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PubMed1993

The role of flow cytometry in carcinoma of the colon and rectum.

Flow cytometry was performed upon 312 patients with adenocarcinoma of the colon and rectum, satisfactory results being obtained with 275 (108 diploid, 130 aneuploid and 37 tetraploid). The proportion of nondiploid instances increased from 28 percent if one, to 80 percent when six specimens were assessed per patient. Reproducibility of the technique showed substantial agreement in the assessment of deoxyribonucleic acid ploidy (Kappa value equals 0.74). Increasing values of cells in the diving (G2/M) phase of the cell cycle were associated with little lymphocytic tumor infiltration (p = 0.0002) and extensive tumor fibrosis (p = 0.003). Univariate survival analysis revealed that, although diploid tumors tended to have a better prognosis than nondiploid tumors (p = 0.06), no flow cytometric variable was significantly related to survival. Flow cytometry similarly was not of prognostic value in instances without lymph node metastases or without distant metastases. Multivariate regression analysis of flow cytometric and clinicopathologic variables identified Dukes' stage, patient age and tumor differentiation as the combination of variables most closely related to survival. No flow cytometric variable could significantly improve on the prognostic model containing these three variables. It is concluded that conventional histologic variables remain the best predictors of prognosis in carcinoma of the colon and rectum.

Adenocarcinoma↗

PubMed1986

Flow cytometry as a tool for the prognostic assessment of human neoplasia.

Flow cytometry permits the quantitative description of neoplastic cell populations from the point of view of their cytogenetic and cytokinetic features. The advances in preparation of cellular monodispersed samples allow the examination not only of in vitro and hematological, but also of surgical, biopsy, endoscopic, and lavage specimens. The analysis of cytometric DNA content has evidenced the importance of (aneu)ploidy as a remarkable tumor marker. Tumors of different sites and, in some cases, stages and/or grades are characterized by a differential occurrence of diploid vs. aneuploid cell subpopulations and by the eventual presence of different stem cell lines within the same tumor. For certain classes of neoplasms, these parameters can be used for the early recognition of neoplasia and related to disease evolution and dissemination and to the results of therapy. Flow cytometry can also be used to evaluate the fraction of (cycling) cells in the S-phase and of proliferating cells (growth fraction). The percent of S cells can be extracted from cytometric DNA content histograms. Furthermore, the method of Bromodeoxyuridine (BrdUrd) incorporation has been recently introduced into flow cytometry. BrdUrd labeling in cycling cells can be detected either by the induction of quenching or enhancement of specific DNA-dye fluorescence or by fluorescent anti-BrdUrd monoclonal antibodies. This approach has been confirmed by preliminary comparative tests on cultured cells, normal and malignant bone marrow, and human solid tumor specimens. These parameters, together with other cytometric parameters of potential importance for the cellular characterization of malignancy, offer a reliable and real time-saving tool for the prognostic assessment of human tumors and the predicting and monitoring of the results of therapy.

Aneuploidy↗

PubMed1994

Retrospective evaluation of flow cytometry as a platelet crossmatching procedure.

We performed a retrospective analysis of flow cytometry as a platelet crossmatching procedure. Sera from 17 alloimmunized refractory patients were tested against 32 donor platelets, which had been stored as platelet-rich plasma for up to 36 months. Overall, 14/32 (44%) crossmatches were positive. The mean 1 h posttransfusion corrected count increments (CCIs) were 9,195 and 2,269 for a negative and a positive crossmatch, respectively. The predictive value of a positive crossmatch was 86%, whereas the predictive value of a negative crossmatch was 56%. When samples with low background fluorescence or with high panel-reactive antibody (PRA) levels were evaluated separately, the accuracy of the crossmatch improved from 69% to 80%. When compared to the platelet adhesion immunofluorescence test (PAIFT) and the standard and antiglobulin-enhanced lymphocytotoxicity tests for the detection of HLA antibodies, flow cytometry appeared to be more sensitive. We conclude that flow cytometry is a useful technique for platelet crossmatching, particularly for alloimmunized patients for whom HLA compatible platelets may not be readily available.

