Standard Flow Cytometry Methods Research Articles

Quality Assessment of Cryopreserved Peripheral Blood Stem Cell Products: Evaluation of Two Methods for Flow Cytometric Viability Testing

ABSTRACTIntroductionThe standard flow cytometry method for viability testing using 7‐aminoactinomycin D (7‐AAD) determines cells in necrosis and late apoptosis. The colony‐forming unit (CFU) assay, which evaluates the proliferation ability of HSCs, is also used in graft quality assessment despite known deficiencies that make this assay impractical in routine clinical settings. The aim was to compare the effectiveness of the flow cytometry 7‐AAD/annexin V method with the 7‐AAD method in assessing the quality of HSCs in autologous and allogeneic peripheral blood stem cell (PBSC) products.MethodsThirty autologous and 30 allogeneic fresh and thawed cryopreserved PBSC products were included in this study. The viability of HSCs was determined using the 7‐AAD method and 7‐AAD/annexin V method on a flow cytometer, while their clonogenic capacity was assessed by CFU assay.ResultsThere was an excellent correlation for CD34+ cell viability between the 7‐AAD and the 7‐AAD/annexin V method for fresh samples (Rs = 0.930, p < 0.001) and a good correlation for thawed PBSC samples (Rs = 0.739, p < 0.001). Excellent correlation was observed for post‐thaw CD34+ cell recovery between the two methods for viability (Rs = 0.980, p < 0.001). Statistical analysis showed a weak correlation between CFU‐GM recovery and CD34+ cell recovery, regardless of which viability testing method was used (7‐AAD method p = 0.021, Rs = 0.298; 7‐AAD/annexin V method p = 0.029, Rs = 0.282).ConclusionsResults of this study showed that in the quality assessment of cryopreserved PBSC product viability, the 7‐AAD/annexin V method had no added value compared to the 7‐AAD method, which was suitable enough for routine quality control of cryopreserved autologous and allogeneic PBSC samples.

Pluripotency retention and exogenous mRNA introduction in planarian stem cells in culture

Planarians possess naturally occurring pluripotent adult somatic stem cells (neoblasts) required for homeostasis and whole-body regeneration. However, no reliable neoblast culture methods are currently available, hindering mechanistic studies of pluripotency and the development of transgenic tools. We report robust methods for neoblast culture and delivery of exogenous mRNAs. We identify optimal culture media for the short-term maintenance of neoblasts invitro and show via transplantation that cultured stem cells retain pluripotency for two days. We developed a procedure that significantly improves neoblast yield and purity by modifying standard flow cytometry methods. These methods enable the introduction and expression of exogenous mRNAs in neoblasts, overcoming a key hurdle impeding the application of transgenics in planarians. The advances in cell culture reported here create new opportunities for mechanistic studies of planarian adult stem cell pluripotency, and provide a systematic framework to develop cell culture techniques in other emerging research organisms.

High-throughput enrichment and isolation of megakaryocyte progenitor cells from the mouse bone marrow

Megakaryocytes are a rare population of cells that develop in the bone marrow and function to produce platelets that circulate throughout the body and form clots to stop or prevent bleeding. A major challenge in studying megakaryocyte development, and the diseases that arise from their dysfunction, is the identification, classification, and enrichment of megakaryocyte progenitor cells that are produced during hematopoiesis. Here, we present a high throughput strategy for identifying and isolating megakaryocytes and their progenitor cells from a heterogeneous population of bone marrow samples. Specifically, we couple thrombopoietin (TPO) induction, image flow cytometry, and principal component analysis (PCA) to identify and enrich for megakaryocyte progenitor cells that are capable of self-renewal and directly differentiating into mature megakaryocytes. This enrichment strategy distinguishes megakaryocyte progenitors from other lineage-committed cells in a high throughput manner. Furthermore, by using image flow cytometry with PCA, we have identified a combination of markers and characteristics that can be used to isolate megakaryocyte progenitor cells using standard flow cytometry methods. Altogether, these techniques enable the high throughput enrichment and isolation of cells in the megakaryocyte lineage and have the potential to enable rapid disease identification and diagnoses ahead of severe disease progression.

