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PubMed1987

Confirmation of Fanconi's anemia and detection of a chromosomal aberration (1Q12-32 triplication) via BrdU/Hoechst flow cytometry.

Peripheral blood lymphocyte cultures of a patient with clinical evidence of Fanconi's anemia (FA) were investigated by means of BrdU/Hoechst flow cytometry. This technique can be used to differentiate cycling from noncycling cells after PHA stimulation; it also permits the quantitative assessment of cell cycle progression and cell-cycle arrest. A characteristic cell kinetic pattern of combined G2 phase prolongation and G2 phase arrest was observed in the present patient, confirming the clinical diagnosis of FA. An additional finding was the presence of a small subpopulation of peripheral blood cells, refractory to PHA stimulation, and displaying a DNA content 5% greater than that of the diploid cells. BrdU/Hoechst flow cytometry was crucial to the unequivocal demonstration of this subpopulation, which would not have been detected without enrichment by sequential depletion of PHA-responsive cells from the G0/G1 compartment during a 96-h stimulation experiment. The parallel finding of a presumptive triplication affecting the q12-32 segment of chromosome 1 in rare spontaneous divisions of a 40-h peripheral blood culture is consistent with the flow-cytometric detection of elevated DNA content if, as is common in FA, the abnormal cell lineage represents a preleukemic clone of monocytic origin.

Adolescent↗

PubMed2003

Diagnosis of invasive septate mold infections. A correlation of microbiological culture and histologic or cytologic examination.

We correlated results of microbiologic culture and histopathologic examination for 2,891 consecutive samples from autopsy tissue, surgical or biopsy tissue, and bronchoalveolar lavage (BAL) or bronchial washing (BW) specimens. For 23 autopsy cases with suspected invasive septate mold infections by histopathologic examination, culture yielded a mold in 12 cases (52%). For 1,683 surgical or biopsy samples, histopathologic evidence of invasive septate mold infection was present in 30 samples, 9 of which also grew mold by culture (30%); 20 additional samples grew mold in culture alone, possibly representing culture contamination. Of 1,185 BAL and BW samples, mold was evident in 28 by cytologic examination and culture, 20 by cytologic examination alone, and 68 by culture alone. These results suggest a positive concordance for culture and histologic-cytologic examination of 23%, although both methods were negative in 96% of surgical and biopsy tissue and BAL and BW samples. The septate molds cultured from these samples were Aspergillus fumigatus (19), Aspergillus flavus (15), Aspergillus terreus (13), Aspergillus niger (7), Fusarium species (3), and Scedosporium apiospermum (2). A flavus was isolated significantly more frequently from tissue than from BAL and BW samples.

Aspergillosis↗

PubMed1993

Detection of intracellular cytokines by flow cytometry.

During the last years it has become increasingly clear that production of most cytokines is not confined to one cell type. Thus, a method to detect cytokines at the single cell level would be a helpful tool to study the contribution of different cells to cytokine production in heterogeneous cell populations. Recently, Sander et al. (1991) demonstrated that it is possible to detect intracellular cytokines by fixation with paraformaldehyde, permeabilization with saponin and subsequent indirect immunofluorescent staining using fluorescence microscopy. Here, we describe a modified method to increase the specific intracellular staining which enables us to detect IFN-gamma, IL-2 and IL-4 producing cells by single laser flow cytometry. The carboxylic ionophore monensin was used to interrupt intracellular transport processes leading to an accumulation of the cytokine in the Golgi complex. This resulting increase of the signal/noise ratio permitted us to detect weakly fluorescent cells such as IL-4 producing cells. While IL-4 was detected in approximately 1-3% of peripheral mononuclear cells from healthy donors, up to 30% of the cells produced IFN-gamma and nearly 50% IL-2 after phorbol ester and ionomycin stimulation. Microscopic and flow cytometric analysis showed a highly significant correlation. Using three-color flow cytometry it was possible to measure intracellular cytokines and cell surface markers simultaneously. Subpopulations of human T cells (e.g., CD4+ CD45R0-) producing a restricted cytokine pattern could be identified by cell surface staining and were characterized by their cytokine production. Consequently, there was no further need for cell sorting to determine cytokine producing subsets in heterogeneous cell populations. We have tested human T cell clones for intracellular cytokine production and found a high concordance to ELISA analysis of the supernatants. We conclude that detection of intracellular cytokines by flow cytometry is a rapid, easy and semiquantitative assay which may be used to study individual cells in heterogeneous populations as well as to screen homogeneous cells for their cytokine pattern. This method is particularly relevant in view of the accumulating evidence of the functional role that subsets of (T) cells may play in various diseases depending on the pattern of cytokines they produce.