Blood Grouping and Crossmatching↗

PubMed1994

Detection of intracellular lymphoid differentiation antigens by flow cytometry in acute lymphoblastic leukemia.

The value of flow cytometric detection of the intracellular lymphoid differentiation antigens CD3 and CD22 in the differential diagnosis of acute leukemia was assessed in cases of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and leukemic cell lines. Cells were fixed in 0.25% paraformaldehyde at 4 degrees C for 60 min, permeabilized with 0.2% Tween 20 at 37 degrees C for 15 min, then stained with CD3 or CD22 monoclonal antibodies by indirect immunofluorescence. Cytoplasmic CD22 was detected on greater than 20% (mean 55%; range 20-87%) of blasts from all 20 cases of precursor-B ALL analyzed. The percentage of cells with cytoplasmic CD22 was greater than that with membrane CD22 in all except 2 cases of precursor-B ALL. Cytoplasmic CD22 was not detected in 8 cases of precursor-T ALL, 4 T-leukemia cell lines, or in 7 cases of AML. In contrast, cytoplasmic CD3 was detectable by flow cytometry in all 8 cases of precursor-T ALL, but not in precursor-B ALL, pre-B leukemia cell lines, or in AML. These results confirm that cytoplasmic CD3 and CD22 are excellent markers of the early T and B lineages in ALL and can be reliably detected by flow cytometry. This technique should be a valuable addition to routine immunophenotyping for classification of acute leukemia.

Antigens, CD↗

PubMed2002

Mixing small volumes for continuous high-throughput flow cytometry: performance of a mixing Y and peristaltic sample delivery.

BACKGROUND: Online mixing for continuous high-throughput flow cytometry has not been previously described. A simple, general high-throughput method for mixing and delivery of submicroliter volumes in laminar flow at low Reynolds numbers would be widely useful. MATERIALS AND METHODS: We describe a micromixing approach that is compatible with commercial autosamplers, flow cytometry, and other detection schemes that require mixing of components that have been introduced into laminar flow. The scheme is based on a previous approach to high-throughput flow cytometry (HyperCyt, Kuckuck et al.: Cytometry 44:83-90, 2001). We showed that samples from multiwell plates that have been picked up by an autosampler can be separated during delivery by the small air bubbles introduced during the transit of the autosampler probe from well to well. Here, a particle sample flowing continuously is brought together in a Y with reagent samples from wells, which have been separated by bubbles. RESULTS: In the effluent stream, the particles and reagents are mixed, most likely as a result of peristaltic action, and reagents from individual wells can be resolved. The sample volumes that can be mixed with this technology are submicroliter in volume, and samples can be mixed at rates up to at least 100/samples per minute. With the current device, carryover between samples can be eliminated if the mixing system is flushed with several volumes of buffer. The anticipated throughput for screening is expected to be at least 20 samples per minute. CONCLUSIONS: The high-throughput approach and peristaltic mixing in HyperCytTM serve to integrate autosamplers with submicroliter detection volumes for analysis in flow cytometry or in microfluidic channels.

Avidin↗

PubMed1983

Method for analysis of cellular DNA content of paraffin-embedded pathological material using flow cytometry.

A method has been developed that allows flow cytometry to be used for measuring the cellular DNA content of paraffin-embedded human tumors. Thick (i.e., 30 micron) sections were cut from tissue blocks using a microtome and dewaxed in xylene. The sections were then rehydrated by sequentially immersing them in 100, 95, 70, and 50% ethanol before finally washing in distilled water. Single cell suspensions were then prepared by incubation in 0.5% pepsin, pH 1.5, at 37 degrees C for 30 min. The cells were counted, washed, and stained with 1 microgram/ml 4',6'-diamidino-2-phenylindole for 30 min, and DNA content was measured using an ICP 22 flow cytometer. There was a good correlation between the DNA histograms produced using this method and those obtained using unfixed tissue from the same tumor stained with ethidium bromide plus mithramycin. This method allows the retrospective study of archival material where the clinical outcome is already known, and it should, therefore, be particularly useful for determining the prognostic significance of abnormal DNA content measured by flow cytometry.

