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PubMed2003

A preliminary comparison of flow cytometry and tube agglutination assays in detecting red blood cell-associated C3d.

This study compared flow cytometric analysis with tube agglutination assays for the detection of red blood cell (RBC)-associated complement and immunoglobulins (Igs). RBCs from 20 patients with reactive tube direct antiglobulin tests (DATs) were evaluated by flow cytometry with anti-C3d, anti-IgG, anti-IgM and anti-IgA. Serial samples were also tested from a patient at risk of passenger lymphocyte haemolysis. Results of flow cytometry and tube assays for anti-IgG were as follows: 12 of 20 samples reactive in both; six of 20 nonreactive in both; two of 20 discordant with a reactive tube and a nonreactive flow cytometry assay. Anti-C3d results showed nine of 20 reactive in both and 11 of 20 discordant with a nonreactive tube and a reactive flow cytometry assay. In the IgM flow cytometry assay, three samples were reactive with anti-IgM. Samples from a group A woman who was transplanted with stem cells from a group B donor showed that on days 3 through 6 post-transplant, the flow cytometry assays for anti-IgG and/or anti-C3d were reactive, whilst the tube assays were nonreactive. In conclusion, flow cytometric analysis is more sensitive than the tube assay for the detection of RBC-associated C3d. Further studies are needed to determine the correlation of C3d levels with clinical sequelae.

Adult↗
PubMed2001

An update on the use of flow cytometry in HIV infection and AIDS.

Flow cytometry has played an invaluable role in recent advances made against HIV and AIDS, and there is every reason to expect it will continue to do so. Newer-generation machines, using three- and four-color panels, make measurements of lymphocyte subsets ever more accurate and potentially cost-effective. Similarly, recent single-platform techniques promise even more efficiencies, freeing CD4 enumeration from parallel determination of the CBC. In other developments, quantitative reporting of immunophenotype may well change the way subsets traditionally defined by qualitative determinations are viewed. The most promising initial candidates for such changes are surrogates of immune activation, such as CD38 expression on CD8 cells. But technical demonstration of reproducibility and reliability, and clinical trial evidence of utility--especially as measured against such established laboratory parameters as CD4 cell and HIV RNA determinations--are needed before a change from qualitative to quantitative immunophenotype measurement can be widely accepted. Although too soon to tell if they will emerge from the research arena into the clinical arena, tetramer binding, intracellular cytokine detection, and assays of cell division are rapidly advancing understanding of HIV-disease, and indeed understanding of all of human immunology. Some of these assays provide often subtly different information, and together they will help determine the most important surrogate measures of clinical immunity. Identifying such surrogates is critical to developing a rational HIV vaccine. The power of flow cytometry is its ability simultaneously to analyze data in four, five, six, or more dimensions. For longitudinal experiments, time adds yet another dimension. Humans struggle to think past three dimensions; interpreting results from experimental permutations and combinations that expand geometrically with each additional parameter studied requires considerable effort. Ultimately computer programs might come to our aid, but for now we have to live with the information overload if we hope to gain insight into the workings of complex biologic systems. In a world of limited resources, and with limited capacity to conceptualize in multiple dimensions, insight, inspiration, and perhaps a little luck are needed to ask the right questions and use the right assays if the recent string of advances against AIDS is to continue.

Acquired Immunodeficiency Syndrome↗
PubMed2001

Determination of differential cell counts in feline bone marrow by use of flow cytometry.