Animals↗

PubMed1990

Differentiation of Naegleria fowleri from Acanthamoeba species by using monoclonal antibodies and flow cytometry.

Monoclonal antibodies to Naegleria fowleri and Acanthamoeba polyphaga were analyzed by enzyme-linked immunosorbent assay, indirect immunofluorescence microscopy, and fluorescence flow cytometry to assess specificity and cross-reactivity with axenically cultured N. fowleri and Acanthamoeba spp. Four monoclonal antibodies to N. fowleri were specific for N. fowleri and had no reactivity to A. polyphaga. Similarly, four monoclonal antibodies to A. polyphaga did not react with N. fowleri. Two of the four monoclonal antibodies to A. polyphaga did not react with other Acanthamoeba spp. tested, while two of the antibodies demonstrated a high degree of cross-reactivity with a putative Acanthamoeba castellanii strain by immunofluorescence microscopy; this was confirmed by fluorescence flow cytometry for one of the antibodies. These monoclonal antibodies were used to identify Acanthamoeba trophozoites in infected brain sections of a patient who died of suspected Acanthamoeba-caused granulomatous amoebic encephalitis, demonstrating potential utility in the direct identification of N. fowleri and Acanthamoeba spp. in clinical specimens.

Acanthamoeba↗

PubMed1988

DNA flow cytometry of endoscopically examined colorectal adenomas and adenocarcinomas.

DNA ploidy of 64 colorectal adenomas and 49 adenocarcinomas, examined endoscopically, was studied by flow cytometry. We found DNA aneuploidy in none of the 105 normal mucosa samples (0%), in 20 adenomas (31%), and in 36 adenocarcinomas (74%). DNA ploidy of adenomas correlated with size (P = 0.02) and degree of dysplasia (P less than 0.01) but not with histologic type. Adenomas had a 45% incidence of DNA aneuploid stem lines in the DNA index range of 0.80-1.20, compared with 8% in the case of adenocarcinomas. The distribution of the DNA index values of adenocarcinomas was approximately normal, with a mean value 1.63 +/- 0.28. The mean DNA index for the three cases of "carcinoma in adenoma" with invasion of the stalk of the adenoma was 1.52 +/- 0.18. These results, using DNA flow cytometry, provide evidence for the progression of colorectal adenoma to adenocarcinoma. The classification of adenomas according to DNA ploidy may be information of considerable practical value to the clinician in predicting risk of further adenomas and/or risk of cancer.

Adenocarcinoma↗

PubMedDate not supplied

Evaluation of circulating tumour cells expressing CD44 variants in the blood of gastric cancer patients by flow cytometry.

The occurrence of circulating tumour cells in the blood of 51 patients with gastric cancer (stages I-IV) was studied using flow cytometry, cell sorting, immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). The lysed whole blood samples were stained with monoclonal antibodies against common leukocyte antigen (CD45), epithelial membrane antigen (EMA), tumour associated glycoprotein 72kD (TAG72), CD44 variants (v5 and v6) and analysed by flow cytometry within ungated or CD45-gated populations. The frequency of detection of TAG72+, CD44v5+ and v6+ cells within CD45- gate was considerably increased in comparison to the ungated population. Furthermore, the presence of tumour cells was directly demonstrated by immunostaining for cytokeratin 18 of sorted CD45- population. The presence of CD44v5+, v6- cells and CD44v-mRNA in the blood was compared to their expression in the primary tumour. The occurrence of circulating CD44v+ cells was associated with their presence in the primary tumour and CD44v-mRNA in the blood. The method described may provide a sensitive tumour marker-independent tool for detection of circulating tumour cells in cancer patients.