DNA, Neoplasm↗

PubMed1993

Whole blood method for simultaneous detection of surface and cytoplasmic antigens by flow cytometry.

Dual parameter analysis of surface antigens in flow cytometry has become a standard method for detection of cell subsets. However, few methods have been described for the extension of multiparameter analyses to include cytoplasmic or intracellular antigens. Here we describe a simple and reproducible method for simultaneous detection of surface and intracellular antigens by flow cytometry in lysed whole blood samples. This method employs the use of digitonin, a mild glycoside detergent, and formaldehyde for permeabilization and fixation. Red blood cells are lysed with 2% acetic acid. Preparation of samples in this manner resulted in altered light scatter characteristics relative to unpermeabilized samples; however, gating issues were overcome using a combination of scatter vs. fluorescence gating. Quantitation of CD3+/CD4+ and CD3+/CD8+ cells using this method was equivalent to counts obtained with the reference method using a commercially available lysis procedure and fluorescence vs. scatter gating. The effectiveness of the permeabilization process was assessed using a monoclonal antibody designated TIA-1, which is specific for a cytoplasmic antigen associated with cytotoxic granules predominantly found in CD8+ cells. The method effectively quantitated TIA-1 positive cells and demonstrated the specificity of the reagent for a subpopulation of CD8+ lymphocytes. Using this simplified procedure for simultaneous identification of surface and cytoplasmic antigens could help in studies of cell activation, proliferation, and other functional characteristics of the immune system.

Antibodies, Monoclonal↗

PubMed1987

Characterization of porcine peripheral blood leukocytes by light-scattering flow cytometry.

As a basis for other experiments using flow cytometry of porcine peripheral blood leukocytes, cell fractions were isolated by various methods and analyzed by forward angle light scatter and 90 degree light scatter. Cytospin smears of cell samples were also studied by leukocyte differential counts and nonspecific esterase staining. Three main populations of peripheral blood leukocytes [lymphocytes, monocytes, and granulocytes (primarily neutrophils)], were defined in the log 90 degree light scatter by forward angle light scatter histogram. Partial overlap was observed between lymphocyte and monocyte, and between monocyte and granulocyte domains. Correlation between leukocyte differential counts and flow cytometric quantification based on bitmap statistics of appropriate domains was between r = 0.872-0.892 for lymphocyte and granulocyte. Percoll density gradients were used for subfractionation of leukocyte populations, especially for the enrichment of granulocytes. The specific densities were calculated for lymphocytes (1.0585-1.0819 g/cc), monocytes (1.0585-1.0702 g/cc), granulocyte (1.0819-1.0936 g/cc), and erythrocytes (greater than 1.0952 g/cc). We suggest that light scatter characterization is a basis for future studies of porcine blood by flow cytometry.

Animals↗

PubMed1988

Flow cytometry and cytology as response indicators to M-VAC (methotrexate, vinblastine, doxorubicin and cisplatin).

We evaluated 23 patients treated with neoadjuvant methotrexate, vinblastine, doxorubicin and cisplatin for muscle invasive bladder carcinoma with serial bladder wash flow cytometry and serial urinary cytology. The sensitivities of bladder wash flow cytometry and cytology in detecting bladder cancer were 74 and 47 per cent, respectively, based on subsequent diagnosis from biopsy and cystectomy specimens. Exclusion of those cases without surface carcinoma increased the sensitivities of bladder wash flow cytometry and cytology to 93 and 60 per cent, respectively. Persistently positive bladder wash flow cytometry and cytology correctly predicted residual disease in 88 and 100 per cent, respectively. However, negative bladder wash flow cytometry and cytology after chemotherapy were not reliable indicators of disease-free status.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1994

Measurement by flow cytometry of genomic AT/GC ratio and genome size.

