Validation of a flow cytometry based G(2)M delay cell cycle assay for use in evaluating the pharmacodynamic response to Aurora A inhibition

Pharmacodynamic assays are important aspects for understanding molecularly targeted anticancer agents to investigate the relationship between drug concentration (pharmacokinetics) and drug “effect” or biological activity. As new drug entities are developed that affect DNA cell cycle, a pharmacodynamic assay which measures cell cycle perturbation would be a valuable clinical trial tool. During recent years, flow cytometry has established itself as a useful method to determine the relative nuclear DNA content and percentage of cycling cells of biological specimens. However to date, the analytical validation of cytometry based assays is limited and there is no suitable guidance for method validation of flow cytometry based cell cycle assays. Here we report the validation of a flow cytometry based cell cycle G₂/M delay assay for use in evaluating the effect of investigational drug MLN8237, a small molecule inhibitor of a mitotic kinase Aurora A, for clinical trial use. The assay method was validated by examining assay robustness, repeatability, reproducibility, precision, and determining the cutoff for a true drug effect based on biostatistical analysis models. Experimental results show that the intra-assay repeatability was less than 20% with an intra-donor variability of less than 40%. The robustness of the assay was less than 30%. Since this is an ex-vivo stimulation assay, variability parameters were expected to be higher. Based on biostatistical modeling, an absolute change in %G₂M of 5.2% (95% CI) was needed in order to detect a true drug effect. Overall, the assay demonstrated acceptable variability to warrant further in vivo testing.     

Improved flow cytometry based cytotoxicity and binding assay for clinical antibody HLA crossmatching

The presence of preformed donor-specific HLA antibodies leads to early antibody mediated kidney allograft rejection. Therefore, detection and avoidance of donor reactive HLA antibodies prior to transplantation is of outmost importance in order to minimize the risk of rejection. Detection of pre-formed HLA antibodies is currently performed using complement-dependent cytotoxicity (CDC) assay alone or together with a flow cytometry based crossmatch (FCXM). This study was initiated to further evaluate our recently developed flow cytometry based procedure for determination of both cytotoxicity of and IgG binding to donor-derived lymphocytes by HLA antibodies. Highly enriched immuno-magnetic bead purified T and B lymphocytes were used as target cells for patient sera using 96-well plates. Importantly, the assay shows high sensitivity and specificity as determined by HLA typed donor cells and serum with defined HLA antibody IgG and C1q. Based on this and additional data generated in this paper, such as evaluation of appropriate serum and complements incubation times and assay reproducibility and stability, will enable us to more rapidly implement this assay in our clinical laboratory routines. In addition, we demonstrate that FCtox crossmatching of deceased donor cells has superior specificity compared to conventional CDC assay especially regarding high frequencies of false-positive reactions.     

A flow cytometry based in vitro micronucleus assay in TK6 cells—Validation using early stage pharmaceutical development compounds

The micronucleus test (MNT) is a well established test for detecting clastogenic and aneugenic compounds. Despite the assay's advantages, the MNT may produce false positive and false negative results in some conditions. This fact may be related to the underestimation of apoptosis or necrosis, the p53 status of the cell system or the cytotoxicity assay, and the top dose selection. The purpose of our studies was to contribute to the validation efforts of the flow cytometry based MNT. To identify the most reliable cytotoxicity assay for the top dose selection five parameters for relative survival were tested: relative cell count, relative population doubling, trypan blue supravital staining, relative ratio of scored nuclei to latex beads, and ethidium monoazide staining. For all compounds the least sensitive method was the relative cell count and the most reliable was the nuclei/beads ratio. The comparative evaluation of micronuclei induction in TK6 cells, analyzed with microscopy and flow cytometry, was performed with reference compounds and internal Novartis early development compounds with positive, weak positive, equivocal, and negative genotoxic effects. Our data document a good correlation between the MNT results obtained by flow cytometry and by microscopy. The results confirm that the method may be applied for routine testing in the pharmaceutical industry for the tested group of compounds, including compounds which require metabolic activation. However, further validation and miniaturization may be required. Environ. Mol. Mutagen., 2011. © 2010 Wiley‐Liss,Inc.     

