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T cell subpopulations defined by monoclonal antibodies after HLA-identical sibling marrow transplantation. II. Activated and functional subsets of helper-inducer and cytotoxic-suppressor subpopulations defined by two-colour fluorescence flow cytometry.

Two-colour fluorescence flow cytometry was utilized to define subsets within the Leu-3+ (T4+) helper-inducer and the Leu-2+ (T8+) cytotoxic-suppressor T cell subpopulations in recipients of unmanipulated HLA-identical sibling bone marrow transplants. The absolute number of activated cytotoxic-suppressor T cells expressing the HLA-DR and OKT10 activation antigens was increased. The proportion (or relative number) of cells bearing each of these activation antigens was also elevated in both the Leu-2+ and the Leu-3+ subpopulations, with up to 60% of Leu-2+ cells and up to 40% of Leu-3+ cells expressing them. Interestingly, absolute numbers of cells expressing two other activation-associated antigens, the interleukin-2 (IL-2) and transferrin receptors, were not increased in either major T cell subpopulation, although the relative number of Leu-3+ cells expressing both these surface structures was increased early post-transplant. Three putative functional subsets were also enumerated: the Leu-3+, Leu-8+ suppressor-inducer subset was depressed in absolute and relative numbers at most time points post-transplant. The Leu-4+, Leu-15+ suppressor-effector subset subpopulation was normal at all time points posttransplant, while the Leu-2+, 9.3+ cytotoxic precursor subset showed low relative and absolute numbers both early and late post-transplant. None of the abnormalities demonstrated in the present study was correlated with presence or absence of graft-versus-host disease. The study further demonstrates the heterogeneity of the abnormalities in the immune system after human marrow transplantation and lays the basis for functional studies involving these cell populations.

Adult↗

Autoantibodies to decondensed sperm nuclear deoxyribonucleic acid in patients with antisperm antibodies and systemic lupus erythematosus detected by immunofluorescence flow cytometry.

OBJECTIVE: To evaluate a flow cytometric method to detect and quantitate serum anti-DNA antibodies using unfixed, swollen and decondensed human sperm nuclei and to examine the relationship between antibodies against sperm surface antigens to the presence of antibodies against nuclear antigens. DESIGN: Serum IgG and IgG subclass antibodies to decondensed sperm nuclei were detected by indirect immunofluorescence (IIF) flow cytometry. Sera were screened by IIF for anti-double-stranded DNA antibodies using the protozoan Crithidia luciliae as the substrate and for antinuclear antibodies using human epithelial (HEp 2) cells, respectively. All sera were assessed for antibodies against the sperm plasma membrane by an indirect immunobead test. SETTING: Infertility laboratory at the University of Oklahoma Health Sciences Center and rheumatology laboratory at the Oklahoma Medical Research Foundation. PATIENTS: Sera from 33 antisperm antibody-positive patients (5 subgroups), 33 patients with systemic lupus erythematosus (SLE; 6 subgroups), and 20 normal controls were selected. RESULTS: IgG antibodies against decondensed sperm nuclear DNA were detected in 11 (33.3%) of 33 antisperm antibody-positive patients versus 14 (42.4%) of 33 patients with SLE. Anti-DNA antibodies were most prevalent in vasectomized men and in antisperm antibody positive women with SLE. In the sera from patients with SLE, the presence of the anti-nuclear ribonucleoprotein antibody was associated with the presence of sperm head-directed antisperm antibodies. Anti-double-stranded DNA antibodies were found in 6 (18.1%) of 33 sera from patients with antisperm antibody and 17 (51.5%) of 33 sera from patients with SLE. Antinuclear antibodies were found in only 9 (27.2%) of 33 sera from patients with antisperm antibody and 30 (90.9%) of 33 sera from patients with SLE. All 20 of the control sera gave negative results in the three tests. Serum IgG reactivity to sperm nuclei was predominantly of the IgG1 and IgG3 subclasses. CONCLUSION: Anti-DNA is frequently found in either patients with antisperm antibodies or patients with SLE. Our results indicated that decondensed sperm nuclei can provide a specific substrate for screening serum anti-DNA antibodies.

Antibodies↗

Comparison of four nuclear isolation buffers for plant DNA flow cytometry.