Toll like receptors (TLRs) in response to human gut microbiota of Indian obese and lean individuals.

Background:The rising incidence of obesity is one of the most serious public health issues in the developed as well as in developing countries like India. Obesity and overweight are most important risk factors for many chronic diseases, including cardiovascular diseases, diabetes and cancer. In this study the body mass index (BMI) cut off was taken as 18.5-22.9 kg/m2 for normal, 23.0-24.9 kg/m2 for Overweight and >25 kg/m2 for obese as per WHO recommendation for Asian Indians, which is different for developed and developing countries. Role of gut microbiota mediated immune response in the development of obesity has been studied but the literature on Indian population are lacking. Therefore, a study was conducted to determine Toll like receptors (TLRs) in response to human gut microbiota of Indian obese and lean individuals using viable colonocytes in a Non invasive technique and Flowcytometry.Methods:A total of 20 healthy volunteer (10 obese and 10 lean) were enrolled in the study as per inclusion and exclusion criteria. Viable colonocytes were isolated from fecal samples using a Non invasive technique (SCSR Method). Toll like receptors (TLRs) and immunoglobulin (IgA &IgG) receptor concentration were measured by standard Flowcytometry methods using specific fluorochrome conjugated antibodies.Results:Average TLR2 receptor concentration was significantly higher in obese (6.35 %) as compared to lean (2.9 %) (P = 0.01). TLR4 receptor concentration was 1.4 % in obese and 1.65 % in lean although the difference was not statistically significant (P = 0.59). IgA & IgG receptor concentration was 49.6 % & 11.2 % in the obese and 67.15 % & 8.05 % in the lean respectively but the differences among both the group were not statistically significant.Conclusion:The results of the present study will be helpful for physicians and researchers to find some biomarkers which can determine predisposition of the obesity in Indian population and helps to use alternative therapeutics such as probiotics to maintain gut homeostasis and immune modulation to prevent obesity.

Performance evaluation of BD FACSPresto™ point of care CD4 analyzer to enumerate CD4 counts for monitoring HIV infected individuals in Nigeria.

BackgroundDespite the upsurge in support and intervention of donor agencies in HIV care and treatment programing in Sub-Sahara African, antiretroviral (ART) programs are still confronted with access and coverage challenges which influence enrolment of new patients. This study investigated the validity of point of care BD FACSPresto™ CD4 analyzer for CD4+ cell count, overall agreement, correlation, sensitivity, and specificity in comparison to a reference standard flow cytometry method. We also assessed the feasibility of use among non-laboratorians.MethodsBlood samples from 300 HIV infected individuals were analyzed for CD4+ T cell and CD4%, using finger prick capillary sample from 150 PMTCT clients and 150 ART clients at Murtala Mohammed Specialist Hospital, Kano, Nigeria. Their venous samples were compared on a flow cytometry reference method using BD FACSCount CD4+ count system. The accuracy of the BD FACSPresto machine in comparison to BD FACSCount was evaluated. Statistical analysis was carried out using STATA (version 12). Bland-Altman method and correlation analysis were used to analyze agreement between both measurements. In addition, sensitivity and specificity of both measurements were determined. Statistical significance was set at p-value <0.05.ResultsThe mean bias and limit of agreement for CD4+ count between BD FACSPresto and BD FACS count machine were 7.49 (95% CI: 2.44 to 12.54) and -8.14 to 96.39 respectively. Further analysis revealed close agreement between BD FACSPresto and BD FACSCount with no significant difference between the two methods (p = .0.95). Using a threshold of 500 cells/μL, sensitivity and specificity of BD FACSPresto were 95.1% and 97.1% respectively, compared to BD FACSCount. There was no statistically significant difference in the misclassification between BD FACSPresto and BD FACSCount results (p = 0.23). Furthermore, sensitivity and specificity were similar when BD FACSPresto machine was operated by a nurse or laboratory scientist, there was no substantial difference in testing variability observed between laboratory and non-laboratory operators using the BD FACSPresto analyzer.ConclusionsOverall, BD FACSPresto Point of Care CD4+ count finger stick capillary blood results is a reliable method in comparison to venous sample cytometry method and no significant difference variability observed between laboratory personnel and non-laboratory operators. The BD FACSPresto is simple, more robust and easy to use equipment without significant variability in reliability by non-laboratory health care workers hence will be a valuable instrument in increasing access and coverage of CD4 estimations in developing countries. The introduction of the BD FACSPresto POC analyzer has a high potential in reducing patients waiting time and improving the overall quality of ART service and clients’ satisfaction especially in rural settings.