CFSE检测马传染性贫血病毒疫苗免疫马T细胞增殖方法的建立

目的:以流式细胞术(FCM)检测马传染性贫血病毒(EIAV)抗原特异性的T淋巴细胞增殖.方法:将新鲜分离的马外周血单个核细胞(PBMC)进行CFSE 染色, 经纯化病毒体外刺激并培养5 d后, 进行T淋巴细胞亚型标记, 检测特异性增殖的CD4 和CD8 细胞比例.结果:对马PBMC的染色浓度、特异性刺激物的种类及反应时间等条件进行了优化.可对马外周血抗原特异性T淋巴细胞不同亚型的增殖水平进行定量评价 .结论:FCM检测方法为研究马传染性贫血病毒疫苗免疫保护机制提供了一个有效的手段; 该法也可以为其他病毒的免疫学研究提供借鉴.     

Polychromatic flow cytometric high-throughput assay to analyze the innate immune response to Toll-like receptor stimulation

Polychromatic flow cytometry allows the capture of multidimensional data, providing the technical tool to assess complex immune responses. Interrogation of the adaptive T cell response to infection or vaccination already has benefited greatly from standardized protocols for polychromatic flow cytometric analysis. The innate immune system plays an important role in health and disease, and presents potentially important therapeutic and diagnostic modalities. We describe here a high-throughput polychromatic flow cytometry-based platform that enables the rapid interrogation and large scale screening of human blood antigen presenting cell responses to Toll-like receptor (TLR) ligands and other innate immune modulators. Using this assay, we found that for certain stimuli (e.g., TLR9 and TLR3 ligands), the general protocol for intracellular cytokine cytometry had to be significantly modified to allow response detection. Furthermore, high concentrations of TLR7/8 and TLR4 stimuli caused substantial changes in lineage markers, potentially confounding analysis if one were to use a conventional "lineage-negative" cocktail. The assay we developed is reproducible and has been used to show that a given individual's TLR response pattern is relatively stable over at least several months. This protocol is in strict compliance with published guidelines for polychromatic flow cytometry, provides a common platform for scientists to compare their results directly, and may be applicable to the diagnostic evaluation of Toll-like receptor function and the rapid screening of promising therapeutic innate immune modulators.     

Combined use of enzyme-linked immunosorbent assay and flow cytometry to detect antibodies to Trypanosoma cruzi in domestic canines in Texas

Canines may be sentinels and/or reservoirs for human Trypanosoma cruzi exposures. This study adapted a method originally designed for human diagnostics to detect serum immunoglobulin G to T. cruzi in canines. The method combined an enzyme-linked immunosorbent assay (ELISA) for screening and flow cytometry detection of anti-live trypomastigote antibodies (ALTA) for confirmation. The assays were optimized by using known positive and negative control canine sera, and cutoff values were established. The ELISA and ALTA assay easily distinguished between reactive (positive controls) and nonreactive (negative controls) sera and were used to test sera collected in a cross-sectional seroprevalence survey of 356 domestic canines from Harris County, Tex., and the surrounding area. Fifty-three (14.9%) of 356 asymptomatic canines in the survey were positive by ELISA, and 5 (1.4%) were confirmed positive with the ALTA assay, with an additional 4 (1.1%) canines classified as "suspect positive." Thus, the overall prevalence of T. cruzi antibodies in this population was 2.6%. This is the first U.S. study to use the combination of ELISA and ALTA to detect serum antibodies to T. cruzi and the first report of the prevalence of T. cruzi infection in domestic canines in the Houston, Tex. (Harris County), region. Our results demonstrate that the combination of ELISA and ALTA has been successfully adapted for use in testing canines for serological evidence of T. cruzi infection. Seroprevalence survey results suggest that T. cruzi antibody-positive domestic canines in the peridomestic setting are present in the Houston, Tex., region and further suggest that T. cruzi is enzootic in the region.     