BACKGROUND AND AIMS: DNA flow cytometry requires preparation of suspensions of intact nuclei, which are stained using a DNA-specific fluorochrome prior to analysis. Various buffer formulas were developed to preserve nuclear integrity, protect DNA from degradation and facilitate its stoichiometric staining. Although nuclear isolation buffers differ considerably in chemical composition, no systematic comparison of their performance has been made until now. This knowledge is required to select the appropriate buffer for a given species and tissue. METHODS: Four common lysis buffers (Galbraith's, LB01, Otto's and Tris.MgCl2) were used to prepare samples from leaf tissues of seven plant species (Sedum burrito, Oxalis pes-caprae, Lycopersicon esculentum, Celtis australis, Pisum sativum, Festuca rothmaleri and Vicia faba). The species were selected to cover a wide range of genome sizes (1.30-26.90 pg per 2C DNA) and a variety of leaf tissue types. The following parameters were assessed: forward (FS) and side (SS) light scatters, fluorescence of propidium iodide-stained nuclei, coefficient of variation of DNA peaks, presence of debris background and the number of nuclei released from sample tissue. The experiments were performed independently by two operators and repeated on three different days. KEY RESULTS: Clear differences among buffers were observed. With the exception of O. pes-caprae, any buffer provided acceptable results for all species. LB01 and Otto's were generally the best buffers, with Otto's buffer providing better results in species with low DNA content. Galbraith's buffer led to satisfactory results and Tris.MgCl2 was generally the worst, although it yielded the best histograms in C. australis. A combined analysis of FS and SS provided a 'fingerprint' for each buffer. The variation between days was more significant than the variation between operators. CONCLUSIONS: Each lysis buffer tested responded to a specific problem differently and none of the buffers worked best with all species. These results expand our knowledge on nuclear isolation buffers and will facilitate selection of the most appropriate buffer depending on species, tissue type and the presence of cytosolic compounds interfering with DNA staining.

Buffers↗

Intracellular cytokine production by human CD4+ and CD8+ T cells from normal and immunodeficient donors using directly conjugated anti-cytokine antibodies and three-colour flow cytometry.

Using three-colour flow cytometry, we have measured intracellular IL-2, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) induced in human CD4+ and CD8+ T cells from normal donors and patients with common variable immunodeficiency (CVID). Since a new range of directly FITC-conjugated anti-cytokine antibodies was used, conditions were optimized for the concentration of antibody, for cell permeabilization and fixation, and for the time of exposure to monensin to retain the cytokines within the cell. Kinetics of intracellular cytokine production were measured for up to 20 h in culture with phorbol myristate acetate (PMA) and ionomycin, or with phytohaemagglutinin (PHA). Kinetic studies of activation with PMA and ionomycin show that a higher proportion of normal CD4+ cells can make IL-2 than the other two cytokines, and that there are more TNF-alpha-positive CD4+ cells than cells with IFN-gamma. For normal CD8+ cells the highest production of cytokine is of IFN-gamma (up to 50% of the cells) especially at longer times (10-20 h) of stimulation. For CD8+ cells, IL-2-positive cells exceed those with TNF-alpha. The other mitogenic stimulus used (PHA) was grossly inferior to PMA and ionomycin in its ability to induce intracellular cytokines. The time of exposure to monensin was also examined. Its continuous presence in the cultures (up to a maximum of 20 h) increased the detection of IL-2-positive cells without apparently reducing the percentage of cytokine-positive CD4+ or CD8+ cells. Finally, using optimal conditions, we compared cytokine production in cells from patients with the disease CVID and showed normal cellular levels of ability to produce IL-2 and TNF-alpha but significantly raised levels of production of IFN-gamma in both CD4+ and CD8+ lymphocytes. This suggests that the pathology of this disease may involve an excessive Th1-type response.

Adult↗

Natural killer cell cytotoxicity: a methods analysis of 51chromium release versus flow cytometry.

