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Results for “Flow Cytometry”

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PubMed1995

The effect of antimicrobial therapy on testicular aspirate flow cytometry.

Many antibiotics have been shown to have adverse effects on spermatogenesis. Deoxyribonucleic acid (DNA) flow cytometry of testicular aspirate has been shown to be an effective method of quantitatively evaluating testicular function. To assess this problem, DNA flow cytometry of testicular aspirate was performed on 8 groups of rats, each of which received antibiotics via daily gavage for 10 days. Aspirations were performed on days 0, 11 and 56. Data thus obtained were analyzed using a two-way ANOVA with repeated measures. Antibiotics studies included ciprofloxacin 15 mg./kg./day, sulfamethoxazole (SMX) 20 mg./kg./day (with trimethoprim (TMP) 4 mg./kg./day), nitrofurantoin 7 mg./kg./day, ofloxacin 10 mg./kg./day, lomefloxacin 6 mg./kg./day, doxycycline 3 mg./kg./day and norfloxacin 10 mg./kg./day. One group received the same SMX/TMP dose, together with folate 0.014 mg./kg./day. A statistically significant change in aspirate content on day 11 as compared with baseline existed in groups receiving TMP/SMX (p = 0.00025), nitrofurantoin (p = 0.0000043), ofloxacin (p = 0.000075) and doxycycline (p = 4.89 X 10(-9). Control rats and the group receiving TMP/SMX with folate showed no significant change. On day 56 the abnormality persisted in groups TMP/SMX (p = 0.042), nitrofurantoin (p = 0.001), ofloxacin (p = 0.036) and doxycycline (p = 0.003). Controls and groups receiving ciprofloxacin, norfloxacin and lomefloxacin continued to show no statistically significant difference from baseline on day 56. These data suggest that SMX/TMP, nitrofurantoin, ofloxacin and doxycycline significantly alter spermatogenesis. Ciprofloxacin, norfloxacin and lomefloxacin had no apparent effect on spermatogenesis as measured by DNA flow cytometry.

Animals↗

PubMed1995

Quantitative analysis of phagocytosis and killing of Cryptococcus neoformans by human peripheral blood mononuclear cells by flow cytometry.

Monocytes may represent an important defense mechanism in disseminated cryptococcosis. We have developed a flow cytometric method to study the interaction of Cryptococcus neoformans with monocytes. For phagocytosis, C. neoformans was labelled with fluorescein isothiocynate (FITC). Monocytes were identified on the flow cytometer by labelling with anti-CD14-R-phycoerythrin. Discrimination between attached cells (association) and internalized cells (uptake) was made by quenching FITC-labelled C. neoformans with trypan blue. Only internalized cells kept their FITC fluorescence after quenching. For comparison under the microscope, specific staining of the cell wall of C. neoformans with Uvitex was used. Internalized C. neoformans cells were not stained, as Uvitex was occluded from phagocytes. To assay killing, C. neoformans was labelled with 0.2 mM 2'-7(1)-bis(2-carboxyethyl)-5-carboxyfluorescein acetoxymethylester. After phagocytosis of labelled cells by monocytes, blood cells were lysed with 25 mM deoxycholate. Viable yeast cells retained the fluorescence, but nonviable cells lost it. Quantitative counts of viable cells on Sabouraud dextrose agar were performed for comparison. The change in the relative fluorescence of green within the monocyte region was used to quantitate association, uptake, and killing of C. neoformans by monocytes on the flow cytometer. The flow cytometry methods showed that 18% +/- 2%, 35% +/- 14%, 50% +/- 11%, 51% +/- 6% of monocytes had become associated with C. neoformans after 0, 30, 60, and 120 min, respectively. After 2 h of phagocytosis time, 30% of C. neoformans-associated monocytes had taken up the cells, and killing rates of 23% +/- 17%, 22% +/- 9%, and 40% +/- 13% were obtained with effector-to-target cell ratios of 1:1, 10:1, and 50:1, respectively. Results with the flow cytometry methods compared favorably with those by the conventional methods used, but the flow cytometry methods are simpler, rapid, more reproducible, and objective.