OBJECTIVE: To evaluate the potential usefulness of 2 flow cytometric methods for determination of differential cell counts in feline bone marrow. SAMPLE POPULATION: 10 bone marrow specimens from client-owned cats. PROCEDURE: Bone marrow specimens were stained with 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and evaluated by use of flow cytometry. Differential counts were also determined by analysis of scatterplots of forward-angle versus side-angle light scatter of unstained specimens, obtained by use of flow cytometry (scatterplot method). Results of both flow cytometric methods were compared with differential cell counts determined by manually counting 1,000 cells on slides of Wright-stained smears. RESULTS: Staining with DiOC6 resulted in identification of mature and immature erythroid and myeloid cells and lymphocytes. Use of the scatterplot method resulted in identification of mature and immature erythroid and myeloid cells and metamyelocytes. However, to identify lymphocytes by use of the scatterplot method, bone marrow specimens were first labeled with an anti-major histocompatability class-II antibody. Comparison of results of the scatterplot method with manual counts yielded higher correlation coefficients and more similar mean values than did comparison of results of the DiOC6 method. CONCLUSIONS AND CLINICAL RELEVANCE: The scatterplot method provided more accurate and precise results than the DiOC6 method for determination of bone marrow differential cell counts in cats by use of flow cytometry. When combined with fluorescent labeling of lymphocytes, the scatterplot method has potential to provide rapid semiquantitative assessment of bone marrow differential cell counts in cats.

Animals↗
PubMed1992

Image cytometric DNA analysis in human breast cancer analysis may add prognostic information in diploid cases with low S-phase fraction by flow cytometry.

Measurements of DNA ploidy can be performed either with image cytometry (ICM) or flow cytometry (FCM); both methods provide independent prognostic information in primary breast cancer. The aim of the present investigation was to compare the two methods and to relate the findings to prognosis (median follow-up 42 months). Concordance in ploidy status (diploid, tetraploid, aneuploid) was obtained in 76% of the samples (168/222). When the fraction of S-phase cells (SPF) from FCM analysis was also taken into consideration, four different groups of samples were obtained (Flow I-IV), which were considered to correspond to the Auer classification (Auer I-IV) of DNA histograms obtained from image cytometry. Complete concordance between the two techniques now was 70% (155/222). Samples classified as Flow I (diploid or near-diploid with low SPF) and Auer I had a distant metastasis rate of 3/60 (5%), as compared to 62/154 (40%) for all other combinations of the Flow and Auer classifications taken together. Thus, the only findings of prognostic importance were that some samples were Flow I but not Auer I, or vice versa. These two groups represent 17 (7.7%) and 14 (6.3%), respectively, of the total number of samples, and had frequencies of distant metastasis similar to those of the other high-risk groups, namely, 7/17 and 5/14, respectively. In a multivariate analysis, flow cytometric S-phase value was a stronger prognostic factor than either the Flow and Auer classification. We conclude that when routine FCM DNA analysis is used, diploid or near-diploid samples with a low S-phase value should be reanalyzed with ICM.

Breast Neoplasms↗
PubMed2005

Detection of Cyclospora cayetanensis oocysts in human fecal specimens by flow cytometry.

A diagnosis of cyclosporiasis typically involves stool examinations for the presence of Cyclospora oocysts by means of microscopy. In recent years, flow cytometry has been gaining in popularity as a novel method of detecting pathogens in environmental and clinical samples. The present study is an evaluation of a flow cytometric method for the detection and enumeration of Cyclospora oocysts in human fecal specimens associated with food-borne outbreaks of cyclosporiasis in Ontario, Canada. Flow cytometry results were generally very comparable to the original microscopy results for these specimens, in terms of both presence or absence of oocysts and relative oocyst concentrations. Of the 34 fecal specimens confirmed positive for Cyclospora by microscopy, 32 were also found positive by flow cytometry, and 2 others were considered equivocal. Of the eight fecal specimens reported to be negative by microscopy, two were found positive by flow cytometry and five others were considered equivocal. These two flow cytometry-positive samples and one of the equivocal samples were confirmed by microscopic reexamination, suggesting that flow cytometry may be more sensitive than microscopy. While the sample preparation time for flow cytometry is similar to or slightly longer than that for microscopy, the actual analysis time is much shorter. Further, because flow cytometry is largely automated, an analyst's levels of fatigue and expertise will not influence results. Flow cytometry appears to be a useful alternative to microscopy for the screening of large numbers of stool specimens for Cyclospora oocysts, such as in an outbreak situation.

Animals↗
PubMed2006

Polychromatic flow cytometry: a rapid method for the reduction and analysis of complex multiparameter data.

