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PubMed1990

The use of flow cytometry to monitor chitin synthesis in regenerating protoplasts of Candida albicans.

Flow cytometry was used to monitor chitin synthesis in regenerating protoplasts of the yeast Candida albicans. Comparisons of cells stained with Calcofluor White, a fluorochrome with known affinity for chitin, and cells incubated in the presence of N-[3H]-acetylglucosamine, the precursor substrate for chitin, showed a linear relationship between fluorescence and incorporation of label over time. Changes in both the fluorescence and light scatter of regenerating protoplasts treated with inhibitors of fungal chitin synthase were also quantitated by flow cytometry.

Aminoglycosides↗

PubMed2003

Normal features of tissue-engineered auricular cartilage by flow cytometry and histology: patient safety.

BACKGROUND: Cytokinetic abnormalities in DNA content, such as aneuploidy, haploidy, and tetraploidy, have been found to occur in human cartilaginous tumors. The high number of chondrocytes needed for tissue-engineered cartilaginous implants requires the cells to be passaged repeatedly. The theoretical risk of changes in the normal diploid state of these cells during their growth in vitro and after generation of tissue-engineered cartilage in vivo is not known. Materials and methods Auricular chondrocytes were obtained from 6 patients and cultured in vitro. Chondrocyte number was increased by repeated passaging. The passaged cells were implanted in nude mice for 8 weeks to generate tissue-engineered cartilage. Fresh control chondrocytes along with the passaged cells and cells obtained from the tissue-engineered constructs were collected and compared for DNA content by flow cytometry. RESULTS: Flow cytometry demonstrated 100% diploidy with no evidence of aneuploidy, haploidy, or tetraploidy in all groups of cells. Histology of the tissue-engineered cartilage also showed no evidence of cellular atypia. CONCLUSION: The number of human auricular chondrocytes can be increased by repeated passaging and passaged chondrocytes can be safely used for implantation to generate tissue-engineered constructs without a change in the normal diploid state of the cells. Histology of the cartilage generated showed normal features without atypia.

Aneuploidy↗

PubMed1990

A prospective comparison of DNA quantitation by image and flow cytometry.

Advances in computer and video technology suggest that image analysis may be practical method of measuring DNA that also allows visual confirmation of cell type. The purpose of this study was to prospectively compare DNA quantitation from 92 solid tumors in which DNA indices had been measured by image analysis of touch preparations (CAS 100) and flow cytometry of cell suspensions (FACScan). For 81 cases, there was excellent correlation between the two methods. For nine cases, however, an aneuploid population, usually near tetraploid, was identified by image but not by flow cytometry. Three cases had aneuploid peaks by flow cytometry that were not identified by image. Although these methods show good correlation, rare populations may be missed by CAS, presumably because of sampling errors in the touch preparation. Aneuploid populations may also be missed by flow cytometry, either because of cell loss during processing or because visual identification by image can increase sensitivity.

Adenocarcinoma↗

PubMed1989

Results of the flow cytometry ACTG quality control program: analysis and findings.

The AIDS Clinical Trial Group's (ACTG) Immunology Committee was charged with initiating a quality control program for all laboratories participating in the ACTG program reporting flow cytometry data. Forty-one laboratories were evaluated. This report defines the goals of this program and the subsequent findings after 19 send-outs were made. Both HIV positive volunteer donors and normal age-matched donors were used. Sample sets included both heparin and EDTA anticoagulated bloods. Laboratories were asked to report hematologic parameters as well as flow cytometry data both in percentages and absolute numbers. Results were evaluated using nonparametric statistical analysis. Robust CVs and interquartile ranges were used to define the performance of individual laboratories for each CD subset analyzed. Intralaboratory reproducibility was analyzed by paired sample sets. All laboratories were found to be able to define normal samples as normal. Seventy-five percent of the laboratories were able to define abnormal samples as abnormal. Twenty-five percent could not identify two abnormal samples as abnormal. Forty percent of the labs were found unable to reproduce paired samples within an absolute of +/- 5%. EDTA was found slightly superior to heparin in bloods evaluated by flow cytometry within 30 hr of collection. The analysis of specific histograms, questionnaires, and data analysis led to a specific set of recommendations for performance of flow cytometry studies.

Acquired Immunodeficiency Syndrome↗

PubMed1994

Analysis of acute myeloid leukemia cells by flow cytometry, introducing a new light-scattering classification.