The purpose of this study is to describe design considerations for the use of flow cytometry (FC) compared to 51chromium (51Cr)-release assays utilizing cryopreserved peripheral blood mononuclear cells (PBMCs) to detect natural killer (NK) cell cytotoxicity. Subjects were 10 healthy women aged 18 to 39 years. Intra-assay variability between methods differed only at the lowest effector-target ratios evaluated. Interassay variability was wide but did not differ between methods. The relationship of lytic unit-10 between methods was strongly positive. Cytotoxicity detected by 51Cr release was higher than that detected by FC for all 10 subjects. Cost was comparable. However, had more assays been performed, technician time would have been greater with flow cytometry. More whole blood was needed to perform the flow cytometry cytotoxicity assay than 51Cr-release cytotoxicity assay. The authors found no compelling reason to adopt NK cell cytotoxicity by flow cytometry over 51Cr release.

Adolescent↗

Liquid chromatography coupled on-line to flow cytometry for postcolumn homogeneous biochemical detection.

The feasibility of flow cytometry as read-out principle for homogeneous cell- or bead-based assays coupled on-line to LC is demonstrated using digoxin-coated beads (Dig-Beads) and fluorescent-labeled anti-digoxin (AD-FITC) as model system. The assay is carried out in a postcolumn continuous-flow reaction detection system where the AD-FITC and Dig-Beads are simultaneously added to the eluate of an LC separation column. Binding of AD-FITC to Dig-Beads results in a constant amount of fluorescence associated with the beads, which is detected by the flow cytometer. The presence of active compounds, such as digoxin and its analogues, in the sample will results in a decrease of the AD-FITC-Dig-Bead complex and, consequently, in the bead-associated fluorescence. Hence, the bead-associated fluorescence detected is inversely related to the digoxin concentration. A data-handling algorithm was developed in-house for adequate analysis of raw data output from the flow cytometer. Various conditions that influence the performance of this novel LC-biochemical detection (LC-BCD) system were investigated to determine the optimal settings of the bead-based biochemical interaction. The optimized flow injection bead-based assay was capable of detecting very low concentrations of digoxigenin (0.5 nmol/L), digoxin (0.1 nmol/L), and gitoxigenin (50 nmol/L). The applicability of LC coupled on-line to flow cytometry was demonstrated by the individual detection of digoxin, digoxigenin, and gitoxigenin in a single LC analysis. The successful coupling of LC on-line to flow cytometry principally enables the use of a wide range of new homogeneous assay formats in LC-BCD, such as membrane-bound receptor assays, cell-binding assays, and functional cell-based assays. Next to the ability to use insoluble targets, and also multiplexing assays, i.e., performing a number of assays simultaneously, using color- or size-coded beads becomes at hand in LC-BCD.

Journal Article↗

DNA-flow cytometry of 207 pituitary adenomas: ploidy, proliferation, and prognosis.

The principal factors involved in pituitary adenoma formation are unknown. DNA-flow cytometry is a useful study providing an estimation of a tumor proliferative rate. In this study, DNA-flow cytometry was performed to evaluate its capability to both assess prognosis and predict recurrence. Two hundred and seven fresh pituitary adenoma specimens were assessed by flow cytometry. Pre-operative endocrine function, previous medical treatment, radiographic appearance, surgical findings and immunohistochemistry were recorded for each patient. Patient outcomes were assessed at a mean follow-up of 5.3 +/- 3.1 yr. Endocrinologically inactive pituitary adenomas were predominantly euploids (50.8%). The highest proliferation rates occurred in Nelson's syndrome and the lowest in Cushing's disease. A significant difference in proliferation was observed with prolactinomas and acromegaly when a medical treatment was performed before primary surgery. Massive histological invasiveness was directly associated with a significant increase in proliferation rate. Radiotherapy did not affect the recurrence rate (4.4%) statistically. In conclusion, DNA-flow cytometry was found to be useful for determining ploidy and obtaining an overview of cell cycle status. It was helpful in identifying patients requiring closer follow-up, such as those with invasive adenomas and Nelson's syndrome. No single parameter revealed by DNA-flow cytometry could predict tumor prognosis or recurrence in the follow-up of 7.5 +/- 1.3 yr.

Adenoma↗

Screening for urothelial malignancies by cytologic analysis and flow cytometry in a community urologic practice: a prospective study.