Blood Bactericidal Activity↗

PubMed2003

Intracellular determination of activated caspases (IDAC) by flow cytometry using a pancaspase inhibitor labeled with FITC.

BACKGROUND: Flow cytometry-based methods of simultaneous detection of multiple activated caspases in single cells are of diagnostic value and remain to be fully explored. METHODS: Genomic DNA fragmentation was determined by agarose gel electrophoresis and flow cytometry. Whole cells were incubated with the cell-permeable pancaspase inhibitor, FITC-Val-Ala-Asp-fluoro-methyl-Rerone (FITC-VAD-FMK), and propidium iodide (PI) and analyzed for intracellular activated caspases and plasma membrane integrity, respectively, by flow cytometry. RESULTS: Two distinct populations of apoptotic cells were identified by flow cytometry: an early apoptotic population indicated by FITC-VAD-FMK binding and the late apoptotic cells characterized by FITC-VAD-FMK staining and permeability to PI. The binding of FITC-VAD-FMK to apoptotic cells was time dependent and concurred with DNA fragmentation. Pretreatment with the pancaspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-methyl-fluoro-methyl-kerone (Z-VAD (OMe)-FMK), coordinately attenuated apoptosis induction and activation of caspases. The pancaspase inhibitor also competitively blocked the binding of FITC-VAD-FMK to early apoptotic cells in vitro. Significantly, the use of FITC-VAD-FMK permitted apoptosis determination in a number of cell lines including the caspase-3 gene-deleted MCF-7 responding to various apoptotic stimuli. CONCLUSIONS: Apoptosis detection based on FITC-VAD-FMK binding is specific and easily performed by flow cytometry in a variety of cells. This novel technique has implications for detecting apoptosis in pathophysiologic conditions.

Amino Acid Chloromethyl Ketones↗

PubMedDate not supplied

Flow cytometry used to distinguish between complete and partial hydatidiform moles.

Cells prepared from fresh tissue from a normal conception, and three complete hydatidiform moles (CHMs), were shown to be diploid by flow cytometry, while triploid populations were identified in tissue from two partial hydatidiform moles (PHMs). Similar results were found in cells from formalin-fixed, paraffin-embedded material. In nine cases where cytogenetic analysis was carried out this correlated with the results from flow cytometry. Using flow cytometry we were able to show that two cases which were pathologically complete were in fact diploid, despite the finding of trisomy at one locus in the molar tissue using biochemical markers. The most likely explanation of apparent trisomy of the enzymes in the molar tissue was contamination by maternal tissue.

Diploidy↗

PubMed1997

Features of DNA oligonucleosomal fragmentation in human tumor cell lines and its detection by flow cytometry: utility and limitations.

Cultured HL-60, HeLa S3 and WiDr cells were treated with various doses of ethanol, then subjected to flow cytometry and gel electrophoresis of cellular DNA. On electrophoresis of DNA from HL-60 cells treated with 0.5 or 1.0 mM ethanol, a ladder pattern was recognized after 3 h. At higher doses of ethanol (2.0 and 5.0 mM), a smear pattern resulted. On flow cytometry, however, A0 cells (lower fluorescence level than G0+G1 cells) were noted from 0.5 to 5.0 mM ethanol. The observation of A0 cells at higher doses indicated loss of DNA after random DNA degradation. HeLa S3 and WiDr cells were partially detached from flasks after administration of ethanol and separated into adherent and non-adherent categories. In DNA from non-adherent HeLa S3 cells treated with 0.5 mM ethanol, a ladder pattern was observed after 24 h. On flow cytometry, prior to the appearance of A0 cells, an accumulation in the G2 + M-phase became obvious after 3 h. Increased mitotic indices indicated that this phenomenon was due to M-phase arrest. Adherent HeLa S3 cells showed no DNA oligonucleosomal fragmentation or A0 cells. These findings indicate that detection of A0 cells by flow cytometry is not proof of cell death by DNA oligonucleosomal fragmentation.

Apoptosis↗

PubMed1997

Flow cytometry in impression cytology specimens. A new method for evaluation of conjunctival inflammation.