A combined flow-cytometric evaluation of light scattering and the immunophenotype of acute myeloid leukemia (AML) cells from 71 newly diagnosed consecutive patients was conducted. Light-scattering characteristic of AML cells examined by flow cytometry and multiple surface markers were also analyzed using the same samples, to enable a comparison with the French-American-British (FAB) classification. Our AML cases could be classified into three light-scattering classification (LSC) types according to their physical properties on flow cytometry. These were type A, where forward light scattering (FSC) of the leukemic cell population was larger than that of lymphocytes, while side light scattering (SSC) was the same or larger than that of lymphocytes but smaller than that of monocytes; type B, where FSC of the leukemic cell population was larger than that of lymphocytes and SSC spread toward that of monocytes; and type C, where both FSC and SSC of the leukemic cell population spread beyond those of monocytes. Although a clear relationship between the FAB classification and LSC classification by the light-scattering profile of AML was not established, we observed the following findings. The majority of cases were classified as type A (58%), while type B comprised 25% and type C comprised 17%. While CD7 expression on AML cells is considered to be an immature characteristic, CD7 was expressed more frequently among LSC type A cases. Furthermore, all but one of the FAB M1 cases were classified as type A. On the other hand, CD7 was not expressed on type C leukemic cells. The percentage of cases in which more than 60% of leukemic cells possessed another immature surface antigen, CD 34ö, was 13/18 (72%) among FAB M1 cases, much higher than among FAB M2 (35%) or FAB M4 (27%) cases. A negative correlation was observed between mature antigen CD33 and CD34 among the FAB M2 cases. The frequency of CD7 expression was 25% among the total cases, and CD7-positive cases were frequent among FAB M1 and M2, but not among FAB M3 cases. These findings concerning LSC and immunophenotyping indicate that the scattergram pattern analysis may contribute towards more precise immunophenotyping, in that it reflects the maturation stage of each AML case.

Acute Disease↗

PubMed1998

Analysis of mutagens with single cell gel electrophoresis, flow cytometry, and forward mutation assays in an isolated clone of Chinese hamster ovary cells.

We investigated the induction of DNA strand breaks in the single cell gel electrophoresis (SCGE or comet) assay with whole cell clastogenicity measured with flow cytometric analysis in cells from an isolated clone of the Chinese hamster ovary (CHO) AS52 cell line. Under identical treatment conditions the responses were compared with forward mutation at gpt using 2-acetoxyacetylaminofluorene (2AAAF), ultraviolet radiation (UV) and ethyl methanesulfonate (EMS). Cytotoxicity for each agent was evaluated in the SCGE and forward mutation assays. Forward mutation was 4-10-fold more sensitive than DNA strand breaks detected in the SCGE assay. For 2AAAF and EMS, the kinetics of the induction of genetic damage were similar for the three assays, although there were differences in sensitivity. With UV, the induction kinetics of gpt mutation differed from that expressed by SCGE and flow cytometric analysis. With the chemical mutagens 2AAAF and EMS, there was a high correlation between the SCGE assay and forward mutation and also between the SCGE assay and flow cytometry. There was no significant correlation between flow cytometry and forward mutation. With UV, only the SCGE assay and flow cytometry were correlated. Agent-specific variations in the intragenomic distribution of DNA damage for each mutagen was measured in the SCGE assay.

Acetoxyacetylaminofluorene↗

PubMed1981

Clinical evaluation of DNA flow cytometry of fine needle aspirates from testes of infertile men.

DNA flow cytometry was performed on fine needle aspirates from the testes of 40 oligozoospermic of azoospermic men under investigation for infertility. The DNA distributions from men with increased FSH serum levels were all abnormal. The values were below the level of detection (or very low) with respect to both haploid (1c) and tetraploid (4c) cells, indicating reduced proportions of spermatids and primary spermatocytes. This confirms that increased FSH serum levels are indicative of severely damaged spermatogenesis. The findings of both normal and abnormal testicular DNA distribution in the large group of oligozoospermic men indicate that the presented method may be of importance for evaluating prognosis, and for selection of men for further investigation and therapy. Many azoospermic men showed normal testicular DNA distribution patterns, suggesting the value of DNA flow cytometry for selection of such cases for surgical treatment (epididymovasostomia).

Antibodies↗

PubMed1998

Sequential flow cytometry and fluorescence in situ hybridization for the study of formalin-fixed, paraffin-embedded breast cancer cells.