The use of flow cytometry to measure neutrophil function

Neutrophils are important professional phagocytic cells that provide the host with a first line of defense against acute bacterial and fungal diseases and recurrent, severe or unusual infections are associated with inherited defects of neutrophil function. Furthermore, abundant evidence links inappropriate neutrophil-mediated tissue damage to the pathogenesis of conditions such as acute respiratory distress syndrome, septicemia with multiorgan failure, ischemia-reperfusion injury and rheumatoid arthritis. Flow cytometry has been increasingly used to evaluate the functional capabilities of neutrophils. In this review, we discuss the use of flow cytometry to assess neutrophil functional responses including calcium mobilization, F-actin assembly, adhesion, aggregation, degranulation, phagocytosis and reactive oxygen species (ROS) production. The use of flow cytometry to identify neutrophil priming is also discussed. The advantage of flow cytometry is that the majority of neutrophil functions can be measured using a small volume of whole blood that reduces artifactual changes in function caused by purification procedures. The advent of numerous new fluorochromes and multiparametric analysis allows the simultaneous measurement of several neutrophil functions in the same population of cells. Flow cytometric analysis provides a rapid screen for abnormalities of neutrophil function and reflects more accurately their behavior in vivo.     

Validation and quality control of immunophenotyping in clinical flow cytometry

Clinical flow cytometry has evolved from two-parameter quantitative assessment of peripheral blood lymphocytes to six-parameter qualitative evaluation of bone marrow for hematopathology. Leukemia and lymphoma immunophenotyping represent an extremely important complement to morphology in the diagnosis and monitoring of hematopoietic malignancies. The complexity of five- and six-parameter analyses and the interpretation of the data rely on standardization and validation of the instrument, the reagents and the procedure. In addition, flow cytometry laboratories in the U.S. are required to document proficiency testing, sample preparation, method accuracy, specificity, sensitivity and precision. NCCLS and the U.S.-Canadian Consensus Conference have provided recommendations, but each laboratory is ultimately responsible for validating its own qualitative and quantitative procedures. This paper reviews procedures for validation and quality control of all aspects of the operation of a clinical flow cytometry service.     

Evaluation of T and B lymphocyte function in clinical practice using a flow cytometry based proliferation assay

The golden standard for functional evaluation of immunodeficiencies is the incorporation of [³H]-thymidine in a proliferation assay stimulated with mitogens. Recently developed whole blood proliferation assays have the advantage of parallel lymphocyte lineage analysis and in addition provide a non-radioactive alternative. Here we evaluate the Flow-cytometric Assay for Specific Cell-mediated Immune-response in Activated whole blood (FASCIA) in a comparison with [³H]-thymidine incorporation in four patients with severe combined immunodeficiency. The threshold for the minimum number of lymphocytes required for reliable responses in FASCIA is determined together with reference values from 100 healthy donors when stimulated with mitogens as well as antigen specific stimuli. Finally, responses against PWM and SEA + SEB stimuli are conducted with clinically relevant immunomodulatory compounds. We conclude that FASCIA is a rapid, stable and sensitive functional whole blood assay that requires small amounts of whole blood that can be used for reliable assessment of lymphocyte reactivity in patients.     

Evidence of a pharmacogenomic response to interleukin-l receptor antagonist in rheumatoid arthritis

Biological activity of the IL-1 system depends on the balance between two proinflammatory proteins (IL-1alpha and IL-1beta) and the related anti-inflammatory protein, the IL-1 receptor antagonist (IL-1Ra). The genes for these proteins lie within 430 kb on human chromosome 2. Based on a clinical trial of human recombinant IL-1ra in rheumatoid arthritis, we tested whether IL-1 genotype might be related to the likelihood of response to anti-IL-1 therapy. A positive response was defined as a reduction of at least 50% in the number of swollen joints by week 24, following treatment with either 150 mg/day IL-1ra or placebo. The response rate to treatment, independent of genotype, was 48% (44/91). A highly significant association was found between carriage of the rarer allele at IL1A(+4845) and response to treatment (P=0.0009; OR=4.85 (1.85,12.70)). The response rate in patients carrying this allele was 63.4% compared with 26.3% in noncarriers. A weaker association was found for IL1B(+3954) (P=0.02). There was a highly significant interaction between treatment (150 mg/day or placebo) and the composite genotype across IL1A(+4845) and IL1B(+3954) (P=7.6 x 10(-5)). No associations with IL-1 genotypes were found in patients receiving placebo. Thus, a significant pharmacogenomic effect was found in the treatment of RA patients with recombinant IL-1ra.     