PURPOSE: To investigate feasibility and potential uses of flow cytometry in impression cytology as a new procedure to assess and quantify conjunctival inflammation. METHODS: Specimens for cytology were collected by impression from 30 patients with various chronic ocular surface disorders and from 10 normal subjects. Two specimens were obtained in each eye: One was transferred onto a glass slide and processed by immunofluorescence with antibodies to human leukocyte antigen (HLA)-DR antigens; cells from the other were suspended in phosphate-buffered saline for flow cytometry. Monoclonal antibodies to HLA-DR antigens and CD23, the low affinity receptor to immunoglobulin E, were used. RESULTS: Abnormal expression of HLA-DR and CD23 by conjunctival cells was found in 13 of 18 dry eyes and in 20 of 22 eyes with chronic conjunctivitis, whereas specimens remained almost negative (less than 10% of cells were positive) in normal eyes. Percentages of positive cells ranged between 20% and 98% of all conjunctival cells. Correlation between the two methods, immunocytology and flow cytometry, was highly significant (coefficient of correlation 0.77, P = 0.0001). Moreover, HLA-DR positivity, at its strongest intensity, was observed in a minority of cells (1% to 12%), most of which were resident class II-expressing dendritic cells. Percentages of those cells expressing high levels of HLA-DR were 3 +/- 1.2% in normal eyes, 5.8 +/- 4% in dry eyes (P = 0.05), and 5.9 +/- 3.5% in eyes with chronic conjunctivitis (P = 0.02). CONCLUSIONS: Results of this preliminary study confirm that conjunctival epithelial cells may abnormally express inflammatory markers in chronic ocular surface disorders. Development of flow cytometry in analysis of cytologic specimens provides a new, sensitive, and objective tool for exploring conjunctival pathology.

Antibodies, Monoclonal↗

PubMed2002

[Immunophenotyping of eighty six children with acute lymphoblastic leukemia by three-color flow cytometry].

OBJECTIVES: To evaluate the specificity of three-color flow cytometry in childhood acute lymphoblastic leukemia (ALL) immunophenotyping. METHODS: Immunophenotyping was performed by three-color flow cytometry analysis using CD(45)/SSC gating. RESULTS: The percentage of blasts was correlated better with leukemic cell count compared with that of FSC/SSC, and the false positive results were low. Among eighty six cases of ALL, 95.3% was B-ALL, in which common-ALL and Pro-B-ALL were 76.8% and 6.1%, respectively, and 2.3% was T-ALL. CD(34)(+) and myeloid-associated antigen expression were observed in 57.0% and 34.9% of the cases, respectively, among which Pro-B-ALL was the commonest. CD(33) was more commonly expressed than CD(13) in Pro-B-ALL cases, but no difference in the expression between these two antigens in other subtypes. CONCLUSION: Gating of CD(45)/SSC eliminated effection of normal cells to blasts in bone marrow, with which the immunophenotyping results were more reliable.

Antigens, CD↗

PubMed1999

Optimisation of HLA-B27 testing by association of flow cytometry and DNA typing.

HLA-B27 typing contributes to the diagnosis of ankylosing spondylitis. The classical technique of microlymphocytotoxicity is costly and can give false-negative results. We have compared 304 samples using two relatively new methods - flow cytometry and PCR-SSP - and evaluated their respective uses in routine analysis. Flow cytometric HLA-B27 testing was performed using three monoclonal anti-B27 antibodies (HLA-ABC-m3, GS145.2 and FD705 clones). Cut-off values were established to differentiate HLA-B27-positive from HLA-B27-negative samples with ROC curves. Although flow cytometric analysis with a reliable monoclonal antibody (mAb) is valuable for HLA screening, none of the HLA-B27 flow cytometry protocols was sufficient on its own to ascertain the HLA phenotype in 100% of samples. Two false negatives were observed with the FD705 mAb and the use of two different monoclonal antibodies did not increase the accuracy of HLA-B27 typing. HLA-B27 typing using molecular biology is a reliable but costly technique. Therefore we suggest that DNA typing could be used as a complementary technique and applied to samples whose HLA-B27 phenotype cannot be determined by flow cytometry. The association of flow cytometry and DNA typing is, in our experience, an economical and reliable approach.