A prospective study was initiated to compare the ability of flow cytometry and cytologic analysis to detect malignant cells in urine obtained at the time of cystoscopy. The population studied consisted of patients from general urologic practices who were undergoing cystoscopy in a single community hospital. Over a 1-yr period, 335 specimens from 317 patients were studied. Nineteen biopsy-proven urothelial malignancies were identified. Cytologic examination of urine obtained at the time of cystoscopy was positive in seven of these cases, and an aneuploid population of cells was identified by flow cytometry in three cases. All three cases of high-grade transitional cell carcinoma and carcinoma in situ were correctly identified by the combination of cytologic examination and flow cytometry; however, only four of 16 low-grade superficial papillary transitional cell carcinomas were recognized cytologically, with only one being aneuploid. The combination of cytologic analysis and flow cytometry did not increase the diagnostic sensitivity above that achieved with cytologic testing alone (overall sensitivity, 37%). We conclude that flow cytometry and cytologic analysis, either individually or in combination, are too insensitive for use in a routine screening program for urothelial malignancy in a community hospital setting because of the inability of either method to detect low-grade transitional cell carcinomas reliably.

Carcinoma, Squamous Cell↗

Steric-hyperlayer sedimentation field flow fractionation and flow cytometry analysis applied to the study of Saccharomyces cerevisiae.

Sedimentation field flow fractionation separation associated with flow cytometry has been used for the characterization of several commercial Saccharomyces cerevisiae yeasts used for wine production. A new type of channel 80 microm thick and new operating conditions, such as sample introduction when field and flow are established and a channel inlet connected to the accumulation wall, were used. Good repeatability (5% RSD) and reduced analysis time (2-10 min) were obtained. The avoidance of the stop-flow relaxation process in conjunction with the use of a channel of reduced thickness has demonstrated that an effective "steric-hyperlayer" mode driving to a major focusing effect of the species in the channel thickness is involved in the elution of the yeast cells. Flow cytometry analyses were performed, and the forward scattering and side scattering yeast characteristics correlation maps were obtained. Field flow fractionation and flow cytometry information obtained indicated that the fractogram profiles of the yeast cell depended not only on the size, but also on the shape and density.

Equipment Design↗

Matching of flow-cytometry histograms using information theory in feature space.

Flow cytometry is a widely available technique for analyzing cell-surface protein expression. Data obtained from flow cytometry is frequently used to produce fluorescence intensity histograms. Comparison of histograms can be useful in the identification of unknown molecules and in the analysis of protein expression. In this study, we examined the combination of a new smoothing technique called SiZer with information theory to measure the difference between cytometry histograms. SiZer provides cross-bandwidth smoothing and allowed analysis in feature space. The new methods were tested on a panel of monoclonal antibodies raised against proteins expressed on peripheral blood lymphocytes and compared with previous methods. The findings suggest that comparing information content of histograms in feature space is effective and efficient for identifying antibodies with similar cell-surface binding patterns.

Animals↗

Comparision of two-flow cytometry methods for basophil degranulation in patients sensitized to grass pollen.

BACKGROUND: Two-flow cytometry methods for quantification of degranulated basophil after allergen-specific activation were discussed. The methods are discerned by used membrane receptors--FcepsilonRI or IL-3Ralpha Our goal was to evaluate the diagnostic potential of the methods and to correlate them to allergen-specific IgE detection and skin prick test (SPT). METHODS: Patient's and control's groups were studied with Bulgarian grass pollen allergen B1 simultaneously by flow cytometry kits: Basotest and BD FastImmune test. Allergy diagnosis was based on clinical history and SPT. The determination of specific IgE was performed by ELISA--RIDASCREEN. RESULTS: There were no significant differences between the patient's results from Basotest and FastImmune (P>0.05). A significant correlation between values, analyzed by Basotest and by FastImmune was found (r=0.88). The sensitivity and specificity of the Basotest, FastImmune, specific IgE and SPT were 85%, 72%, 92% and 92% sensitivity and 100%, 92%, 100% and 85% specificity respectively. The efficiency was between 82% and 97%. There were a significant correlation between the specific IgE and flow cytometry tests: tau=0.92 (Basotest) and tau=0.71 (FastImmune) and a moderate significant correlation between the SPT and the in vitro tests: tau=0.26 (Basotest) and tau=0.31 (FastImmune). CONCLUSION: The successful use of the Bulgarian grass pollen allergen B1 and both flow cytometry tests was presented. These methods could be as specific tools for IgE-mediated diagnosis especially FastImmune in the case of low IgE receptor expression.

