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PubMed1991

A simple diagnostic test for Fanconi anemia by flow cytometry.

A simple diagnostic test for Fanconi anemia (FA) by flow cytometry is proposed. It is based on the cell cycle disturbances of FA cells and their sensitisation by alkylating agents. Following PHA-stimulation of whole blood cell cultures in the presence or absence of nitrogen mustard, the accumulation of cells in G2/M phase was measured. A sharp increase of cells in G2/M was observed in cultures from FA patients when nitrogen mustard was added. This increase allows one to distinguish FA patients from patients with anemias of other origin, healthy controls, and FA heterozygotes, as effectively as chromosome breakage studies. The rapidity of the test and its reliability as demonstrated on the ten FA patients studied, will make the diagnosis of FA easier in centers without cytogenetic laboratory facilities.

Cell Cycle↗

PubMed1993

The uses and limitations of DNA flow cytometry in stage IB or II cervical carcinoma.

BACKGROUND: The role of flow cytometry in predicting prognosis for cervical carcinoma remains unclear. METHODS: Flow cytometric analysis was performed on tissues, fixed in formaldehyde solution and embedded in paraffin, from 411 patients with Stage IB or II cervical carcinoma who had been treated with radical abdominal hysterectomy and bilateral pelvic lymphadenectomy. RESULTS: DNA aneuploid-multiploid tumors were found in 37.5%, tetraploid in 4.6%, and diploid-peridiploid in 57.9%. Five-year recurrence-free survival rates of the three groups were 74.3%, 77.8%, and 76.4%, respectively (P > 0.05). DNA aneuploidy and DNA index (DI) of greater than 1.3 were highly correlated to parametria extension. In univariate analysis, pelvic lymph node metastases, stage, parametrial extension, depth of cervical stromal invasion, tumor size, and DI (1.3, 1.4, 1.5 as breakpoint) were significant prognostic factors. DNA ploidy, S-phase fraction, and S-G2M fraction were not significant. In multivariate analysis, DI of greater than 1.3, pelvic node metastases, clinical Stage II, and depth of stromal invasion greater than two-thirds of full cervical thickness were independent and significant variables. The prognostic index (PI), defined by the model, was able to categorize the patients into three distinct risk groups. The 5-year recurrence free survival rates of the low-, intermediate-, and high-risk groups were 89.5%, 73.0%, and 58.9%, respectively (P < 0.0001). CONCLUSIONS: The prognostic value of the DI as a single variable is promising and warrants additional investigation to establish its appropriate use.

Adult↗

PubMed1998

Methodological aspects of assessing phagocytosis of Vibrio anguillarum by leucocytes of gilthead seabream (Sparus aurata L.) by flow cytometry and electron microscopy.

In this paper we optimize a flow cytometric method for evaluating the phagocytic activity of leucocytes in gilthead seabream (Sparus aurata L.) and characterize the phagocytic cells observed. Optimal conditions were established for the fluorescein-labelling and analysis of the bacterium Vibrio anguillarum by flow cytometry. Head-kidney leucocytes were incubated with the heat-killed fluorescein isothiocyanate (FITC)-labelled bacteria for different periods, during which the kinetics of phagocytosis was studied. Attached and interiorized bacteria were distinguished. Although phagocytic ability reached a maximum after 60 min, phagocytic capacity reached its maximum at 20 min. The amount of ingested bacteria per phagocyte was estimated from the mean fluorescence of the leucocytes. Cytochalasin B or colchicine was used to inhibit phagocytosis. Monocyte-macrophages and acidophilic granulocytes showed phagocytic activity as demonstrated by transmission electron microscopy. In conclusion, the technique presented allows the screening of thousands of cells, and individual cell evaluation, by quantifying interiorized particles in fish phagocytes. Our ultrastructural results demonstrate that V. anguillarum is actively phagocytized by seabream macrophages and acidophilic granulocytes.

Animals↗

PubMed1988

DNA content and genetic evolution of human colorectal adenocarcinoma. A study by flow cytometry and cytogenetic analysis.

We have conducted in parallel DNA flow cytometry (FCM) and cytogenetic (CG) analysis of a series of surgical specimens from 35 human colorectal adenocarcinomas. An excellent quantitative correlation was observed (r = 0.99) between modal peak values of FCM histograms and chromosome counts. This observation confirms that aneuploidy, as defined by FCM, accurately reflects the deviation from diploidy of the genomic DNA. FCM-derived DNA patterns have been analyzed in the context of the clonal chromosomal evolution determined by CG analysis. In the metaphases of a given tumor, even if karyotypes of different ploidy exist, the presence of identical marker chromosomes suggests a common origin for the multiple populations observed by FCM. Thus, heterogeneity in DNA content within a tumor, including the polyploidization step, would be indicative of genetic evolution.