Pitfalls in the use of multicolour flow cytometry in haematology

Multicolour flow cytometry in haematology has developed considerably in recent years. The ability to analyse eight or more colours of fluorescence on millions of cells in a matter of minutes has enabled the provision of rapid and reliable measures of minimal residual disease for clinicians. The use of multicolour analysis has also enabled more specific characterisation of presenting leukaemias and lymphomas. However, there has not been a concomitant increase in the knowledge and experience of the flow cytometrists to deal with certain problems associated with this more complex analysis.     

The use of flow cytometry to detect the biosynthesis of complement components

Agarose beads, an activator of complement, were incubated with MRC-5 or He 9 fibroblast cell lines under serum-free conditions. The beads were tested for binding of anti-complement antibodies by flow cytometry with a FACS 440 using FITC-labelled anti-Ig detection antibodies. Controls consisted of co-cultured beads incubated with irrelevant antibody or albumin, beads maintained in cell cultures containing cycloheximide, and beads which were not exposed to cells. The histograms demonstrated positive staining with anti-C3c, -C5, -C7 and -C9, but not with anti-C6 and -C8. Flow cytometry with multiple histogram analysis confirmed that the differences between the positive curves and the controls were statistically significant. The results show that cell-derived complement components (C3, C5, C7 and C9) were deposited on the beads and could be detected by flow cytometry.     

Development and application of a biomarker assay for determining the pharmacodynamic activity of an antagonist candidate biotherapeutic antibody to IL21R in whole blood

Background

In preparation for potential clinical development of Ab-01, an antagonistic antibody directed against the IL21R, studies were undertaken to address translational medicine needs that fall into four categories: 1) development of a pharmacodynamic biomarker assay suitable for use in the clinic, 2) demonstration that Ab-01 has the desired biological activity in vitro and in vivo in cynomolgus monkeys, the preferred safety study species, 3) pre-clinical in vivo proof-of-concept that the assay can be used to detect Ab-01 pharmacodynamic (PD) activity in treated subjects, and 4) comprehensive assessment of the agonistic potential of Ab-01 when cross-linked. This report and a recently published companion report address the first three of these needs. The fourth has been addressed in a separate study.

Methods

Genes that change RNA expression upon ex vivo rhIL21 stimulation of whole blood were identified in human and cynomolgus monkey. The inhibitory effects of exogenously added Ab-01 were measured ex vivo in human and monkey, and the in vivo inhibitory effects of Ab-01 treatment were measured in monkey.

Results

Stimulation of whole human blood for 2 hours with rhIL21 induced robust increases in RNA expression of 6 genes. This response was blocked by Ab-01, indicating that the assay is suitable for measuring Ab-01 activity in blood. rhIL21 induced expression of a similar set of genes in cynomolgus monkey blood. This response was blocked with Ab-01, thus demonstrating that Ab-01 has the desired activity in the species, and that safety studies done in cynomolgus monkeys are relevant. Proof -of-concept for using this assay system to detect PD activity in vivo was generated by measuring the response in monkey blood to ex vivo rhIL21 stimulation before and 5 minutes following in vivo Ab-01 administration.

Conclusions

A robust PD biomarker assay suitable for clinical use has been developed in human whole blood. The successful adaptation of the assay to cynomolgus monkeys has enabled the demonstration of Ab-01 activity both in vitro and in vivo in monkey, thus validating the use of this species in safety studies and establishing proof-of-concept for using this PD assay system to aid in dose selection in clinical studies.     