Both flow cytometry and fluorescence in situ hybridization (FISH) are useful techniques in the analysis of cancer tissues. When the two are used in the study of the same specimens, they are usually performed in parallel, separately. This is problematic where there is a scarcity of material, making completion of both studies impossible. Fluorescence in situ hybridization procedures that will utilize excess material discarded from flow cytometry would be advantageous. The present report describes an optimized protocol for performing sequential flow cytometry and FISH using formalin-fixed paraffin-embedded archival material. Although breast cancer tissues were used in this initial study, the protocol is applicable to other cancer tissues as well.

Breast Neoplasms↗

PubMedDate not supplied

Clinical applications of flow cytometry in hematology and immunology.

OBJECTIVE: To review the major applications of flow cytometry in the diagnosis of hematologic and immunologic disease. DATA SOURCES: Professional literature and authors' experience. DATA SYNTHESIS: Flow cytometry is evolving as a major diagnostic tool for evaluating hematologic disease, monitoring HIV infection and assessing peripheral blood and bone marrow for CD34 positive stem cells in marrow transplantation. This review highlights the opportunities and pitfalls of this area of diagnostic laboratory medicine. CONCLUSION: The applications of flow cytometers in diagnostic hematology and immunology are expanding, providing new opportunities for enhanced precision in diagnosis. As in any relatively new field much work remains in order to optimize accuracy and efficiency while minimizing cost.

Cost-Benefit Analysis↗

PubMed1993

Image analysis versus flow cytometry for DNA ploidy quantitation of solid tumors: a comparison of six methods of sample preparation.

With the availability of user-friendly interactive image analysis instruments for DNA analysis, there is a growing need for comparison with the established methodology of flow cytometry. We have compared the results of DNA ploidy quantitation in 12 solid tumors prepared by six different techniques of sample preparation: flow cytometry of fresh cell suspensions and of nuclei isolated from formalin-fixed, paraffin-embedded tissue; and image analysis of touch preparations, of disaggregated cells from paraffin-embedded tissue as well as of 3- and 7-microns-thick tissue sections. Complete agreement in DNA ploidy results obtained by the six methods was found in six out of 12 solid tumors. Image analysis of touch preparations detected most tetraploid and multiple aneuploid peaks. Sections of 7-microns-thick tissue gave better histogram quality than 3-microns-thick sections, however tetraploid peaks were not resolved in one case. Image analysis of disaggregated paraffin-embedded tumor showed comparable ploidy to fresh touch preparations in seven out of 12 cases, the discrepancies being due to loss of tetraploid or multiple aneuploid peaks. Flow cytometry gave good histograms, but tetraploid and multiple aneuploid peaks were occasionally not detected. Each method presents advantages and disadvantages. Flow cytometry and image analysis are complementary methods for DNA quantitation, and more than one method may be necessary to confirm the DNA content of solid tumors.

Cell Nucleus↗

PubMed1989

Clinical importance of DNA content in rectal cancer measured by flow cytometry.

The DNA content of 369 rectal cancers was measured by flow cytometry. One hundred and four (28%) were diploid, 252 (68%) were aneuploid, and 13 (3.5%) were tetraploid. Diploid cancers were associated with an improved 5 year survival (p less than 0.001) and were more likely to present at an early stage. DNA content, however, did not confer independent prognostic information in a Cox model based on four discrete pathological variables. Patients were classified by a new system of prognostic grouping and those with a very good or a very poor outlook were removed leaving 137 prognostic group III patients. No further substratification of this group by DNA content or by four additional pathological variables could be achieved. As the new prognostic system is not improved by the addition of ploidy, routine adoption of flow cytometry in the assessment of rectal cancer cannot be recommended.

DNA, Neoplasm↗

PubMed2006

Prognostic value of flow cytometry in surgically treated primary gastric lymphoma.

AIM: To investigate whether flow cytometry could help to define the optimal therapeutic strategy of primary gastric lymphomas. MATERIAL AND METHOD: Retrospective study of 46 patients having primary gastric lymphoma--according to Dawson criteria--in Ann Arbor stage IE and IIE, who were surgically treated. From selected paraffin-embedded tissue blocks of the tumor, DNA content was studied by flow cytometry (FC). Other pathological tumor features were analysed by hematoxiline-eosine and Giemsa stains as well as immunohistochemical study; any possible influence on postoperative survival was investigated through statistical analysis. RESULTS: The DNA ploidy pattern was diploid in 40 cases (87%) and aneuploid (hyperdiploid) in 6 (13%). Postoperative survival probability (PSP) was 62.7% at 5 years. Statistical analysis showed significant prognostic value for Ann Arbor classification--with higher PSP for stage IE (p = 0.009)--and FC parameters: diploid tumors had higher PSP than aneuploid tumors. Also tumors having S-phase (p = 0.044) or G2-M phase values (p = 0.023) under the respective mean values had higher PSP. No influence on PSP was found for wall invasion, Helicobacter pylori infection, Isaacson's histologic type or resection margin involvement. No significant relationship was appreciated between Isaacson's histologic type and DNA ploidy patterns. CONCLUSION: FC could be useful in assessing gastric lymphoma prognosis.