Adult↗

PubMed1999

High affinity binding of fluorescein isothiocyanate to eosinophils detected by laser scanning cytometry: a potential source of error in analysis of blood samples utilizing fluorescein-conjugated reagents in flow cytometry.

BACKGROUND: In samples of peripheral blood cells processed using the commercial kits for detection of apoptosis based on DNA strand break labeling, a subpopulation of cells characterized by high green fluorescence, similar in intensity to that of apoptotic cells but more uniform, was consistently observed by flow cytometry. The labeled cells had no other features of apoptosis. The labeling was observed regardless of the fixative used and was evident in control samples lacking terminal deoxynucleotidyltransferase. Common to all the kits that generated this labeling pattern was the presence of fluorescein (f) conjugated reagents, f-dUTP, f-avidin, or f-antibody. METHODS: Laser scanning cytometry was used to identify the labeled cells and study the mechanism of labeling. Because it was suspected that the traces of unconjugated f-isothiocyanate (FITC) that may contaminate the reagents were responsible for the labeling, FITC binding affinity to white blood cells was studied. Gel electrophoresis was used to detect the presence of unconjugated FITC in the reagents. RESULTS: After staining with Giemsa, the strongly fluorescent objects were identified as eosinophils with normal morphology and no evidence of apoptosis. The fluorescence was localized exclusively within the cytoplasmic granules. Labeling of eosinophils was observed at 2 nM concentration of FITC, which was over three orders of magnitude lower than that needed to label neutrophils, monocytes, or lymphocytes. Gel electrophoresis of the f-conjugated reagents revealed only minor contamination with FITC. CONCLUSIONS: (1) Trace amounts of unconjugated FITC contaminating the reagents are adequate to strongly label eosinophils thereby introducing experimental bias in analysis of apoptosis and in other studies on blood cells utilizing f-labeled antibodies, e.g., in detecting cytokines. (2) FITC at concentration 2-500 nM can be used as a marker of eosinophiles; (3) Because of high affinity to FITC, eosinophiles (or the protein from these cells) may serve as a means of removing traces of unconjugated FITC from the reagents during their manufacture or prior to use.

Apoptosis↗

PubMedDate not supplied

Flow cytometry study of the role of superoxide anion and hydrogen peroxide in cellular photodestruction with 5-aminolevulinic acid-induced protoporphyrin IX.

Flow cytometry was used to investigate the participation of reactive oxygen species, other than singlet oxygen, in the cytotoxic effect of photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) in vitro in A-431 squamous cell carcinoma (SCC) cells and human skin fibroblasts (HSF). We used propidium iodide to determine cellular cytotoxicity, hydroethidine to measure intracellular superoxide anion (O2-) and dihydrorhodamine 123 to assess intracellular hydrogen peroxide (H2O2) content. Our data support the importance of the incubation time with ALA in the selectivity of PDT with ALA against SCC cells, inducing minimum damage on normal HSF. Photoradiation mortality curves of the response of these cell lines to ALA-induced PpIX photosensitization correlated with the extent of photosensitizer accumulation. Intracellular O2- production correlated with cell death, increasing both in a light dose-dependent fashion in ALA treated cells. This correlation was not observed with H2O2-intracellular production. These results suggest the effectiveness of PDT with ALA in vitro in SCC, the significant participation of O2- in its phototoxic mechanism, and the usefulness of flow cytometry in the study of the cytotoxic effect of ALA-induced PpIX PDT.

Adult↗

PubMed2000

Capture ELISA and flow cytometry methods for toxicologic assessment following immunization and cyclophosphamide challenges in beagles.