Rapid whole-blood flow cytometry assay for diagnosis of chronic granulomatous disease.

Chronic granulomatous disease (CGD) is characterized by defective killing of intracellular microorganisms due to mutations in one of the four known components of the NADPH oxidase system. This system is responsible for the generation of superoxide and related antimicrobial oxidants. Diagnosis of CGD requires the demonstration of an abnormal oxidase system in the leukocytes of affected patients. Recently, several flow cytometry-based procedures which measure various reactive oxygen intermediates generated by the NADPH oxidase system have been developed. Most of the procedures developed to date require time-consuming granulocyte isolation, washing, and counting procedures, or they lack sensitivity. We have modified an existing procedure such that cell labelling and stimulation are performed directly in whole blood. Optimization of this procedure and its use in the diagnosis of patients with CGD or X-linked carriers are presented.     

应用CompBeads建立流式多色分析补偿的方法

目的 探讨一种快速简便的流式细胞仪多色分析时的关键参数(荧光补偿)设置方法.方法 以BD CompBeads、实验样品(本实验为慢性乙型肝炎患者外周全血)分别与单克隆荧光抗体染色,结合流式细胞仪Diva软件调节细胞仪光电倍增管(PMT)电压,计算建立荧光补偿矩阵.同时以获得的参数设置仪器,运行慢性乙型肝炎患者外周全血6色荧光标记实验样品.结果 用CompBeads建立的补偿参数稳定可靠,能有效消除荧光光谱之间的交叉重叠.6色荧光标记实验样品检测的结果,可见细胞亚群分布清晰,荧光强度比例表达适当,与通常采用的实验样品单染色方法补偿结果一致(P>0.05).结论 该方法能快速方便进行流式细胞仪检测时仪器参数最佳化设置调整,在多色免疫标记流式检测中有较大的应用价值.     

Validation of a urine-based PCR-enzyme-linked immunosorbent assay for use in clinical research settings to detect Trichomonas vaginalis in men

Trichomonas vaginalis infection is highly prevalent worldwide and is associated with urethritis, prostatitis, and urethral strictures in men. However, the natural history and importance of T. vaginalis in men are poorly understood, in part because of difficulties in diagnosing infection. Traditional detection methods rely on culture and wet-mount microscopy, which can be insensitive and time consuming. Urethral swabs are commonly used to detect T. vaginalis in men, but discomfort from specimen collection is a barrier to large studies. One thousand two hundred twenty-five Malawian men attending sexually transmitted disease and dermatology clinics were enrolled in this cross-sectional study to validate detection by urine-based PCR-enzyme-linked immunosorbent assay (ELISA) with urine and urethral swab culture as the reference standard. This assay for detection of amplified T. vaginalis DNA in first-catch urine (< or = 30 ml) performed with a sensitivity of 92.7%, a specificity of 88.6%, and an adjusted specificity of 95.2% compared to culture of urethral swabs or urine sediment. For clinical research settings in which urethral swabs are not available and culture is not feasible, the urine-based PCR-ELISA may be useful for detection of trichomoniasis in men.     

The chemokine receptor D6 limits the inflammatory response in vivo

How the inflammatory response is initiated has been well defined but relatively little is known about how such responses are resolved. Here we show that the D6 chemokine receptor is involved in the post-inflammatory clearance of beta-chemokines from cutaneous sites. After induction of inflammation by phorbol esters, wild-type mice showed a transient inflammatory response. However, in D6-deficient mice, an excess concentration of residual chemokines caused a notable inflammatory pathology with similarities to human psoriasis. These results suggest that D6 is involved in the resolution of the cutaneous inflammatory response.     