Adult↗

PubMed2002

A small-volume technique for simultaneous immunophenotyping and apoptosis detection in rat whole blood by four-color flow cytometry.

BACKGROUND: Cell permeabilization for the detection of intracellular molecules by flow cytometry is usually incompatible with whole blood. This article describes a new technique for the simultaneous detection of surface antigens and DNA content in rat whole blood. METHODS: In 20 microl of rat whole blood, DNA staining is obtained by permeabilization of cells using a standard red blood cell lysing reagent (Erythrolyse). Immunophenotyping and apoptosis detection by flow cytometry are achieved by using a combination of three surface markers (CD3, CD4, and CD8alpha) and a DNA binding dye (TO-PRO-3). RESULTS: After a 24-h incubation of whole blood with 1 microM dexamethasone, apoptotic lymphocytes were clearly distinguishable from normal lymphocytes by their reduced size and DNA content. The dexamethasone-induced percentage of apoptotic cells was 58.9 +/- 4.6 for CD4+ and 77.4 +/- 2.9 for CD8+ T cells, compared with 12.6 +/- 2.7 for CD4+ and 17.2 +/- 3.5 for CD8+ T cells in the absence of dexamethasone (data from 10 animals with duplicate samples). CONCLUSIONS: We have developed a new technique to permeabilize nucleated cells in microsamples of rat whole blood. The methodology allows simultaneous immunophenotyping and apoptosis detection in rat whole blood.

Animals↗

PubMed2004

[Detection of organic autotrophic particles by FCM (flow cytometry) in an oligotrophic lake].

Organic autotrophic particles were detected by flow cytometry (FCM) in an oligotrophic lake. Utilizing scattering signals and fluorescence of their natural pigments, different populations could be rapidly discriminated and enumerated. We found that they belonged to three populations. R1 were the smallest population, while R3 were the largest one. R1 contained phycoerythrin but lacked chlorophyll. On the contrary, R3 contained a large amount of chlorophyll but lacked phycoerythrin. R2 contained not only chlorophyll but also a great deal of phycoerythrin. Organic autotrophic particles account for the most proportion of the total particles that were smaller than 50 microm. They represented 70.24% of the total particles. In this study, we found that flow cytometry is particularly suited to the analysis of organic autotrophic particles that are difficult to study with traditional methods.

Autotrophic Processes↗

PubMed1995

A comparison of flow cytometry and microcytotoxicity for the evaluation of alloimmune therapy in patients with recurrent spontaneous abortions.

PROBLEM: There is substantial data that support the efficacy of paternal leukocyte immunization (PLI) for the treatment of alloimmune mediated miscarriage; however, there is confusion regarding the laboratory test that should be performed to determine levels of maternal anti-paternal leukocyte antibodies (MAPLA). METHOD: Popular methodologies employed include: 1) microcytotoxicity (MCX), 2) mixed lymphocyte culture (MLC), and 3) cell flow cytometry crossmatch (FCXM). Cell flow cytometry crossmatch correlates well with the more difficult MLC assay although the former proves the more sensitive study. This work compares the MCX assays with FCXM. The study group consisted of ten women who had a history of three or more spontaneous abortions (SABs). All ten had very low levels (< 10%) of MAPLA as measured by FCXM. Following PLI all subjects demonstrated elevated levels (> 50%) of MAPLA by FCXM. At 12 weeks gestation, sera were simultaneously measured for MAPLA by MCX and FCXM. RESULTS: Although all ten patients had very high levels of MAPLA by FCXM during pregnancy, five of ten had antibodies to HLA Class I and two of ten had antibodies to HLA Class II paternal antigens by MCX. Furthermore, all patients who were positive by MCX to paternal Class I antigens were also positive to Class I antigens not seen in either parent. Both patients who were positive by MCX to paternal Class II antigens were also positive to maternal Class II antigens. Notable is that all ten women eventually delivered healthy infants. CONCLUSION: Based on this preliminary study, the MCX assay is neither sensitive or reliable enough to determine the need and/or to monitor the effectiveness of PLI. Flow cytometry should be the modality of choice when determining the need for alloimmunotherapy and to monitor the effectiveness of treatment.