Monitoring drug induced apoptosis and treatment sensitivity in non-small cell lung carcinoma using dielectrophoresis

Non-invasive real time methods for characterizing biomolecular events that contribute towards apoptotic kinetics would be of significant importance in the field of cancer biology. Effective drug-induced apoptosis is an important factor for establishing the relationship between cancer genetics and treatment sensitivity. The objective of this study was to develop a non-invasive technique to characterize cancer cells that are undergoing drug-induced apoptosis. We used dielectrophoresis to determine apoptotic cells as early as 2h post drug treatment as compared to 24h with standard flow cytometry method using non-small cell lung cancer (NSCLC) adenocarcinoma cell line (HCC1833) as a study model. Our studies have shown significant differences in apoptotic cells by chromatin condensation, formation of apoptotic bodies and exposure of phosphatidylserine (PS) on the extracellular surface when the cells where treated with a potent Bcl-2 family inhibitor drug (ABT-263). Time lapse dielectrophoretic studies were performed over 24h period after exposure to ABT-263 at clinically relevant concentrations. The dielectrophoretic studies were compared to Annexin-V FITC flow assay for the detection of PS in mid-stage apoptosis using flow cytometry. As a result of physical and biochemical changes, inherent dielectric properties of cells undergoing varying stages of apoptosis showed amplified changes in their cytoplasmic and membrane capacitance. In addition, zeta potential of these fixed isolated cells was measured to obtain direct correlation to biomolecular events.

Analysis of Nucleocytoplasmic Protein Shuttling by Imaging Flow Cytometry.

Many intracellular signal transduction events involve the reversible shuttling of proteins between the cytoplasm and the nucleus. Study of these processes requires imaging information on the protein localization in a given cell and a large number of measurements to obtain sufficient statistics on the protein localization in the whole population. The protocol describes method for quantitative imaging flow cytometry analysis of intracellular distribution of NF-kappaB in ARPE-19 cells stained with specific fluorochrome-conjugated antibodies. The described technique alone or in combination with standard flow cytometry methods can be applied to study any protein undergoing translocation from cytoplasm into the nucleus in a variety of cell lines as well as in heterogeneous primary cell populations.

Genome size variation in the Fagaceae and its implications for trees

Polyploidization is a major source of diversification among plants, particularly during cladogenesis, but most evidence involves herbaceous temperate species. The prevalence of polyploidy among woody taxa is largely unknown, especially among tropical groups. In this study, we examined genome size variation globally and at several taxonomic levels within the Fagaceae. This family has diversified in the northern temperate zone (Quercus) and at least twice in the Asian tropics (Lithocarpus and Castanopsis), allowing us to examine genomic size evolution across a broad latitudinal range. We compared nuclear DNA contents from 78 species in six genera, including new measurements for 171 individuals from 47 Chinese species using standard flow cytometry methods. No evidence suggests that polyploidization or whole genome duplication has occurred in the family. Genome size varied among genera, but limited variation was present in each genus and species. In general, tropical species had larger genomes than temperate species, but the ancestral state cannot be determined given current evidence. Partial duplication does seem to occur among species as within genus variation was larger than within species variation. A review of the literature suggests that genome size and even chromosome structure is highly conserved among woody plants and trees. We propose that ploidy level and genome size are conserved among trees because they participate in diverse syngameons. This behavior would provide similar benefits to polyploidization but avoid exclusion from the syngameon. This conservatism in genome size and structure should enhance ongoing whole genome studies.