The purpose of this subacute 22-day study was to evaluate methods for canine circulating immunoglobulins (IgM, IgG, and IgE) and select B- and T-lymphocyte populations (CD4-helpers, CD8-suppressors, pan-T and pan-B) for immunotoxicity testing using an organ system (concordance) approach. The challenge substance for immunoglobulin testing was repeated immunization with six-way distemper vaccination (DHLAPP), while the challenge substance for leukocyte subpopulations was treatment with cyclophosphamide. Immunoglobulin measurements were made by capture enzyme-linked immunosorbent assay (ELISA), and leukocyte immunophenotyping by fluorescein isothiocyanate/phycoerythrin conjugation (flow cytometry). A battery of parameters that would be used in a typical regulatory study were taken to aid interpretation of the data generated by these methods. Body weights, food consumption, clinical observations, complete clinical chemistry and urinalysis measurements were taken. Gross pathology and micropathology of sternal bone marrow, spleen, mesenteric and retropharyngeal lymph nodes, thymus, liver and kidney were completed. The ELISA method demonstrated acceptable intra-assay reproducibility for IgM, IgG and IgE, with values in good agreement as reported for radial immunodiffusion. The immunologic challenge demonstrated a biological trend of an increase in IgM that preceded an increase in IgG with no discernible trend in IgE response, and no abnormalities in lymphocyte subpopulations. Principle flow cytometry findings related to cyclophosphamide were that the relative percent of B cells decreased dramatically and progressively after compound administration; being statistically decreased in males on day 22 compared with day -5. The relative percent CD4 and CD8 contribution increased, but the CD4/CD8 ratio remained relatively unchanged as total white blood cells decreased progressively. The increase in relative percent CD4 (males only) was statistically significant according to a two-sample t-test on days 17, 20 and 22 when compared with the pre-treatment day -5. There was a relative percent increase in CD5-panT, but absolute numbers were dramatically decreased. We conclude that an organ system approach to assessment of the immune system which incorporates humoral antibody, enumeration of lymphocyte populations and pathologic evaluation of the lymphoreticular organs assists in the interpretation of an adverse toxicological response. The ELISA method for measurement of Igs detected the expected levels of IgG, IgM and IgE due to repeated vaccinations and to cyclophosphamide treatment. The flow cytometry method was acceptable for measuring select canine lymphocyte populations and detecting the expected decrease in B cells due to cyclophosphamide treatment. Both methods may be added to a testing battery for assessing immunotoxicityl in canine regulatory studies.

Adenoviridae↗

PubMed1996

[Measurement of proliferation of allergen-stimulated peripheral blood lymphocytes by flow cytometry].

The percentage of S-phase cells in peripheral blood mononuclear cells (PBMC) from 14 atopic and 6 non-atopic subjects was measured by flow cytometry after stimulation with mite allergen (Der f) for 6 days and converted to the stimulation index (S.I.F.: stimulation index measured by flow cytometry). S.I. F. showed a close positive correlation with the serum IgE level (r = 0.67, p = 0.001). S.I.F. in the atopic subjects was significantly higher than in the non-atopic subjects (165.6 +/- 49.8 vs 113.2 +/- 12.2, p < 0.05). S.I.F. in subjects with a high (> or = 100 PRU/ml) IgE-RAST level for mite allergen Der p was significantly higher than that in subjects with a negative RAST level (178.4 +/- 53.8 vs 113.2 +/- 12.2, p < 0.02). Flow-cytometric measurement of the proliferation of PBMC in response to allergenic stimulation may be a useful method for studying the immunological abnormality present in atopic subjects.

Adolescent↗

PubMed2000

The effects of heparin, protamine, and heparinase 1 on platelets in vitro using whole blood flow cytometry.