Abortion, Habitual↗

PubMed1997

Application of flow cytometry and fluorescent in situ hybridization for assessment of exposures to airborne bacteria.

Current limitations in the methodology for enumeration and identification of airborne bacteria compromise the precision and accuracy of bioaerosol exposure assessment. In this study, flow cytometry and fluorescent in situ hybridization (FISH) were evaluated for the assessment of exposures to airborne bacteria. Laboratory-generated two-component bioaerosols in exposures chambers and complex native bioaerosols in swine barns were sampled with two types of liquid impingers (all-glass impinger-30 and May 3-stage impinger). Aliquots of collection media were processed and enumerated by a standard culture technique, microscopy, or flow cytometry after nucleic acid staining with 4',6-diamidino-2-phenylindole (DAPI) and identified taxonomically by FISH. DAPI-labeled impinger samples yielded comparable estimates of bioaerosol concentrations when enumerated by microscopy or flow cytometry. The standard culture method underestimated bioaerosol concentrations by 2 orders of magnitude when compared to microscopy or flow cytometry. In the FISH method, aliquots of collection media were incubated with a probe universally complementary to eubacteria, a probe specific for several Pseudomonas species, and a probe complementary to eubacteria for detection of nonspecific binding. With these probes, FISH allowed quantitative identification of Pseudomonas aeruginosa and Escherichia coli bioaerosols in the exposure chamber without measurable nonspecific binding. Impinger samples from the swine barn demonstrated the efficacy of the FISH method for the identification of eubacteria in a complex organic dust. This work demonstrates the potential of emerging molecular techniques to complement traditional methods of bioaerosol exposure assessment.

Bacterial Typing Techniques↗

PubMed2000

Determination of aneuploids in hop (Humulus lupulus L.) using flow cytometry.

In order to study the possibility that high-resolution flow cytometry can be used for determination of aneuploids, different genotypes of Humulus lupulus were analyzed. Triploid cultivars are bred by hybridization between diploid and tetraploid lines, and as the result of this process, some aneuploids are occasionally also formed. We analyzed eight triploid cultivars and seven putative aneuploids. Triploid cultivars Cerera, Cicero, Celeia, Cekin, Blisk, Mt. Hood, Huller Bit. and Willamette (3x = 30) were measured for nuclear DNA content using Trifolium repens as reference. No significant differences among peak positions of triploid cultivars (having an average CV value per peak of 1.94%) were found. Measurement of nuclear DNA content was also performed for seven lines: 175/75, 89/113, 89/154, 91/215, 175/17, 89/87 and 91/74 previously determined by chromosome counting to be aneuploids (CV per peak was 1.41%). A statistically lower DNA content was found for line 175/75 and higher values were measured for lines 89/154, 89/113 and 91/215. Repeated chromosome counting revealed that the number of chromosomes in line 175/75 was 29, while lines 89/154, 89/113 and 91/215 possessed 31 chromosomes. The other lines were identified as triploids. We conclude that flow cytometry can be efficiently used for determination of aneuploidy in Humulus lupulus.

Aneuploidy↗

PubMed1991

[Study on the objective identification of the origin of urinary erythrocyte using flow cytometry].

We examined fresh urine samples from patients with hematuria using flow cytometry to evaluate its clinical usefulness and accuracy in objectively identifying glomerular and non-glomerular hematuria. Two parameter analysis was applied for forward light scattering (0LS), chiefly representing cell volume, and perpendicular light scattering (90LS), chiefly representing cell structure. In patients whose diagnosis were established by renal biopsy or diagnostic procedures, the patterns of the distribution curves of 0LS and 90LS were divided into three groups: (1) glomerular, (2) non-glomerular and (3) debris patterns. After a prospective blind study was undertaken, the results of flow cytometry were later compared with the results of clinical diagnosis. Glomerular hematuria above 30/high power field could be detected with high sensitivity (92.9%) and specificity (90.5%). Glomerular diseases showed a significantly lower peak channel number than non-glomerular diseases did (0LS: 53.8 +/- 16.4 versus 97.5 +/- 26.5, p less than 0.01, 90LS: 48.4 +/- 17.0 versus 85.5 +/- 32.9, p less than 0.01, respectively). It is suggested that flow cytometric analysis of hematuria is an useful diagnostic procedure as a rapid, non-invasive, accurate and objective test for determination of origin of hematuria.

Adolescent↗