Adenocarcinoma↗

PubMed1993

Rapid diagnosis of malignancy using flow cytometry.

The rapid and accurate diagnosis of childhood malignancy is important both in the planning of appropriate treatment and in relieving the inevitable family anxiety. The use of flow cytometry to analyse monoclonal antibody coated single cell suspensions is widely accepted as having increased the speed and accuracy of diagnosis in leukaemias, though its use in solid tumour diagnosis is not widely reported. Ten cases of childhood malignancy in whom the diagnosis was initially made by flow cytometry and subsequently confirmed histologically are described. The technique has a number of advantages. Only a small sample is required as the analysis is carried out on a single cell suspension, the method is rapid, a diagnosis being reached within three hours of receipt of the sample, and information is obtained on cell lineage and stage of differentiation. Diagnostic accuracy is good when compared with histological results.

Adolescent↗

PubMed2005

Safe susceptibility testing of Mycobacterium tuberculosis by flow cytometry with the fluorescent nucleic acid stain SYTO 16.

The time needed to obtain susceptibility results of Mycobacterium tuberculosis using classical methodologies is still too long, and flow cytometry is a promising technique in the setting of the clinical laboratory, giving fast results. A safe, reliable and rapid method to study susceptibility to streptomycin, isoniazide, rifampicin and ethambutol is described. Isolates of mycobacteria, grown for 72 h in the absence or presence of antimycobacterial drugs in the mycobacteria growth indicator tube (MGIT), were heat-killed, stained with SYTO 16 (a nucleic acid fluorescent stain that only penetrates cells with severe lesion of the membrane) and then analysed by flow cytometry. Sixteen strains with different susceptibility patterns were tested and an excellent correlation with the BACTEC MGIT 960 protocol for susceptibility was shown. In contrast to resistant strains, sensitive strains lose their cellular integrity after incubation with antimycobacterial drugs, allowing SYTO 16 to penetrate the cells. Comparing the intensity of fluorescence of Mycobacterium cells incubated with antimycobacterial drugs with that of drug-free cells, after staining with SYTO 16, it was possible to distinguish between sensitive, intermediate and resistant phenotypes. Other cytometric assays have been described for mycobacteria susceptibility testing but these have lower accuracy and safety. The described flow cytometric assay is a simple, fast, safe and accurate way to determine susceptibility of M. tuberculosis.

Antibiotics, Antitubercular↗

PubMed1987

Trout and salmon erythrocytes and human leukocytes as internal standards for ploidy control in flow cytometry.

Control of technique and use of biological standards in flow cytometry have become increasingly important due to the wider use of the method for ploidy determination of malignant tumors in clinical research. Trout (TRBC) and salmon erythrocytes and human buffy coat leukocytes were selected for a study of factors influencing the DNA stainability. Whether standard and test cells were mixed before or after enzymatic treatment and staining was found to be critical for the ploidy comparisons. Otherwise, artifactual differences of at least 20% may be noted, leading to an overestimation of DNA aneuploidy. The time from staining to analysis had minimal effect, with some exceptions. The proportions of different cells in the sample had no influence, and nonlinearity of measurements was negligible. Diploid cells in normal endometrium and benign ovarian tumors, as well as the diploid fraction of aneuploid tumor cells, were systematically measured to have a DNA staining 5-7% above human leukocytes.

Aneuploidy↗

PubMed1989

Characteristics of rat hepatocytes sorted by fluorescence-activated flow cytometry. Effects of mixed function oxidase inducers.

Fluorescence-activated flow cytometry has been used to separate rat liver parenchymal cells into five equal computer-generated regions on the basis of their mixed function activities towards diethoxyfluorescein. Enzymic activities in the sorted fractions showed 2.3-2.9-fold enrichment of 3-cyano-7-ethoxycoumarin O-deethylase, lactate dehydrogenase and pyruvate kinase activities in the cells of region 5. Cytochrome P-450 PBla and glutamine synthase activities were enriched 1.8- and 7-fold respectively in region 2 hepatocytes. Pretreatment of rats with phenobarbitone or 3-methylcholanthrene did not change the pattern of enrichment with respect to monooxygenase or lactate dehydrogenase activities. However, cytochrome P-450 PB3a and MC1a respectively were concentrated in the cells from region 5. In contrast, ethoxyquin caused enrichment of cytochrome P-450 in the hepatocytes in region 2. This fraction also contained the highest percentage of gamma-glutamyltranspeptidase positive cells.