BACKGROUND: Recent advances in flow cytometry have resulted in the development of reliable techniques for performing polychromatic (5-17 color) flow cytometry analysis. However, the data reduction and analysis involved in the resolution of hundreds of possible cellular subphenotypes identified, using a single polychromatic flow cytometry staining panel, presents a major obstacle to the successful application of this technology. METHODS: To generate two distinct collections of T cell populations with differentially expressed surface markers, cryopreserved lymph node cells from 5 melanoma patients vaccinated with the modified gp100(209-2M) melanoma peptide were stimulated with cognate peptide and cultured in either IL-21 + low-dose IL-2 or IL-15 + low-dose IL-2. In vitro stimulated (IVS) cells were interrogated using 8-color flow cytometry. Data were analyzed using Winlist Hyperlog and FCOM software, and 32 T cell subsets were resolved for each culture condition. Hierarchical clustering analysis was applied to the relative percentages of each subphenotype for both IVS conditions to determine if unique cell surface marker expression signatures were produced for each IVS culture. RESULTS: Sequential data analysis using Hyperlog and FCOM demonstrated that lymphocytes cultured in IL-21 + IL-2 had a distinctively different set of subphenotype signatures compared to cells grown in IL-15 + IL-2 for all 5 patients. Importantly, subsequent cluster analysis of all 32 subphenotype frequencies in each IVS test condition for all 5 patients reproducibly demonstrated that cellular subphenotypes produced after IL-21 + IL-2 IVS partitioned separately from subphenotypes produced by IL-15 + IL-2 IVS. CONCLUSIONS: The integrated sequential use of Hyperlog and FCOM software with cluster analysis algorithms for the reduction and analysis of polychromatic flow cytometry data produces an effective, rapid technique for the assessment of complex patterns of subphenotype expression between and within multiple test samples. This approach to data analysis may enhance the use of polychromatic flow cytometry for both research and clinical applications.

Algorithms↗
PubMed2004

In vitro evaluation of the effects of candidate immunosuppressive drugs: flow cytometry and quantitative real-time PCR as two independent and correlated read-outs.

BACKGROUND: Immune monitoring may use flow cytometry or molecular biology techniques. Flow cytometry assays cells that are phenotypically characterized, whereas TaqMan RT-PCR starts with RNA extraction from unfractionated heterogeneous cell populations. We therefore wondered how the effects of immunosuppressive drugs on cytokine production in stimulated whole blood, as determined by flow cytometry, would correlate with those obtained with quantitative real-time PCR (TaqMan RT-PCR). METHODS: Blood drawn from naive cynomolgus monkeys was exposed to incremental amounts of cyclosporine (CsA; 300, 600, 900 and 1200 ng/ml) or tacrolimus (TRL; 8, 20, 40 and 80 ng/ml) before lectin stimulation in vitro. Blood was then either stained for CD3, IFN-gamma, IL-2, IL-4, and TNF-alpha and analyzed on a flow cytometer with various gating strategies, or submitted to RNA extraction for analysis of the above mentioned cytokines mRNA transcripts using TaqMan RT-PCR. RESULTS: Both methods revealed a parallel dose-dependent inhibition of cytokine production in stimulated blood. The 50% inhibitory concentrations (IC(50)'s) ranged from 511-771 ng/ml (CsA) and 15-29 ng/ml (TRL) with flow cytometry, and from 275-529 ng/ml (CsA) and 11-48 ng/ml (TRL) with TaqMan RT-PCR for T-helper 1 cytokines. Both assays correlated well with a Pearson product moment correlation of 0.76. Extending gating from a CD3(+) gate to a lymphocyte gate improved correlation (r = 0.85) for all cytokines investigated (except IL-2; unchanged) whereas further extending gating resulted, to the contrary, in lower correlations. Independent of gating strategy a high correlation (r = 0.97) was observed when drug IC(50)'s were considered. CONCLUSIONS: Flow cytometry and TaqMan RT-PCR may be used interchangeably to monitor the effects of candidate immunosuppressive drugs on cytokine mRNA production in lectin-stimulated whole blood.