Antibodies, Monoclonal↗

PubMed2001

The clinical utility of flow cytometry in the study of platelets.

Flow cytometry has emerged in the past few years as an important technology for the study of platelets. It offers the ability to make measurements on platelets with little or no isolation or manipulation. Most flow cytometric platelet studies can be carried out on whole blood, thus eliminating a host of artifacts. In addition, flow cytometric techniques have been developed that allow the measurement of nearly all of the functional capabilities of platelets, such as activation and aggregation and to identify new functions by permitting observation of platelets interacting with leukocytes and measurement of platelet microparticles. Several of these measurements have already reached the stage of clinical utility and others offer considerable promise for practical applications. This review describes each of the flow cytometric techniques used to study platelets and summarizes their current state of clinical utility. Semin Hematol 38:160-168.

Animals↗

PubMed2001

Flow cytometry in the diagnosis of acute leukemia.

Acute leukemias are a heterogeneous group of malignancies with varying clinical, morphologic, immunologic, and molecular characteristics. Many distinct types are known to carry predictable prognoses and warrant specific therapy. Distinction between lymphoid and myeloid leukemias, most often made by flow cytometry, is crucially important. Several advances in flow cytometry, including availability of new monoclonal antibodies, improved gating strategies, and multiparameter analytic techniques, have all dramatically improved the utility of flow cytometry in the diagnosis and classification of leukemia. Acute leukemias reflect the pattern of antigen acquisition seen in normal hematopoietic differentiation, yet invariably demonstrate distinct aberrant immunophenotypic features. Detailed understanding of these phenotypic patterns of differentiation, particularly in myeloid leukemia, allows for more precise classification of leukemia than does morphology alone. However, morphologic and differentiation-based classifications of leukemia are limited in their prognostic value; cytogenetics and molecular genetics appear to be most important for identifying entities with distinct prognoses and clinical behavior. Increasingly, many of these genetically distinct subgroups of leukemia have been found to be closely associated with distinct immunophenotypes. For example, translocations such as t(8;21), t(15;17), and inv(16) in acute myeloid leukemia (AML), and t(1;19) and t(12;21) in acute lymphoblastic leukemia (ALL) have distinctive immunophenotypic profiles. Thus, in addition to classification into differentiation-based subtypes, detailed flow cytometric studies can define complex antigenic profiles that are associated with specific molecular defects and well-defined biology. In summary, multiparameter flow cytometry is an invaluable tool in the diagnosis, classification, and monitoring of patients with acute leukemia. Semin Hematol 38:124-138.

Acute Disease↗

PubMed2001

Using flow cytometry to follow the apoptotic cascade.

Flow cytometry has been extensively used to follow the apoptotic cascade and to enumerate apoptotic cells, both in cell cultures and, to a lesser extent, in tissue biopsies. An overview of the apoptotic cascade and how flow cytometric measurements can be used to observe the different elements of this process is presented.

Animals↗

PubMed1989

[Acute biphenotypic leukemia followed by two color flow cytometry].

In this paper is reported a case of acute biphenotypic leukemia who was treated by chemotherapy and pursued its effect by two color flow cytometry. A 33-year-old male patient was admitted due to fever and general fatigue and diagnosed as acute leukemia by hematological findings. Surface markers were investigated to find positive reaction of Leu 12 (CD19), J 5 (CD10), My 7 (CD13) and My 9 (CD33), in which Leu 12 and My 9 were simultaneously expressed on the same blast cells by two color flow cytometry. He was treated with daunorubicin, enocitabine, mercaptopurine, vincristine, and prednisolone to obtain partial remission. Then, he was administered L-asparaginase, doxorubicin, vincristine and prednisolone to reach complete remission. The effect of chemotherapy was investigated by not only bone marrow puncture, but also by two color flow cytometry. From the findings in this case, the two flow cytometry was proved to be a useful tool for not only diagnosis of acute mixed leukemia, aut also the judgement of the effect of treatment.

Acute Disease↗

PubMed2004

Quantitation of platelet aggregation and microaggregate formation in whole blood by flow cytometry.