Adolescent↗

Use of flow cytometry to extend and improve in vivo determinations of microvessel hematocrit and cell flux.

The use of fluorescent-erythrocyte tracer methods for estimating microvascular variables has been extended and improved by using flow cytometry. With cell tracer methods, the precision of estimates of cell flux, hematocrit, etc. is dependent on the labeled-cell fraction (p). In larger arterioles, high cell flux and the requirement to identify each labeled cell mandate the use of very low labeled-cell fractions. Precise microvascular estimates require high total cell counts in the estimation of p. This is not practical to do manually, but can be accomplished by flow cytometry. Determinations of p in experimental samples were made by both flow cytometry and microscopy. These values were compared to each other and to calculated values. In the range 0.0045 less than p less than 0.075, where manual counting statistics give CV(p) less than 0.12, counts by microscopy and flow cytometry were not significantly different. This confirms that flow cytometry identifies the same cell population as seen in vivo. Flow cytometry counts will resolve p greater than 0.00001 for rhodamine-labeled-cell fractions and p greater than 0.0002 for fluorescein-labeled-cell fractions. Rhodamine-labeled-cell fraction samples analyzed when fresh or after 1-3 weeks storage in RPMI 1640 media (GIBCO) gave p values which agreed to within 4%. We identify the use of rhodamine-labeled cells as a simple yet powerful method of rare-event analysis for many in vivo applications.

Animals↗

Fringe-scan flow cytometry.

We describe the development of a scanning flow cytometer capable of measuring the distribution of fluorescent dye along objects with a spatial resolution of 0.7 micron. The heart of this instrument, called a fringe-scan flow cytometer, is an interference field (i.e., a series of intense planes of illumination) produced by the intersection of two laser beams. Fluorescence profiles (i.e., records showing the intensity of fluorescence measured at 20 ns intervals) are recorded during the passage of objects through the fringe field. The shape of the fringe field is determined by recording light scatter profiles as 0.25 micron diameter microspheres traverse the field. The distribution of the fluorescent dye along each object passing through the fringe field is estimated from the recorded fluorescence profile using Fourier deconvolution. We show that the distribution of fluorescent dye along microsphere doublets and along propidium iodide stained human chromosomes can be determined accurately using fringe-scan flow cytometry. The accuracy of fringe-scan shape analysis was determined by comparing fluorescence profiles estimated from fringe-scan profiles for microspheres and chromosomes with fluorescence profiles for the same objects measured using slit-scan flow cytometry.

Chromosomes↗

Detection of anti-HLA antibodies with flow cytometry in needle core biopsies of renal transplants recipients with chronic allograft nephropathy.

The aim of this study was to assess the feasibility of detecting anti-HLA antibodies in eluates from needle core biopsies of renal transplants with chronic allograft nephropathy. Two methods of screening, the enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FlowPRA) were compared. Twenty renal transplants with CAN were removed after irreversible graft failure. To assess the feasibility of detecting anti-HLA antibodies in small samples, needle core biopsies were sampled at the same place as surgical samples and at a second cortical area. Antibodies were eluted with an acid elution kit and anti-class I and class II IgG HLA antibodies detected using ELISA and flow cytometry. Flow cytometry was found to be more sensitive than ELISA for detecting anti-HLA antibodies in eluates from renal transplants with CAN (95% vs. 75% of positive cases). Detection of anti-HLA antibodies showed good agreement between surgical samples and needle core biopsies performed at the same place for anti-class I (80% vs. 65%, r=0.724 P<0.01) and anti-class II HLA antibodies (70% vs. 55%, r=0.827 P<0.01). In addition, differences in the detection of anti-class I HLA antibodies in needle core biopsies sampled at different sites suggests that immunization to class I donor antigen could be underestimated in needle core biopsy samples. These data indicate that anti-HLA antibodies can be detected in needle core biopsies from renal transplants. Provided further evaluation is done, elution might be a complementary method to detect anti-HLA antibodies when they are bound to the transplant.