UNLABELLED: The effects of heparinization and the reversal of heparin activity on platelet function after cardiopulmonary bypass have not been well defined. Flow cytometry has become a convenient and powerful technique for characterizing platelets. We examined the expression of a secretion marker (P-selectin) and an aggregation marker (activated fibrinogen receptor GP IIb-IIIa) on normal platelets in response to heparin, heparinase 1, and protamine in vitro using whole blood flow cytometry. Unfractionated heparin increased adenosine diphosphate-induced expression of P-selectin and GP IIb-IIIa in a dose-dependent manner. Heparinase 1 alone decreased both markers of platelet activation. Protamine alone increased P-selectin expression but had no effect on GP IIb-IIIa expression. Heparinase 1 antagonized the stimulatory effect of heparin on both markers. In contrast, protamine antagonized the effect of heparin on GP IIb-IIIa expression but potentiated the effect of heparin on P-selectin expression. These in vitro observations suggest that 1) both heparin and its reversal agents affect platelet secretion and aggregation, and 2) heparinase 1 reverses heparin-induced platelet preactivation more effectively than protamine. IMPLICATIONS: This experimental in vitro study demonstrates that heparin and its reversal agents affect platelet secretion and aggregation.

Adult↗

PubMed1997

The use of isotypic control antibodies in the analysis of CD3+ and CD3+, CD4+ lymphocyte subsets by flow cytometry. Are they really necessary?

OBJECTIVE: Isotypic control reagents are defined as irrelevant antibodies of the same immunoglobulin class as the relevant reagent antibody in a flow cytometry panel. The use of the isotypic control antibody has been advocated as a necessary quality control measure in analysis of flow cytometry. The purpose of this study was to determine the necessity of an isotypic control antibody in the analysis of CD3+ and CD3+, CD4+ lymphocyte subsets. MATERIALS AND METHODS: We performed a prospective study of 46 consecutive patient samples received for lymphocyte subset analysis to determine the need for the isotypic control. For each sample, a sham buffer (autocontrol) and isotypic control reagent were stained for three-color immunofluorescence, processed, and identically analyzed with Attractors software. The Attractors software allowed independent, multiparametric, simultaneous gating; was able to identically and reproducibly process each list mode file; and yielded population data in spreadsheet form. RESULTS: Statistical analysis (Fisher's z test) revealed no difference between the CD3+ autocontrol and CD3+ isotypic control (correlation = 1, P < .0001) or between the CD3+, CD4+ autocontrol and the CD3+, CD4+ isotypic control (correlation = 1, P < .0001). The elimination of the isotypic control reagent resulted in a total cost savings of $3.36 per test. Additionally, the subtraction of isotypic background can artifactually depress population enumeration. CONCLUSIONS: The use of an isotypic control antibody is not necessary to analyze flow cytometric data that result in discrete cell populations, such as CD3+ and CD3+, CD4+ lymphocyte subsets. The elimination of this unnecessary quality control measure results in substantial cost savings.

Antibodies↗

PubMed1984

Chromosome analysis by flow cytometry: a review.

The latest developments in the application of flow cytometry to the analysis and fractionation of metaphase chromosomes is reviewed. This includes the preparative techniques, staining and machine technology used in flow karyotype production. In particular, the application of this technology to the study of chromosomal changes and to the construction of chromosomal specific DNA libraries is reviewed.

Chromosome Mapping↗

PubMed1993

Detection of HIV-1 DNA and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry.

Human immunodeficiency virus type-1 (HIV-1) DNA and messenger RNA sequences in both cell lines and blood obtained directly from HIV-1-infected patients were amplified by polymerase chain reaction and hybridized to fluorescein-labeled probes in situ, and the individually labeled cells were analyzed by flow cytometry. After flow cytometric analysis, heterogeneous cell populations were reproducibly resolved into HIV-1-positive and -negative distributions. Fluorescence microscopy showed that the cellular morphology was preserved and intracellular localization of amplified product DNA was maintained. Retention of nonspecific probe was not observed. Analysis of proviral DNA and viral messenger RNA in cells in the blood of HIV-1-infected patients showed that the HIV-1 genome persists in a large reservoir of latently infected cells. With the use of this technique it is now possible to detect single-copy DNA or low-abundance messenger RNA rapidly and reproducibly in a minor subpopulation of cells in suspension at single-cell resolution and to sort those cells for further characterization.

Base Sequence↗

PubMed1990

A rapid technique for lymphocyte preparation prior to two-color immunofluorescence analysis of lymphocyte subsets using flow cytometry. Comparison with density gradient separation.