Animals↗
PubMedDate not supplied

Flow cytometry: basic concepts and clinical applications in immunodiagnostics.

OBJECTIVE: To review current and possible future clinical applications of flow cytometry in immunodiagnostic procedures. DATA SOURCES: Recent research and review articles and textbooks on laboratory diagnosis and flow cytometry. STUDY SELECTION: Not applicable. DATA EXTRACTION: Performed by the author. DATA SYNTHESIS: Flow cytometry is the measurement of cellular and fluorescent properties of stained cells in a fluid stream as they move past a set of fixed detectors. The instruments are capable of measuring these properties on thousands of cells in but a few seconds. Many specimen types can be analyzed on the flow cytometer. CONCLUSION: Flow cytometry is especially useful in monitoring immunodeficiencies, leukemias, and other malignancies, but it has other uses, as well. Improvements in instruments, reagents, and methods will cause an increase in the number of applications for flow cytometry. More laboratories will be able to purchase the equipment as it becomes less expensive. Flow cytometry provides the clinician valuable information in the diagnosis and treatment of disease.

Antibodies, Monoclonal↗
PubMed1998

Clinical Usefulness of Flow Cytometry in Detection and Quantification of Fetomaternal Hemorrhage.

> Objective: To compare the technique of immunofluorescence flow cytometry against traditional Kleihauer testing with respect to the sensitivity and specificity of clinical detection of fetomaternal hemorrhage. Methods: Blood samples from D-negative unsensitized postpartum women were analyzed by flow cytometry (customized, directly conjugated monoclonal anti-D) and Kleihauer testing using commercial kits as well as a manual technique. Results: Both flow cytometry and manual Kleihauer tests performed well in vitro against known standards, giving similar results with detectable fetal cells in the range of 2-10%. Correlation of in vivo results obtained by flow cytometry and either Kleihauer method was poor. The percentage of fetal red cells detected by flow cytometry was approximately twice that of the manual Kleihauer. Commercial Kleihauer testing proved unreliable in the in vivo setting. Conclusions: Flow cytometry offers a reliable alternative to traditional Kleihauer testing for detecting fetomaternal hemorrhage and may, with standardized methodology, help optimize immunoprophylaxis against erythroblastosis fetalis.

Journal Article↗
PubMed1989

"Going with the flow": a perspective on flow cytometry in gastroenterology.

In this perspective on the applications of flow cytometry in gastroenterology are described the use of flow cytometry in the study of normal physiology, in normal anatomy, and in premalignant and malignant conditions of the digestive tract. The basic principles and applications for cell counting, sorting, cell cycle analysis, and quantitation of cellular characteristics are addressed to emphasize the great potential and availability of flow cytometry.

Cell Separation↗
PubMed1995

Flow cytometry in colon cancer: does flow cytometric cell cycle analysis help predict for short-term recurrence in patients with colorectal carcinoma?

The purpose of this prospective study was to determine whether tumor ploidy or S-phase fraction measurements were of prognostic value in predicting short-term recurrence for colorectal carcinoma. A total of 52 patients underwent curative resection of colorectal carcinoma. Fresh suspensions of tumor cells were used for flow cytometric analysis. Patients underwent follow-up for possible recurrence, which then was related to ploidy status, S-phase fraction measurement, adjuvant therapy received, and Dukes stage. Disease-free probability was determined using Kaplan-Meier actuarial curves for various subgroups of the study populations. Results revealed that elevated S-phase fraction (>16%) did predict for a higher probability of recurrence in those patients receiving no postoperative adjuvant treatment. Among patients receiving postoperative adjuvant therapy, however, a higher S-phase fraction predicted for a lower probability of recurrence. These data suggest that the S-phase fraction does predict for disease-free probability and also predict response to adjuvant therapy in patients with colorectal carcinoma.

Adenocarcinoma↗
PubMed1992

Deoxyribonucleic acid flow cytometry and semiquantitative histology of spermatogenesis: a comparative study.