Antibodies↗

Aspiration flow cytometry of the testes in the evaluation of spermatogenesis in the infertile male.

Deoxyribonucleic acid (DNA) flow cytometry of testicular tissue has been demonstrated to be a quantitative means of assessing spermatogenesis. This study evaluates testes aspirates and DNA flow cytometry in the evaluation of the infertile male. Testicular tissue obtained from 12 men who underwent bilateral orchiectomy for prostate cancer (group 1) were examined by both flow cytometry and standard histologic technique to assess the correlation between these two modalities. Thirteen men evaluated for infertility (group 2) and requiring histologic evaluation of spermatogenesis underwent both open biopsy and fine needle aspiration of their testes. Histology was independently examined and grouped according to standard nomenclature. Flow cytometric analysis revealed characteristic patterns in the relative numbers of haploid (1C), diploid (2C), and tetraploid (4C) cells. These patterns correlated reproducibly with the histologic diagnoses. DNA flow cytometry of testicular aspirates provides a rapid and reliable quantitative means of assessing spermatogenesis.

Biopsy, Needle↗

Evaluation of reticulocyte counts by flow cytometry in a routine laboratory.

Reticulocyte counting using flow cytometry and a membrane-permeable fluorescent dye, thiazole orange, was evaluated as an alternative to the conventional manual method. The flow cytometric method was more precise (mean c.v. of 4.3%) than the imprecise manual technique (mean c.v. of 22.4%). Linearity was highly acceptable (r = 0.99) over the reticulocyte count range of 1.8-30.1%. Flow cytometry allows processing of large numbers of samples with reduction in technologist time, and the improved precision and tenfold increase in the number of cells counted should considerably improve the clinical utility of the reticulocyte count.

Adult↗

Flow cytometry for automated analysis of milk containing Listeria monocytogenes.

This paper describes the application of flow cytometry to the analysis of milk for the presence of Listeria monocytogenes. A total of 939 raw milk samples from 54 farms were analyzed for the presence of L. monocytogenes by cold enrichment vs flow cytometry, which incorporated a selective enrichment step. Analyzed samples consisted of milk from individual farm bulk tanks; string samples which represented milk from 25 to 40 cows; and combination samples representing milk from 200 cows. Raw milk samples were directly enriched for 24 h at 37 degrees C and analyzed by flow cytometry following fluorescence labeling of bacterial populations, or were cold enriched for 1 month at 4 degrees C. Following cold enrichment, samples were streaked to McBride's Listeria agar, and suspect colonies were biochemically identified as L. monocytogenes. L. monocytogenes was isolated from only 15 of the analyzed samples, all of which were common to one farm. Results of flow cytometry analysis yielded a 5.86% false positive rate and a 0.53% false negative rate when compared with culture procedures.

Animals↗

Contribution of flow cytometry in the diagnosis of cutaneous lymphoid lesions.

The histologic diagnosis of cutaneous lymphoid lesions remains one of the most challenging areas of dermatopathology and is augmented by incorporation of immunophenotypic and genotypic data. To improve the analysis of surface Ig light chain expression and to increase the yield of immunophenotypic data obtained from skin biopsies, we evaluated the utility of flow cytometry in cutaneous lymphoid infiltrates. Flow cytometric immunophenotypic analyses were performed on skin specimens of 19 patients as a part of diagnostic procedures. We found that skin biopsy specimens, including a routine punch biopsy, yield sufficient material for diagnostic flow cytometry. One reactive lymphoid hyperplasia showed polyclonal B cells and no aberrant T cell populations. Ig light chain restriction was detected by flow cytometry and contributed to the diagnosis in 88% (15 of 17) of cutaneous primary or secondary B cell lymphomas, compared to 37% (three of eight) by immunohistochemistry. Nearly one-third of these cases were histologically suspicious but difficult lesions due to processing artifact, mixed cellular infiltrate, or paucity of abnormal cells. Additional markers (3-23) were analyzed by flow cytometry on 15 specimens, and contributed to subclassification of the lymphomas. Our experience demonstrates that flow cytometry can be successfully applied to routine skin biopsies and contributes to the diagnosis and subclassification of cutaneous lymphoid lesions.

Adult↗