The use of flow cytometry to discriminate avian lymphocytes from contaminating thrombocytes

Evaluation of peripheral blood mononuclear cells (PBMC) in avian species by flow cytometry is complicated by the presence of large numbers of nucleated thrombocytes. With light scattering properties similar to those of lymphocytes, variations in the proportion of thrombocytes in PBMC can result in apparent shifts in percentages of lymphocyte subpopulations. We have applied a dual-labeling procedure for flow cytometric analyses to exclude thrombocytes from the analyzed population by labeling with K55 monoclonal antibody (MAb), which differentially labeled leukocyte and thrombocyte populations. Flow cytometric analyses with K55 labeled chicken PBMC differentiated leukocytes into three populations according to their intensity of fluorescence. Using the Kl MAb, the K55-low population was shown to consist of thrombocytes. Dual-labeling with K55 MAb and MAb specific for B lymphocyte, CD4 or CD8 markers indicated that the K55 intermediate population consisted of lymphocytes. Therefore, concentrations of CD4+ and CD8+ T lymphocytes could be determined from the lymphocyte fraction by gating specifically on the K55 intermediate cells. Selecting cross-reactive chicken MAbs that included K55, this protocol was shown to identify CD4+ and CD8+ T lymphocytes in PBMC of another avian species, the endangered Attwater's prairie chicken.     

Quantification of mucosal mononuclear cells in tissues with a fluorescent bead-based polychromatic flow cytometry assay

Since the vast majority of infections occur at mucosal surfaces, accurate characterization of mucosal immune cells is critically important for understanding transmission and control of infectious diseases. Standard flow cytometric analysis of cells obtained from mucosal tissues can provide valuable information on the phenotype of mucosal leukocytes and their relative abundance, but does not provide absolute cell counts of mucosal cell populations. We developed a bead-based flow cytometry assay to determine the absolute numbers of multiple mononuclear cell types in colorectal biopsies of rhesus macaques. Using 10-color flow cytometry panels and pan-fluorescent beads, cells were enumerated in biopsy specimens by adding a constant ratio of beads per mg of tissue and then calculating cell numbers/mg of tissue based on cell-to-bead ratios determined at the time of sample acquisition. Testing in duplicate specimens showed the assay to be highly reproducible (Spearman R=0.9476, P<0.0001). Using this assay, we report enumeration of total CD45(+) leukocytes, CD4(+) and CD8(+) T cells, B cells, NK cells, CD14(+) monocytes, and myeloid and plasmacytoid dendritic cells in colorectal biopsies, with cell numbers in normal rhesus macaques varying from medians of 4486 cells/mg (T cells) to 3 cells/mg (plasmacytoid dendritic cells). This assay represents a significant advancement in rapid, accurate quantification of mononuclear cell populations in mucosal tissues and could be applied to provide absolute counts of a variety of different cell populations in diverse tissues.     

Characterization and use of a rabbit-anti-mouse VPAC1 antibody by flow cytometry

Vasoactive intestinal peptide receptor-1 signaling in lymphocytes has been shown to regulate chemotaxis, proliferation, apoptosis and differentiation. During T cell activation, VPAC1 mRNA is downregulated, but the effect on its protein levels is less clear. A small number of studies have reported measurement of human VPAC1 by flow cytometry, but murine VPAC1 reagents are unavailable. Therefore, we set out to generate a reliable and highly specific α-mouse VPAC1 polyclonal antibody for use with flow cytometry. After successfully generating a rabbit α-VPAC1 polyclonal antibody (α-mVPAC1 pAb), we characterized its cross-reactivity and showed that it does not recognize other family receptors (mouse VPAC2 and PAC1, and human VPAC1, VPAC2 and PAC1) by flow cytometry. Partial purification of the rabbit α-VPAC1 sera increased the specific-activity of the α-mVPAC1 pAb by 20-fold, and immunofluorescence microscopy (IF) confirmed a plasma membrane subcellular localization for mouse VPAC1 protein. To test the usefulness of this specific α-mVPAC1 pAb, we showed that primary, resting mouse T cells express detectable levels of VPAC1 protein, with little detectable signal from activated T cells, or CD19 B cells. These data support our previously published data showing a downregulation of VPAC1 mRNA during T cell activation. Collectively, we have established a well-characterized, and highly species specific α-mVPAC1 pAb for VPAC1 surface measurement by IF and flow cytometry.