Flow cytometry with the AT-specific fluorochrome Hoechst 33258 (HO) and the GC-specific fluorochrome olivomycin (OM) was used for measurement of base pair specific DNA content in 20 species of vertebrates. The results were found to be in good correlation with the biochemical literature on base pair frequencies (r = 0.972, P < 1 x 10(-8). This correlation allows one to determine the percent of GC/AT-pairs and genome size from flow cytometric data. The genome sizes obtained were compared with the literature data on flow cytometric genome size values determined with the use of propidium iodide (PI) that is usually believed to be non-base pair specific. The results were found to be in general agreement; however, the previously reported slight GC-preference of PI is confirmed. The optimal conditions for flow cytometry of AT/GC ratio and genome size with the use of OM and HO are discussed. The approach can be useful for research in ecology, fisheries science, species conservation, and other environmental studies as a tool for rapid survey of a vast array of specimens.

Animals↗

PubMed1984

7-Amino-actinomycin D as a specific fluorophore for DNA content analysis by laser flow cytometry.

A technique for DNA amount determination by flow cytometry based on the use of 7-amino-actinomycin D (7-amino-AMD), a fluorescent analogue of antibiotic actinomycin has been investigated, and a particular staining procedure has been developed. The procedure includes short fixation in 70% ethanol and staining for 20 min in 10(-5)M solution of 7-amino-AMD at pH7. The results of DNA content measurements are very reproducible. The histograms obtained have a coefficient of variation less than 3%. The absorption maximum of the complex of 7-amino-AMD with DNA is situated in the green spectrum region, making this stain particularly suitable for argon laser flow cytometry.

Animals↗

PubMed1989

[Flow cytometry. Principles and methods for cell studies].

Through flow cytometry cells are investigated by optical, electrical and electronic components. On a rate of several hundred cells per second, quantitative multiparametric cells studies are realized on a cell by cell basis. Cells in monodisperse suspension are drawn by a stream of fluid which aligns them before their interception by a beam of light, generally produced by Laser. The generated optical signals (diffused lights and emitted fluorescences), related to biological properties of the cells, are conveyed to photomultipliers which transform light energy into electric currents. The signals are quantified by electronic and processing components which can drive cell sorting. Flow cytometry is playing an increasingly significant role in the practice of the clinical laboratory in several areas i.e. oncology (DNA content measurement) or immunology (lymphocytes phenotyping).

Electronic Data Processing↗

PubMed1998

O-Polysaccharide epitopic heterogeneity at the surface of Brucella spp. studied by enzyme-linked immunosorbent assay and flow cytometry.