Particle Focusing in Staged Inertial Microfluidic Devices for Flow Cytometry

Microfluidic inertial focusing has been demonstrated to be an effective method for passively positioning microparticles and cells without the assistance of sheath fluid. Because inertial focusing produces well-defined lateral equilibrium particle positions in addition to highly regulated interparticle spacing, its value in flow cytometry has been suggested. Particle focusing occurs in straight channels and can be manipulated through cross sectional channel geometry by the introduction of curvature. Here, we present a staged channel design consisting of both curved and straight sections that combine to order particles into a single streamline with longitudinal spacing. We have evaluated the performance of these staged inertial focusing channels using standard flow cytometry methods that make use of calibration microspheres. Our analysis has determined the measurement precision and resolution, as a function of flow velocity and particle concentration that is provided by these channels. These devices were found to operate with increasing effectiveness at higher flow rates and particle concentrations, within the examined ranges, which is ideal for high throughput analysis. Further, the prototype flow cytometer equipped with an inertial focusing microchannel matched the resolution provided by a commercial hydrodynamic focusing flow cytometer. Most notably, our analysis indicates that the inertial focusing channels virtually eliminated particle coincidence at the analysis point. These properties suggest a potentially significant role for inertial focusing in the development of inexpensive flow cytometry-based diagnostics and in applications requiring the analysis of high particle concentrations.

One‐tube HLA‐B27/B2708 typing by flow cytometry using two “Anti‐HLA‐B27” monoclonal antibody reagents

Flow cytometry-based methods are widely used to detect the ankylosing spondylitis-associated HLA-B27/B2708 antigens. However, the generally used "HLA-B27" monoclonal antibodies (moabs) cross-react with many HLA specificities, including the common HLA-B7 antigen. Thus, using two "B27" moabs is highly recommended. The assay used two "HLA-B27" reagents, FITC and PE conjugated, respectively and a PE-Cy5 anti-CD3 antibody. Assay verification used 51 reference subjects possessing B*2705, B*2702, and B*2708 and a range of cross-reactive HLA antigens. A total of 1,006 consecutive patients' samples, referred for "HLA-B27 typing", were assayed alongside our standard flow cytometry method. A further 12 low frequency HLA-B*27 specificities were tested. Samples reacting with one "B27" moab only were B*27 allele typed by PCR using sequence-specific primers. All patient B27/B2708 positives (28.3%) were identified by our one-tube method which detected B*2705, B*2702, B*2708, and 8/12 other B*27 specificities. It was unaffected by HLA-B7 and other cross-reactive antigens but required a minor adjustment, a reduction in the volume of one of the "B27" moabs used, to avoid detecting a minority of HLA-B57 subjects. Our one-tube B27/B2708 assay is simple, robust, uses two "B27" moabs for typing precision and security, does not suffer from interference by HLA-B7 or other cross-reactive antigens and has the obvious advantage of using a single tube per typing.

