets of high throughput flow cytometry data. Results: We found that graphical representations can reveal substantial non-biological differences in samples. Empirical Cumulative Distribution Function and summary scatterplots were especially useful in the rapid identification of problems not identified by manual review. Conclusions: Graphical exploratory data analytic tools are quick and useful means of assessing data quality. We propose that the described visualizations should be used as quality assessment tools and where possible, be used for quality control

Flow cytometry is a promising and rapid method for differentiating between freely suspended Escherichia coli and E. coli attached to clay particles

Aim: A standard procedure does not exist to distinguish between attached and unattached micro-organisms. In this study, we compared two methods to quantify between Escherichia coli attached to clay particles and E. coli freely suspended in solution: flow cytometry (attachment assay and viability assay) and settling (or centrifugation followed by settling). Methods and Results: Methods were tested using three environmental strains collected from swine facilities (A, B and C) and one purchased modified pathogenic strain (ATCC 43888); four clay particles: Hectorite, Kaolinite, Ca-Montmorillonite, Montmorillonite K-10; and a range of surface area ratios (particle surface area to E. coli surface area). When comparing the two methods, the per cent attached obtained from the flow cytometry was lower, but not significantly different from the per cent attached obtained from the settling method for all conditions except when the particle was Hectorite or Montmorillonite K-10; when the strain was C; and when the surface area ratio was below 100. Differences between the methods are likely because traditional culture-based methods cannot detect the viable but nonculturable (VBNC) population, whereas flow cytometry can detect the fraction of VBNC with intact membranes. Conclusion: Our results indicate that flow cytometry is a rapid and culture-independent method for differentiating between attached and unattached micro-organisms. Significance and Impact of the Study: Flow cytometry is useful for laboratory-based studies of micro-organism–particle interactions

Using Flow Cytometry to Analyze Cryptococcus Infection of Macrophages.

Flow cytometry is a powerful analytical technique, which is increasingly being used to study the interaction between host cells and intracellular pathogens. Flow cytometry is capable of measuring a greater number of infected cells within a sample compared to alternative techniques such as fluorescence microscopy. This means that robust quantification of rare events during infection is possible. Our lab and others have developed flow cytometry methods to study interactions between host cells and intracellular pathogens, such as Cryptococcus neoformans, to quantify phagocytosis, intracellular replication, and non-lytic expulsion or "vomocytosis" from the phagosome. Herein we describe these methods and how they can be applied to the study of C. neoformans as well as other similar intracellular pathogens

Using Flow Cytometry to Analyze Cryptococcus Infection of Macrophages.

Flow cytometry is a powerful analytical technique, which is increasingly being used to study the interaction between host cells and intracellular pathogens. Flow cytometry is capable of measuring a greater number of infected cells within a sample compared to alternative techniques such as fluorescence microscopy. This means that robust quantification of rare events during infection is possible. Our lab and others have developed flow cytometry methods to study interactions between host cells and intracellular pathogens, such as Cryptococcus neoformans, to quantify phagocytosis, intracellular replication, and non-lytic expulsion or "vomocytosis" from the phagosome. Herein we describe these methods and how they can be applied to the study of C. neoformans as well as other similar intracellular pathogens

Procoagulant and platelet-derived microvesicle absolute counts determined by flow cytometry correlates with a measurement of their functional capacity

Background: Flow cytometry is the most commonly used technology to measure microvesicles (MVs). Despite reported limitations of this technique, MV levels obtained using conventional flow cytometry have yielded many clinically relevant findings, such as associations with disease severity and ability to predict clinical outcomes. This study aims to determine if MV enumeration by flow cytometry correlates with a measurement of their functional capacity, as this may explain how flow cytometry generates clinically relevant results. Methods: One hundred samples from healthy individuals and patients with obstructive sleep apnoea were analysed by conventional flow cytometry (FACSCalibur) and by three functional MV assays: Zymuphen MP-activity in which data were given as phosphatidylserine equivalent, STA® Phospholipid Procoag Assay expressed as clotting time and Endogenous Thrombin Potential (ETP) reflecting in vitro thrombin generation. Correlations were determined by Spearman correlation. Results: Absolute counts of lactadherin+ procoagulant MVs generated by flow cytometry weakly correlated with the results obtained from the Zymuphen MP-activity (r=0.5370, p<0.0001); correlated with ETP (r=0.7444, p<0.0001); negatively correlated with STA® Phospholipid Procoag Assay clotting time (−0.7872, p<0.0001), reflecting a positive correlation between clotting activity and flow cytometry. Levels of Annexin V+ procoagulant and platelet-derived MVs were also associated with functional assays. Absolute counts of MVs derived from other cell types were not correlated with the functional results. Conclusions: Quantitative results of procoagulant and platelet-derived MVs from conventional flow cytometry are associated with the functional capability of the MVs, as defined by three functional MV assays. Flow cytometry is a valuable technique for the quantification of MVs from different cellular origins; however, a combination of several analytical techniques may give the most comprehensive information on the role of MVs in health and disease

Absolute bacterial cell enumeration using flow cytometry

Aim: To evaluate a flow cytometry protocol that uses reference beads for the enumeration of live and dead bacteria present in a mixture. Methods and Results: Mixtures of live and dead Escherichia coli with live:dead concentration ratios varying from 0 to 100% were prepared. These samples were stained using SYTO 9 and propidium iodide and 6 {\mu}m reference beads were added. Bacteria present in live samples were enumerated by agar plate counting. Bacteria present in dead samples were enumerated by agar plate counting before treatment with isopropanol. There is a linear relationship between the presented flow cytometry method and agar plate counts for live (R2 = 0.99) and dead E. coli (R2 = 0.93) concentrations of ca. 104 to 108 bacteria ml-1 within mixtures of live and dead bacteria. Conclusions: Reliable enumeration of live E. coli within a mixture of both live and dead was possible for concentration ratios of above 2.5% live and for the enumeration of dead E. coli the lower limit was ca. 20% dead. Significance and Impact of the Study: The ability to obtain absolute cell concentrations is only available for selected flow cytometers, this study describes a method for accurate enumeration that is applicable to basic flow cytometers without specialised counting features. By demonstrating the application of the method to count E. coli, we raised points of consideration for using this FCM counting method and aim to lay the foundation for future work that uses similar methods for different bacterial strains.Comment: 31 pages, 14 figure