Animals↗

PubMed1996

Measurement of glycoprotein IIb/IIIa blockade by flow cytometry with fluorescein isothiocyanate-conjugated crotavirin, a member of disintegrins.

The blockade of platelet membrane glycoprotein IIb/IIIa by a monoclonal antibody, 7E3, was measured by flow cytometry using a fluorescein isothiocyanate-conjugated disintegrin, FITC-crotavirin, as the probe. After treatment of platelets with 7E3 or 7E3 F(ab')2, there is a good correlation between the inhibition of platelet aggregation and the blockade of FITC-crotavirin's binding to platelets. The content of glycoprotein IIb/IIIa for the subsequent binding of FITC-crotavirin to the 7E3-pretreated platelets highly correlated to the extent of glycoprotein IIb/IIIa, remaining available. It was evidenced by the observation that the sum of glycoprotein IIb/IIIa occupation by 7E3 and that of FITC-crotavirin approached the total amount of glycoprotein IIb/IIIa expressed on the platelet membrane. This indicates that the percentage inhibition of FITC-crotavirin's binding at the saturation dose reflects the extent of glycoprotein IIb/IIIa blockade by 7E3. At the saturation binding concentration (5 micrograms/ml), FITC-crotavirin did not displace platelet bound 7E3. Gating the light-scattering profile for platelets, the binding of FITC-crotavirin to platelet glycoprotein IIb/IIIa could be easily determined in diluted whole blood by direct stain method. The available unoccupied glycoprotein IIb/IIIa of platelets in the 7E3 or 7E3 F(ab')2-pretreated whole blood were measured by flow cytometry at the saturation binding dose of FITC-crotavirin (4 micrograms/ml) and the data showed that the higher deconcentration of antibody added into whole blood, the lower debinding of FITC-crotavirin to platelets. This technique may provide an alternative rapid method for measuring the blockade of glycoprotein IIb/IIIa by 7E3, a promising anti-thrombotic agent, thus providing a monitoring method for adjusting the therapeutic dose of 7E3 or its related derivatives.

Antibodies, Monoclonal↗

PubMed2001

Detection by flow cytometry of antibodies against surface and intracellular granulocyte antigens.

A flow cytometric technique was used to detect granulocyte antibodies, with attention to the distinction between antibodies directed against surface and intracellularly expressed antigens. Ten serum samples with positive results and 10 with negative results detected by the granulocyte immunofluorescence test (GIFT), together with 10 positive serum samples detected by an indirect immunofluorescence test (IIF) were analyzed against leukocytes from healthy blood donors tested by flow cytometry (FC). Unpermeabilized and permeabilized cells were used to allow identification of surface and intracellular binding, respectively. The results of testing by GIFT corresponded with those by FC, with the exception of the results for four sera: one serum sample was FC negative, GIFT positive, and three samples were FC positive, GIFT negative. The IIF-positive sera were all FC positive, analyzed against permeabilized granulocytes, and one serum was also positive against non-permeabilized granulocytes. Analysis by FC is a readily performed technique, which can be used for the routine detection of antibodies against leukocyte antigens. Screening for granulocyte-specific antibodies can be carried out with pools of granulocytes from three donors. Analysis by FC allows detection of both HLA antibodies and granulocyte-specific antibodies and by using both unpermeabilized and permeabilized cells, antibodies against surface and intracellular antigens, respectively, can be identified.

Adult↗

PubMed1996

Measurement of intracellular pH in cultured cells by flow cytometry with BCECF-AM.

This study evaluates the suitability of flow cytometry with the fluorochrome BCECF for measuring the intracellular pH (pHi) of cultured cells, and monitors the changes in pHi in murine hybridoma in batch culture and chick embryo fibroblast in monolayer culture (5th passage). The technique produced highly reproducible, repeatable results. The theoretical sensitivity from the calibration curve was 0.0004 pH units. But analysis of the standard deviation of the histogram of the green/red fluorescence ratios indicated a mean sensitivity of 0.08 (0.07-0.09) pH units. Interference due to cell size, fluorochrome incorporation and esterases were minimized by establishing a calibration curve with the cells whose pHi was to be measured using the 525/610 nm fluorescence ratio after excitation at 488 nm. The pHi of exponentially growing, batch cultured hybridomas was 7.50 at the start of culture. pHi increased during the exponential growth phase and dropped towards cell death. The pHi of the chick fibroblasts in monolayer culture was 7.30.