OBJECTIVE: To compare deoxyribonucleic acid (DNA) flow cytometry with a semiquantitative histologic method for the assessment of spermatogenesis and to investigate the influence of various preparative techniques on the results of DNA measurements. DESIGN: Testicular tissue from 52 autopsies was examined for spermatogenesis by DNA flow cytometry and by conventional semiquantitative histology. Flow cytometry was performed on single nuclei suspensions from fine needle aspiration biopsies, resuspended imprints, as well as on specimens obtained by mechanical disaggregation of surgical biopsies of the same testis. RESULTS: In all cases, flow cytometry revealed a characteristic distribution pattern of haploid (n), diploid (2n), and tetraploid (4n) cells independent of the preparation technique used. The percentage of haploid cells of the testes showed statistically highly significant correlations (P = 0.0002) with the results of semiquantitative histology. Fine needle aspiration biopsy always contained more haploid cells compared with resuspended imprints and mechanically disaggregated samples. CONCLUSION: Our results confirm the suitability of DNA flow cytometry as a fast and reliable quantitative method for the assessment of spermatogenesis and demonstrate its optimal feasibility in combination with fine needle aspiration biopsy.

Adult↗
PubMed2004

Current applications of flow cytometry in the diagnosis of primary immunodeficiency diseases.

CONTEXT: To review the applications of flow cytometry in the diagnosis and management of primary immunodeficiency disease. DATA SOURCES: Articles describing the use of flow cytometry in the diagnosis of several primary immunodeficiency diseases were obtained through the National Library of Medicine database. STUDY SELECTION: Publications that described novel and known applications of flow cytometry in primary immunodeficiency disease were selected. Review articles were included. Articles describing the different immunodeficiency diseases and methods of diagnosis were also selected. DATA EXTRACTION: Approximately 100 data sources were analyzed, and those with the most relevant information were selected. DATA SYNTHESIS: The diagnosis of many primary immunodeficiency diseases requires the use of several laboratory tests. Flow cytometry has become an important part of the workup of individuals suspected to have such a disorder. Knowledge of the pathogenesis of many of these diseases continues to increase, hence we acquire a better understanding of the laboratory tests that may be helpful in diagnosis. CONCLUSIONS: Flow cytometry is applicable in the initial workup and subsequent management of several primary immunodeficiency diseases. As our understanding of the pathogenesis and management of these diseases increases, the use of many of these assays may become routine in hospitals.

Antigens, CD↗
PubMed2003

Validation of flow cytometry to quantify the potency of anti-D immunoglobulin preparations.

BACKGROUND AND OBJECTIVES: Flow cytometry has been recommended as an alternative to that of autoanalyser methodology for estimation of anti-D potency. This investigation was performed to validate the flow cytometry method based on a quality assurance system. MATERIALS AND METHODS: A flow cytometry method based on indirect labelling of Rh(D)-positive red blood cells was validated using the parameters precision and accuracy and was compared to the autoanalyser data of manufacturers of anti-D immunoglobulin preparations. RESULTS: The experiment first investigated the possible differences between assays from single donors compared with a pooled assay. Red blood cells of four individual donors and their pooled red blood cells were interchangeable. There was no significant difference between donors, on one hand, and between the use of a single donation and the pooled red blood cells, on the other hand (P = 0.695). The two-sided 95% confidence intervals (CIs) of the difference between single donors and the pool ranged from -4.6% to 4.7%. The intermediate variability was determined by standard deviation (SD) = 48.4 IU/ml [coefficient of variation (CV) = 3.8%]; the repeatability was SD = 34.3 IU/ml (CV = 2.7%). Using a spiking experiment, the second part of the experiment investigated recovery of a known anti-D potency. The recovery of samples spiked with defined amounts of reference preparation was 97.7-101%, with a mean bias of -1.3 (95% CI: -4.1 to 1.6). The results of the flow cytometry assay, as compared to those of the autoanalyser performed by the manufacturers of anti-D immunoglobulin preparations for those manufacturers who have their method validated in-house, ranged from 87 to 129%, indicating good correlation. CONCLUSIONS: Flow cytometry is a suitable quality control method for polyclonal anti-D immunoglobulins, which can be standardized in a quality control laboratory using a quality assurance system.