Inhibition of human head and neck squamous cell cancer growth by modulation of heat shock proteins and induction of the mitochondrial apoptotic pathway with withaferin A

e17003 Background: Epithelial cancers, particularly lung cancer and head and neck squamous cell carcinoma (HNSCC), continue to pose formidable challenges in clinical practice. Novel chemotherapeutic agents have been developed, but even those have limited long-term benefits in the treatment of these tumors, illustrating the need to continue to improve systemic therapy for affected patients. The objective of the present study was to investigate the effect of withaferin A (WA), a plant-derived small molecule, on cancer cell growth, heat shock protein expression and induction of apoptosis in human HNSCCs. Methods: MDA1986 and JMAR HNSCC cells were used in all experiments. The effect of WA on cell viability was determined by MTS assay. Apoptosis and mitochondrial membrane potential changes were assessed by annexin V/propidium iodide and JC-1 staining respectively using standard flow cytometry methods. Effect of WA on modulation of heat shock proteins was determined by Western blot analysis. Results: Withaferin A reduces cell viability in both MDA1986 and JMAR cells with IC50 levels of 265 ± 5 nM by MTS assay, which is 5 fold higher than its reported activity in breast cancer cells. WA completely down-regulates HSP90beta, GRP94, and TRAP-1 expression at 250 nM concentration in HNSCC cells at 24 hours treatment. In addition, WA markedly increased HSP70 levels and mildly increased HSP27 levels in a dose-dependent manner at 24 hours treatment. Flow cytometry with Annexin V/PI staining shows that 5 μM WA treatment for 24 hours induced apoptosis in 63% of MDA1986 and 60% of JMAR cells. WA at 5 μM also reduced mitochondrial membrane potential by JC-1 staining with flow cytometry to less than 10% of controls in both JMAR and MDA1986 cells at 24 hours treatment. Conclusions: Withaferin A is a potent novel inhibitor of HSP90 in human HNSCCs. In addition to HSP modulation, its anticancer mechanistic effects involve apoptotic cell death through the mitochondrial pathway. This molecule shows promise for further in vivo studies to establish preclinical proof of concept as a novel anticancer therapy in this disease. No significant financial relationships to disclose.

A sensitive and rapid alternative to HLA typing as a genetic screening test for abacavir hypersensitivity syndrome

Abacavir hypersensitivity reaction (ABC HSR) is a potentially life-threatening adverse reaction that affects approximately 8% of patients that initiate this antiretroviral drug. Independent groups have shown a strong predictive association between ABC HSR and HLA-B*5701, indicating that exclusion of HLA-B*5701 positive individuals from abacavir treatment would largely prevent ABC HSR. However, the limited availability and relatively high cost of human leukocyte antigen (HLA) typing represent barriers to the widespread implementation of this pharmacogenetic approach to abacavir prescribing. To facilitate routine screening, we have developed a rapid flow cytometry method for HLA-B57 phenotyping using commercially available B17 monoclonal antibodies. Whole blood samples from 84 human immunodeficiency virus (HIV) patients were examined by standard flow cytometry methods, using a two-colour B17-specific immunofluorescence assay in the CD45 lymphocyte population. All eight HLA-B57 individuals examined tested positive, while HLA-B57/58 negative individuals (n=74) tested negative for this flow cytometry test. Two non-HLA-B57 individuals showed weak cross-reactivity. In our predominantly Caucasian population, B17/CD45 dual staining was sufficient to identify individuals carrying B17 cell surface antigens. This approach, utilizing flow cytometry methods that are widely available in HIV laboratories, therefore offers a sensitive, rapid and cost-effective screening assay prior to abacavir prescription. Following risk stratification with this assay, it would be anticipated that identification of HLA-B*5701 using molecular HLA typing methods would be required in <10% of the screened population.