Animals↗

PubMed1990

[Analysis of cellular DNA-RNA content using flow cytometry between primary and metastatic foci in lung carcinoma].

By flow cytometry, cellular DNA-RNA content was analyzed with Acridine Orange staining for primary tumor and the metastatic foci in 12 cases of primary lung carcinoma. Five (42%) of the primary and metastatic foci showed heterogeneous cellular DNA content, with high cellular DNA content of metastatic foci in all cases. Cellular RNA content was significantly higher in metastatic foci than in primary foci, indicating possible higher proliferative activity of the former foci. Three different metastatic foci in one patient showed essentially the same cellular DNA content. From the above it is evident that primary and metastatic foci of lung carcinoma show considerable heterogeneity not only in cellular DNA content but RNA content as well, thus indicating proliferative activity of the metastatic focus. It is consequently advisable to use a combination of various supplementary medications differing in cytocidal effect. Cases with higher cellular DNA content may likely have higher metastatic potential.

Adenocarcinoma↗

PubMedDate not supplied

Prognostic significance of DNA-analysis by flow cytometry in non-Hodgkin's lymphoma.

DNA analysis by flow cytometry was performed on lymph node cells obtained from 65 untreated patients with non-Hodgkin's lymphoma. According to the Kiel classification 41 cases were of low grade malignancy and 24 cases belonged to the high grade malignancy group. 47 out of 61 evaluable cases were diploid/near-diploid, 12 were aneuploid and 2 cases showed polyploidy. No difference in survival was found between diploid/near-diploid and aneuploid cases. The percentage of S-phase cells was found to be a valuable prognostic parameter. Using a cut point at 4 per cent S-phase cells between low and high S-phase lymphomas a highly significant difference in survival was demonstrated (p = 0.0004). The actuarial survival up to 3 years was about 77 per cent for lymphomas with less than 4 per cent S-phase cells to be compared with 20 per cent for patients with high S-phase (greater than or equal to 4 per cent) lymphomas. Subdivision according to the percentage of S-phase cells seemed to be a better prognostic predictor than morphologic subclassification.

DNA, Neoplasm↗

PubMed1988

DNA analysis of stimulated lymphocytes by automatic sampling for flow cytometry.

A microsample delivery system (MSDS) was tested for automatic flow cytometry (FCM) analysis of DNA synthesis in stimulated human peripheral blood lymphocytes (PBL) cultivated in wells of microtiter plates. After incubation, either for 1-3 days with phytohemagglutinin, concanavalin A, and pokeweed mitogen, or for 7 days with allogenic PBL, the cells, while in the wells, were washed in hypotonic Tris buffer and stained with ethidium bromide-RNAse solution. The results obtained from quintuplicate replicated wells, each of the five containing the same control or stimulated cultures, were reproducible in terms of the number of nuclei counted in each histogram of control, mitogen-stimulated PBL, and mixed lymphocyte cultures (MLC). Using a computer program that superimposes histograms and calculates their differences on the scale of fluorescence intensity, it was possible to quantify the intensity of the response to the mitogenic stimuli. This approach to the study of lymphocyte proliferation offers not only a simpler and faster analysis of DNA synthesis than the method of 3H-thymidine incorporation, but it also allows for the analysis of other FCM parameters, such as forward and 90 degrees light scatter and double fluorescence labelling of PBL nuclei.

Cell Cycle↗

PubMed1989

DNA flow cytometry as a predictor of outcome of stage I renal cell carcinoma.

The features most frequently used in predicting the outcome of renal cell carcinoma are stage at presentation and nuclear grade. Recently DNA ploidy pattern, as detected by DNA flow cytometry has also been shown to be predictive. In this study DNA flow cytometry was performed on formalin-fixed paraffin-embedded tissue from 50 patients with Stage I renal cell carcinoma for whom long-term follow-up data were available. Two were eliminated for technical reasons. Of the 48 evaluable tumors, 25 (52%) were diploid, 19 (40%) were nondiploid, and in four, (8%) the ploidy was uncertain. The ploidy pattern was statistically significantly associated with nuclear grade (P less than 0.02), and primary tumor size (P less than 0.05) but did not correlate with cell type, microscopic growth pattern, or the presence or absence of mitotic activity. In the group as a whole, ten patients (21%) died of renal cell carcinoma, seven of 19 (37%) with nondiploid tumor patterns, and two of 25 (8%) with a diploid pattern (P less than 0.03). One of four patients (25%) with tumors of uncertain ploidy also died. However, only two factors, nuclear grade and primary tumor size, were independent predictors of outcome. For Stage I renal cell carcinoma, ploidy can significantly predict patient outcome and correlates with nuclear grade and tumor size, but is not an independent predictive variable.