Platelet aggregation and microaggregate formation were measured in samples of stirred whole blood by flow cytometry. Blood samples were stirred in a multi-sample agitator with ADP, fixed and labelled with a platelet-specific CD42a-FITC fluorescent antibody. The blood was then diluted and applied directly to a flow cytometer. Platelets were identified using a gating procedure based on their expression of CD42a and then quantified. Aggregation was monitored as a fall in the number of single platelets. Both reversible and irreversible aggregation responses to ADP were determined and these were found to correlate directly with aggregation responses determined using a well-established single platelet counting technique using the Ultra-Flo 100 Whole Blood Platelet Counter. We found from flow cytometry that ADP-induced aggregation was coupled with a transient formation of platelet microaggregates over the initial 60 s following ADP addition. Assessment of single platelet loss by flow cytometry was found to be a reliable way of monitoring aggregation responses and provided new information on rapid microaggregate formation in ADP-stimulated blood.

Adenosine Diphosphate↗

PubMed2001

Medicaid and Medicare reimbursement for flow cytometry.

Medicaid, a program administered by individual states but involving federal funding, is the source of medical coverage for many low-income patients. This method of reimbursement is crucial for many flow cytometry laboratories, but is not well understood by many laboratory professionals. Conversely, flow cytometry often is not well understood by administrators in Medicaid offices. The potential exists for great variation in Medicaid reimbursement for flow cytometry services from state to state. As a first step toward elucidating the extent of this variation and bringing more information about Medicaid to laboratory professionals, state Medicaid offices were asked to provide the fee-for-service reimbursement for flow cytometry services. These services included Current Procedural Terminology (CPT) codes 85045 (reticulocyte counts), 86359 (total T-cell count), 86360 (absolute CD4 and CD8 counts, with ratio), 86361 (absolute CD4 count), 86812 (HLA typing, single antigen [B27]), 88180 (immunophenotyping, per surface marker), and 88182 (DNA, cell cycle analysis). Data were collected on technical and professional components and on global reimbursement. Wide variation exists in reimbursement amounts for these tests. Variation for CPT code 88180 was markedly pronounced.

Diagnostic Services↗

PubMed2002

Flow cytometry as a new method to quantify the cellular content of human saliva and its relation to gingivitis.

BACKGROUND: Determining the cellular content of saliva by means of conventional microscopy chamber counting is a very time-consuming and operator-sensitive procedure. This study concentrated on the use of flow cytometry to examine the cellular content of saliva. Erythrocytes, leukocytes, epithelial cells and bacteria were quantified and the results were compared with caries experience and the presence of gingivitis. METHODS: 258 uncentrifuged vortexed paraffin-stimulated saliva samples (112 males and 146 females) were analyzed with the UF-100 flow cytometer. Salivary reference values were established for erythrocyte, leukocyte, epithelial cell and bacterial count. Caries experience (DMF) and the presence of gingivitis were recorded. RESULTS: Caries experience or caries risk could not be assessed with flow cytometry. However, salivary flow cytometry may be useful in determining an individual's risk for gingivitis: a significant increase in salivary leukocytes was observed in individuals with gingivitis. At a cut-off level of 10(3) leukocytes micro l(-1) saliva, a sensitivity of 76% and a specificity of 45% was obtained. Other analytes were not significantly different between individuals with and without gingivitis. CONCLUSION: Flow cytometry of paraffin-stimulated human saliva seems a promising diagnostic or predictive tool and further investigations of diseases of the oro-pharyngeal loge, such as tonsillitis and periodontitis, should be carried out in the future.

Adult↗

PubMed1988

Comparison of fresh and paraffin-embedded tissue as starting material for DNA flow cytometry and evaluation of intratumor heterogeneity.