Automation↗
PubMed1999

Flow cytometry in the exploration of the physiopathology of occupational lung disease.

Flow cytometry is a simple analytical technique used for the investigation of cells taken from various sources. Cells are identified by both their physical characteristics and the presence or absence of specific molecules on the cell surface. These molecules may be either phenotypic, or induced by a specific stimulus. Flow cytometry has been used to identify the nature and extent of the immune response in several occupational respiratory conditions including occupational asthma, irritant induced respiratory problems, and asbestos related lung disease. Also, it may be of value in monitoring workplace exposure to some hazardous materials. Although of limited diagnostic value at present, the technique has provided an insight into the modulation of immune cells, and their function, in people exposed to hazardous materials in the workplace. In this review, the principals of flow cytometry will be explored and the use of flow cytometry to investigate occupational respiratory disease will be discussed.

Asbestosis↗
PubMed1986

Flow cytometry versus urinary cytology in the evaluation of patients with bladder cancer.

We compared the roles of urinary cytology and flow cytometry in the evaluation of patients with bladder cancer in clinical practice situations at a large general hospital. Specimens included 105 bladder washings from patients being followed for urothelial carcinomas and 28 control washings from individuals undergoing cytoscopy for other reasons. Flow cytometry and cytology were performed on aliquots of the same specimen in all bladder cancer samples. When carcinoma was present at the time of specimen collection it was detected by positive cytology in 75 per cent and deoxyribonucleic acid aneuploidy in 78 per cent of the cases. Combination of flow cytometry and urinary cytology increased the diagnostic yield to 95 per cent. Flow cytometry was slightly more sensitive than urinary cytology for detection of abnormalities in specimens from noninvasive and untreated tumors but the only statistically significant difference between the 2 procedures occurred among specimens from treated invasive cancers in which flow cytometry was a less sensitive method than cytology. Abnormal deoxyribonucleic acid ploidy was documented in a few specimens from noncancer-bearing patients having diseases associated with high urothelial cell turnover rates but the concomitant urinary cytology was negative for neoplasia. When used in conjunction with urinary cytology, flow cytometry was a valuable procedure in the followup of patients with bladder cancer. The diagnostic yield with this combination was such that flow cytometry and cytology may be used to reduce the frequency of cystoscopy and biopsy during clinical management in selected situations.

Aneuploidy↗
PubMed2000

The role of flow cytometry in the study of cell growth in the rat anterior pituitary gland.

Flow cytometry is a suitable technique for studying in vivo and in vitro the cell cycle kinetics of different animal and human tissues, both in normal and tumoral conditions. The rat anterior pituitary gland is a model to investigate cell growth and replication of differentiated, neuroendocrine cells, and we report current evidence on its cell cycle kinetics as well as on the role played by flow cytometry in this type of study. The proliferation potential of normal anterior pituitary cells is related to a number of different conditions, including heterogeneity of cell types, age and sex of donors, and circadian influences. In addition, the trend of cell proliferation in both in vivo and in vitro studies is similar, suggesting that cultured anterior pituitary elements may, at least in part, retain growth features analogous to those of the intact gland. Sorting of selective cell types and analysis of the relation between proliferating anterior pituitary cells and the light-dark cycle have shown that flow cytometry may be useful to investigate the replication process of the gland. By using a combination of flow cytometry, light microscopic immunocytochemistry and morphometry, we have reported a peculiar trend of proliferation in primary monolayer cultures of rat anterior pituitary gland, characterized by a non-linear reduction in their proliferation rate with advancing age, primarily dependent on a reduced transition of cells from the G0/G1- to the early S-phase pool. These studies indicate that flow cytometry offers insights into cell cycle check points of anterior pituitary cells, and suggest that it might be applied to the study of growth of selective pituitary elements, both in normal and tumoral conditions.

Animals↗