Carcinoma, Renal Cell↗

PubMed1985

DNA and RNA determination in 111 cases of childhood acute lymphoblastic leukaemia (ALL) by flow cytometry: correlation of FAB classification with DNA stemline and proliferation.

Flow cytometry with acridine orange was used in 111 cases of childhood acute lymphoblastic leukaemia (ALL) to determine simultaneously DNA index, cell cycle distribution and cellular RNA content in bone marrow samples. The overall incidence of DNA aneuploidy in the population was 40%. Significantly higher incidence of DNA aneuploidy was present in the L2 (70.6%) as compared to the L1 (34.1%) group (P = 0.007), using the FAB classification. No difference in the frequency of normal and abnormal DNA stemlines was found between newly diagnosed and relapsed patients. The L2 group had significantly higher proliferation (S phase = 12.4%) than L1 (S phase = 5.8%) (P = 0.01), but RNA content was not significantly different. The aneuploid group had significantly more (P = 0.01) cases with L2 morphology (28.6%) compared to the diploid group (8%) and proliferation was higher in DNA aneuploid (S-phase = 9.5%) compared to DNA diploid (S-phase = 4.7%) leukaemias. Likewise, RNA content was significantly higher in aneuploid than in diploid ALL (P = 0.006). These correlations between morphology, cell kinetics and DNA index elucidate biological properties of leukaemic cells with potentially prognostic value.

Adolescent↗

PubMed1983

DNA-flow cytometry of blood and bone marrow in chronic myelogenous leukemia.

Flow cytometric analysis of bone marrow aspirates and blood samples in 42 patients with chronic myelogenous leukemia (CML) at various disease stages was performed to determine the size of the S-phase compartment of bone marrow and blood. 25 healthy controls were studied for comparative information with both DNA-flow cytometry (DNA-FCM) and 3H-thymidine autoradiography. A correction procedure was applied for peripheral nucleated cell admixture in bone marrow aspirates. The fraction of peripheral nucleated cells in bone marrow aspirates (Fpb) in individual patients was considerable, especially in those with a very high white blood cell count (greater than 100 x 10(9)/l). The size of the S-phase compartments of bone marrow (% Sbm) in patients with CML at diagnosis and in patients at apparent hematological remission was of the same order of magnitude as in normal bone marrow. However, in 3 out of 4 patients at malignant metamorphosis in which the % Sbm could be reliably determined, this percentage was significantly higher than normal (p = 0.013). In 4 out of 11 patients at malignant metamorphosis aneuploidy was noticed. From these findings it is concluded that bone marrow cell proliferation in CML patients at diagnosis and during apparent remission is not essentially different from normal. However, at malignant metamorphosis changes occur in ploidy level and proliferative activity, which can be detected by DNA-FCM already in an early phase.

Adult↗

PubMed1995

Cellular immunity in hemodialysis patients: a quantitative analysis of immune cell subsets by flow cytometry.

Immune cell subsets, when measured by two-color flow cytometry in a population of 129 hemodialysis patients, showed significant variance from normal values. Lymphopenia, decreased absolute counts, and altered percentage values of immune cells were found. Increased proportions of CD3+, T cell receptor (TCR) alpha beta + cells and CD4+ T lymphocytes were present. An abnormally high percentage of a subset of activated TCR alpha beta + cells (alpha beta + DR+) was also seen in hemodialysis patients. The proportion of B lymphocytes was found to be significantly lower as compared with controls. Relative values for TCR gamma delta+cells, both for activated (gamma delta + DR+) and nonactivated (gamma delta + DR-) subsets, as well as for CD8+ lymphocytes and natural killer cells did not vary from those of normal controls. Also, the CD4+/CD8+ ratio showed no significant change. Analysis of absolute counts of the investigated immune cell populations revealed significantly decreased numbers for the majority of subsets, as a result of the preexisting lymphocytopenia, characteristic of end-stage renal disease. We conclude that profound quantitative alterations of immune cells, including TCR+T cells subsets, exist in hemodialysis patients. These account, at least in part, for the immune dysregulation associated with chronic renal failure.

B-Lymphocyte Subsets↗