Smooth Brucella strains are classified into three serotypes, i.e., A+M-, A-M+, and A+M+, according to slide agglutination with A and M monospecific polyclonal sera. The epitopes involved have been located on the O-polysaccharide (O-PS) moiety of the smooth lipopolysaccharide (S-LPS), which represents the most exposed antigenic structure on the surface of Brucella spp. By use of monoclonal antibodies (MAbs) a number of epitope specificities on the O-PS have been reported: A, M, and epitopes shared by both A and M dominant strains, which have been named common (C) epitopes. The latter have been further subdivided, according to relative MAb binding in enzyme-linked immunosorbent assays (ELISA) to A- and M-dominant Brucella strains and to cross-reacting Yersinia enterocolitica O:9, into five epitopic specificities: C (M>A), C (M=A), C/Y (M>A), C/Y (M=A), and C/Y (A>M). In the present study, we studied the occurrence of these epitopes at the surface of representatives of all Brucella species and biovars including the live vaccine strains by analyzing the levels of MAb binding to whole Brucella cells in ELISA and flow cytometry assays. In ELISA, the level of MAb binding correlated well with the previously defined epitope specificity and the serotype defined by polyclonal sera for each Brucella species, biovar, or strain. However, MAbs to the C (M=A) and C (M>A) epitopes showed insignificant binding to B. suis biovar 2 strains and bound at lower titers to B. suis biovar 3 and B. neotomae than to the other Brucella strains. Some of the flow cytometry results were contradictory to those obtained by ELISA. In fact, it appeared by flow cytometry that all O-PS epitopes, including the A and M epitopes, are shared to different degrees by Brucella spp. which nevertheless show a high degree of O-PS heterogeneity according to MAb binding intensities. The subdivision of MAb specificities and Brucella serotypes was therefore less evident by flow cytometry than by ELISA. Whereas in ELISA the MAb specific for the A epitope showed insignificant binding to Y. enterocolitica O:9, this MAb bound strongly to Y. enterocolitica O:9 in flow cytometry. One of the two MAbs specific to the C (M=A) epitope also bound at a low but significant level to B. suis biovar 2 strains. However, as in ELISA the MAb specific for the C (M>A) epitope did not bind at all to B. suis biovar 2 strains in flow cytometry. Flow cytometry provided new information regarding specificity of the MAbs and may further explain some aspects of the capacity of passive protection of some MAbs against smooth Brucella infection in mice. As shown in the present study the occurrence of Brucella strains apparently completely devoid of one specific C O-PS epitope (e.g., B. suis biovar 2 devoid of the C [M>A] epitope) offers the possibility of obtaining vaccine strains devoid of a diagnostic O-PS epitope, which could further help to resolve the problem of discriminating infected from vaccinated animals that remains a major goal in brucellosis research.

Animals↗

PubMed1992

Isolation and characterisation of a subpopulation of human chorionic cytotrophoblast using a monoclonal anti-trophoblast antibody (NDOG2) in flow cytometry.

Human cytotrophoblast cells, isolated from term amniochorion by enzymic digestion and Percoll gradient centrifugation, were characterised by flow cytometry. A panel of 12 anti-trophoblast monoclonal antibodies was screened for labelling of these cells in flow cytometry and the results compared with immunoperoxidase labelling of cytospin preparations and tissue sections. All 12 antibodies were positive for trophoblast on tissue sections, 11/12 were positive on cytospins but only two (NDOG2 and GB25) gave consistent results in flow cytometry. Two-colour labelling with NDOG2 and W6/32, an antibody to HLA-A, -B, -C, demonstrated that 88% of the NDOG2-positive cells also express Class I major histocompatibility complex (MHC) antigens. The NDOG2-positive cytotrophoblast subpopulation was isolated by flow cytometry in sufficient purity (greater than 95%) and yield (3.1 x 10(6)) for use in functional studies in vitro.

Antibodies, Monoclonal↗

PubMed1996

Flow-cytometry analysis of sheep-nematode egg populations.

Flow cytometry was applied to the analysis of nematode populations. Three strains of Haemonchus contortus susceptible or resistant to anthelmintics were studied. Eggs were chosen for these analyses. Data on light-scatter emissions and native green fluorescence were collected. In addition, the size of the eggs (image analysis), the hatching rate, and the susceptibility to benzimidazoles were measured. The results showed that nematode eggs are a suitable material for multiparametric flow-cytometry analyses. Forward-scatter emission is a discriminating parameter for the egg size. The hatching rate and side-scatter emission have a significantly positive relationship. For resistant strains, the rate of resistance shows a significant regression on the native greenfluorescence pulses that might reflect the state of oxidation of associated flavin molecules.

Animals↗

PubMed1998

Detection of minimal residual disease in B-lineage acute lymphoblastic leukaemia by quantitative flow cytometry.