A technique is described for lymphocyte preparation which permits analyses by two-color immunofluorescence and flow cytometry. It consists, briefly, of the lysis of red blood cells and washing of white blood cells prior to labeling. We tested this technique with a large panel of monoclonal antibodies in mono- and dual immunofluorescence. By comparing these results to those obtained after density gradient separation, we found the following statistically significant differences: the count of the phenotype B1+ was higher after whole blood lysis preparation than after density gradient separation; whereas, the corresponding counts of OKT4+ and Leu-4-Leu-7+ phenotypes were lower. No difference was detected with OKT8+, Leu-4+, OKT8+Leu-4+, OKT8+Leu-4-, OKT8-Leu-4+, OKT8+Leu-7+, Leu-4+Leu-7+, Leu-4-Leu-11c+, OKT8+Leu-11c+ and OKT8+Leu-15+ phenotypes. We have studied the reproducibility of both methods and the correlation between them. The disparity of the lymphocyte subset count between these two methods, though statistically significant, was relatively weak and seems to be due to the density gradient separation. Since the preparation of lymphocytes using the density gradient method is time consuming, we propose whole blood lysis as an alternative lymphocyte separation method when assessing immune status in human disease by flow cytometry. It offers the following advantages: (i) it does not require additional steps, (ii) it permits two-color immunofluorescence through the labeling of white blood cells after washing, (iii) it is reliable, (iv) it is reproducible, and (v) it is helpful in studies of lymphopenia since it offers the possibility of lymphocyte enrichment.

Antigens, CD↗

PubMed2006

A comparison of the sensitivity of flow cytometry and bone marrow biopsy in the detection of minimal residual disease in chronic lymphocytic leukemia.

We compared the sensitivity of bone marrow biopsy to blood flow cytometry in detecting minimal residual disease (MRD) in 29 patients with chronic lymphocytic leukemia (CLL) in clinical remission after treatment. These results demonstrate that flow cytometry is more sensitive than bone marrow biopsy in detecting MRD and in predicting relapse in CLL.

Biopsy↗

PubMedDate not supplied

Correlation of (S+G2)-phase fractions determined by flow cytometry and Ki-67 labelling indices in colorectal carcinomas.

Samples from 14 human colorectal carcinomas were analysed by means of classical (one-parameter) flow cytometry. (S+G2)-phase fractions were calculated from DNA histograms with the help of two different algorithms. At the same time, the percentage of cells positive for the Ki-67 antibody, which recognizes proliferating cells, was determined in cytocentrifuge preparations. Significant correlations between the two indices were found only if the histogram analysis included background subtraction and aggregate correction. This finding may throw some light on the reliability of prognosis based on flow cytometry.

Aged↗

PubMed1998

Phagosomal pH determination by dual fluorescence flow cytometry.

Several methods have been developed to measure the pH of phagosomes, using fluorescein derivatives as reporter of pH, and spectrofluorimetry, fluorescence microscopy, or flow cytometry as quantification technique. All have major disadvantages, including either a slow or inaccurate response. In the present study, pH determination was achieved on J774-cell phagosomes containing dual-labeled zymozan particles using dual fluorescence flow cytometry with an argonion laser excitation wavelength at 488 nm. This allowed zymozan-containing macrophages to be distinguished from other cells and their fluorescence to be measured rapidly. The use of a new probe, namely Oregon Green 488 which has a pKa lower than carboxyfluorescein with the same maximum excitation and emission wavelengths, allowed investigation of pH value below 5. The dual labeling with Oregon Green 488 and carboxytetramethylrhodamine as pH-sensitive and pH-insensitive probes, respectively, overcame the absence of an isobestic point in the Oregon Green 488 spectrum. The phagosomal pH was determined using a calibration curve of phagosomal pH established by adding ionophores in phagocyte suspension and measuring the fluorescence intensity ratio (535 nm/585 nm) for different pHs. A phagosomal pH of 4.5 +/- 0.1 can be accurately determined. This method permits pH measurements down to 4, even in the presence of nonengulfed reporter particles.

Animals↗