Comparative Assessment of Five Alternative Methods for CD4+ T-Lymphocyte Enumeration for Implementation in Developing Countries

This study compares five alternative methods with regard to the accuracy of CD4+ cell counts measured at the same time by standard flow cytometry (FC). The alternative methodologies studied included: FACSCount; VCS Technology/Coulter Cytospheres; Dynabeads MPC-Q; Capcellia; and Opti-CIM. These methods were performed simultaneously on the blood samples of 40 HIV-infected patients and 20 healthy adult employees collected on 4 separate days. Overall findings revealed that the correlation coefficients between results obtained by FC and each alternative method were similar whatever the range of CD4+ cell count (> or < 500/cu. mm) except for Capcellia. The median differences between absolute numbers of CD4+ T lymphocytes obtained by standard FC and each alternative method were 45 (n = 45) for FACSCount 56 (n = 46) for Dynabeads 195 (n = 55) for Cytospheres 134 (n = 49) for Capcellia and 126 (n = 27) for Opti-CIM. The study showed that FACSCount equipment was lowest compared with the other methods with regard to the median difference in absolute numbers of CD4+ T lymphocytes. Moreover it was also determined that with Dynabeads when compared with the standard FC method an excellent correlation coefficient was found without regard to the CD4+ cell number. In conclusion the FACSCount and Dynabeads methods compared well with standard FC irrespective of CD4+ cell numbers. The Dynabeads method appears to be less expensive hence it has potential in countries with limited economic resources.

Evaluation of Four Alternative Methodologies for Determination of Absolute CD4+ Lymphocyte Counts

Standard methods for determining absolute CD3+CD4+ T-lymphocyte concentrations using combined results from flow cytometry and hematology analyzers are expensive and require fresh (< 18 h old) blood. We evaluated four new "alternative" methods for determining absolute CD4+ T-lymphocyte counts; (a) the FACSCount System (Becton Dickinson Immunocytometry Systems), (b) VCS Technology/Coulter Cyto-Spheres (Coulter Corporation), (c) Zymmune CD4/CD8 Cell Monitoring Kit (Zynaxis, Inc.), and (d) TRAx CD4 Test Kit (T Cell Diagnostics). EDTA-anticoagulated whole-blood specimens from HIV-seropositive patients and anti-HIV-negative adult blood donors and staff were tested by the standard flow cytometry method on fresh blood (0-6 postphlebotomy) and by the four alternative methods at 0 to 6 h and 24 to 30 h postphlebotomy. Representative specimens (< 6 h old) were tested five times with each system to evaluate reproducibility. Correlation coefficients for absolute CD4+ counts between standard flow cytometry and the alternative methods ranged from 0.84 to 0.92 for both fresh and 24- to 30-h-old specimens. Average coefficient of variation for reproducibility ranged from 4.5 to 7.1%. The four alternative CD4+ T-lymphocyte counting methods performed well relative to standard methods. Each alternative method offers advantages over standard flow cytometry with respect to sample throughout, required technical expertise, and cost per result. These methods should facilitate wider availability of low-cost CD4 counts.

A rapid manual method for CD4+ T-cell quantitation for use in developing countries

To evaluate a manual method (Cytosphere) for quantifying CD4+ T-cell numbers. Cross-sectional study of HIV-1-seronegative and HIV-1-seropositive individuals evaluated for absolute CD4 counts by both standardized flow cytometric measurements and manual Cytosphere technology using a hemacytometer. University research hospitals in both the United States and Africa. Blood specimens from 382 patients were evaluated. These were broken down into 294 samples obtained from HIV-1-seropositive patients and 88 samples obtained from HIV-1-seronegative patients. None. Absolute CD4 cell number. Evaluation of samples obtained from HIV-1 patients in both the United States and Africa demonstrated an overall correlation of the Cytosphere assay with flow cytometry of 0.912 (95% confidence interval, 0.895-0.928; P < 0.001). When samples were stratified based on CD4+ T-cell counts determined by flow cytometry, the Cytosphere assay had a 96% predictive value for correctly identifying individuals with CD4 T-cell counts > 200 x 10(6)/l and a 92% predictive value for correctly identifying individuals with CD4 T-cell counts < 200 x 10(6)/l. This assay appears to have the potential for the quantitation of CD4 cells in the limited laboratory facilities in developing countries and to have a strong correlation with standard flow cytometric technology.