The clinical significance of detecting minimal residual disease (MRD) in B-lineage acute lymphoblastic leukaemia (ALL) was evaluated by quantitative flow cytometry using a combination of TdT with CD10 and CD19. 53 patients with B-cell precursor ALL were followed during and after completion of treatment (median follow-up 23 months). Nine patients relapsed and MRD had been detected in six of them, 5-15 weeks before relapse despite morphological complete remission. 43 patients remain in clinical remission and in none of these was MRD detected. Disease-free survival based on the detection of MRD by flow cytometry showed a statistically significant difference between both groups (P<0.0001). The absence of MRD correlates with a low relapse rate, whereas the presence of MRD predicted early relapse. This study has shown that flow cytometry can improve the morphologic assessment of bone marrow (BM) remission status in B-lineage ALL. The finding of < 5% blasts in BM aspirates did not correlate with 'true' remission in a proportion of cases as residual leukaemic blasts were detected by flow cytometry in nine samples from six patients. On the other hand, the presence of > 5% blasts assessed by morphology was not necessarily a feature of relapse in five patients as these cells were shown to have a phenotype identical to normal TdT-negative B-cell precursors. Quantitative flow cytometry was more informative than conventional morphology to assess remission status and showed a strong correlation with clinical outcome. This methodology is useful to define MRD in the majority of patients with B-lineage ALL and should be tested in prospective clinical trials.

Adult↗

PubMed2000

Terminal deoxynucleotidyl transferase expression in acute myelogenous leukemia and myelodysplasia as determined by flow cytometry.

The significance of terminal deoxynucleotidyl transferase (TdT) expression in acute myelogenous leukemia (AML) remains controversial. Therefore, we studied TdT expression by flow cytometry in 120 previously untreated patients with AML or myelodysplastic syndrome (MDS) to determine the distribution of TdT-positive blasts and the intensity of TdT expression and to seek clinically significant associations. TdT expression measured by flow cytometry (flow TdT%) was heterogeneous, ranging from 0.1% to 87% (median, 8.5%), and 74 patients (62%) had at least 5% TdT-positive blasts. TdT positivity was associated with the M0 or M1 subtype and with expression of CD34 and CD7. No significant correlation was found between TdT expression and type of cytogenetic abnormality or rearrangement of immunoglobulin or T-cell receptor genes. Remission lasted longer in patients with a flow TdT% < 5 than in patients with a flow TdT% > 5 (median, 95 weeks vs 55 weeks, p = 0.02); however, complete remission rates did not differ when patients were classified by initial flow TdT%. Survival was slightly better for patients with flow TdT% less than 5%. Among patients with a flow TdT% > 5%, those with a higher TdT intensity survived longer than those with a lower intensity. These data suggest that quantitative TdT measurement may contribute to prognostic estimate in AML patients.

Adult↗

PubMed1993

Nuclear DNA content of human breast carcinoma: a comparison of results obtained by microspectrophotometry and flow cytometry of paraffin embedded tissue.

This study compares 2 techniques for estimating the nuclear DNA content of tumor cell lines: (i) static cytometry of smears taken from fresh tissue and (ii) flow cytometry of cells extracted from paraffin embedded tissue. Parallel determinations of DNA content, using both techniques, were made on samples of tissue taken from 130 female patients with breast carcinoma. Using a simple classification into diploid and non-diploid groups, the 2 techniques yielded discrepant results in 11% of cases. The most frequent causes of disagreement were (a) the inability of static cytometry to distinguish between a diploid and a near-diploid peak and (b) for flow cytometry, the difficulty of determining whether a minor peak in the tetraploid region represented the G2 peak of a diploid cell line or the G0/G1 peak of a tetraploid cell line. If it is deemed necessary to accurately assess ploidy status, flow cytometry on paraffin embedded tissue, using modern statistical programmes, would seem to be most practical for routine use, but some neoplasms, particularly those with an equivocal ploidy peak in the tetraploid range by this method, will require static cytometry to accurately assess nuclear DNA content. Using this approach, it appears that the disagreement between the 2 techniques would be less than 5%.

Adult↗