Flow cytometric analysis of DNA ploidy and S-phase fraction (SPF) from paraffin-embedded tumors has become an important diagnostic and prognostic tool in clinical pathology and investigative oncology. The present study aimed at elucidating the reliability of the method. About 90% of the 1,400 paraffin-embedded tumors analyzed were evaluable for DNA index and about 70% for SPF, although considerable differences between various tumor types were detected. The within-assay coefficients of variation for determination of tumor DNA index and SPF were 2% and 6%, respectively. Intratumor variation in DNA index was observed in 24% of breast and in 21% of ovarian carcinomas and variation in SPF in 36% and 29% of the respective tumors. Intratumor variation in SPF was greatest in DNA-diploid tumors, which may indicate that SPF values in these tumors may be less reliable owing to variations in the proportions of tumor and nontumor cells. If the methodological variation and the intratumor variation were taken into account, there was a good correlation between DNA indices (r = 0.980) and between SPF values (r = 0.794) obtained from fresh and paraffin-embedded tumors. It is concluded that accurate determination of DNA index and SPF from paraffin-embedded tumors is possible in the majority of cases. Regardless of the type of starting material used for DNA flow cytometry it is advantageous to study several samples from each tumor to account for the intratumor heterogeneity.

Breast Neoplasms↗

PubMed1986

A comparison of static cytofluorometry and flow cytometry for the estimation of ploidy and DNA replication in human breast cancer.

332 primary invasive breast carcinomas were analysed by static cytofluorometry and flow cytometry. The ploidy distributions were similar, and 54% of the tumors were judged DNA aneuploid by both methods. The coefficient of variation of the G0-G1 peaks ranged from 2.0 to 8% with both techniques, but the mean was somewhat lower with flow cytometry--4.1%, compared to 4.9% for the static measurements. The proportion of S-phase cells was possible to estimate from 80% of the flow histograms and 70% of the static histograms. S-phase was not estimated from the static histograms if less than 150 tumor cells were measured. With 160 tumors S-phase was measured by both methods. The range was 0 to 27% with the static measurements and 0.7 to 25% with flow cytometry. Corresponding mean values were 7.6% and 8.2%, which are similar to thymidine labeling index results with breast cancers reported in some studies. A close correlation was obtained (r = 0.927) comparing S-phase fractions estimated from aneuploid tumors with flow cytometry and static cytofluorometry if more than 200 cells were measured with the latter. The proportion of S-phase cells was significantly lower for the diploid tumors. We conclude that both techniques can be useful for the estimation of DNA ploidy and replication in human breast cancer.

Aneuploidy↗

PubMed2002

Detection of alloreactive T cells by flow cytometry: a new test compared with limiting dilution assay.

BACKGROUND: Frequencies of alloreactive T cells determined by limiting dilution assays (LDA) may not adequately reflect the donor-reactive immune status in transplant recipients. To reevaluate LDA frequencies, we developed a flow cytometry test for direct determination of alloreactive T-cell frequencies and compared these frequencies with classical LDA estimates of frequencies. METHODS: For determination of frequencies by flow cytometry, peripheral blood lymphocytes (or lymphocytes taken from primary mixed lymphocyte culture) were stimulated with either Epstein-Barr virus-transformed lymphoblastoid cell lines or T cell-depleted spleen cells and stained for intracellular interferon (IFN)-gamma production and CD69. In lung transplant recipients, frequencies of IFN+ alloreactive T cells were compared with LDA frequencies, that is, cytotoxic T lymphocyte precursors and helper T lymphocyte precursors. RESULTS: With flow cytometry, alloreactive T cells were detected after overnight allostimulation as IFN-gamma CD69bright cells (range, 0.1-0.58% and 0.1-0.66% of total CD4 and CD8 cells, respectively). Frequencies increased 25-fold or more when lymphocytes were prestimulated in primary mixed lymphocyte culture before testing. After lung transplantation, mean donor-specific IFN+ CD8 T-cell frequencies did not decrease as mean donor-specific LDA cytotoxic T lymphocyte precursor frequencies, whereas no difference was seen in pretransplantation samples or third-party-specific frequencies at both time points. Mean frequencies of IFN+ CD4 did not differ from helper T lymphocyte precursors at both time points, but frequencies did not correlate. CONCLUSIONS: The flow cytometry test allows a direct measurement of alloreactive T-cell frequencies and demonstrates a discrepancy between donor-specific IFN+ CD8 T-cell frequencies and LDA CLTp after transplantation. This may be a result of the existence of "functional diverse" alloreactive T cells or of activation-induced cell death of donor-reactive T cells during long (LDA) culturing, which is avoided in the flow cytometry test.

Flow Cytometry↗