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PubMed2000

Combined analysis of DNA ploidy, proliferation, and apoptosis in paraffin-embedded cell material by flow cytometry.

A flow cytometric assay was developed for correlated measurement of DNA content and apoptotic DNA strand breaks in cell nuclei of formalin-fixed, paraffin-embedded tissues. The assay allows a combined analysis of cell ploidy, proliferation, and apoptosis in sections of fixed paraffin-embedded archival or fresh tissue/cell specimens. It is based on (a) proteolytic release of cell nuclei from deparaffinized and rehydrated 90-microm thick sections of the fixed embedded specimen, (b) the inactivation of the protease, (c) FITC-labeling of DNA strand breaks by the terminal deoxynucleotidyl transferase (TdT)-mediated FITC-dUTP nick end-labeling (TUNEL) reaction, and (d) DNA staining with 4'6-diamidino-2-phenyleindole. The fluorescence was recorded with a double-beam flow cytometer equipped with a mercury arc lamp and an argon ion laser. Cytograms obtained with this assay correlated closely with those produced using nonembedded material from the same specimen. Furthermore, a significant correlation was found between flow cytometric analysis of apoptosis in cell nuclei released from paraffin blocks and conventional evaluation of TUNEL on (corresponding) sections (p < 0.001). Since necrotic cells can stain positively by TUNEL, the possibility to microscopically select nonnecrotic tumor regions for flow cytometric analysis is an important advantage of the assay.

Apoptosis↗

PubMed1996

Interference of blood leucocytes in the measurements of immature red cells (reticulocytes) by two different (semi-) automated flow-cytometry technologies.

Flow cytometrical methods have been introduced recently as an alternative to the enumeration of reticulocytes by microscopy. Two of these methods have gained widespread use in haematological practice; the multiparametric flow cytometer using thiazole orange staining (Retic-Count, FACScan) and the single-application reticulocyte counter using auramine-O staining (R-series, Sysmex). Several studies have emphasized the excellent correlations between microscopy and these techniques. The purpose of our study has been to examine the specificity of these automated devices with regard to cells classified as 'reticulocytes' and the effect that this may have on measures of reticulocyte maturity. Our results indicate that the specificity of reticulocyte measurements by both the Sysmex R-1000/-3000 and the Retic-Count system is relatively low. This is due to the presence of leucocytes amongst cells classified as reticulocytes. These leucocytes display intense staining with either dye, leading to an erroneous estimation of RMI (thiazole orange) and high fluorescence count (R-1000/-3000). This error is directly correlated with the leucocyte count. The basis for reticulocyte identification should be improved before automated estimation of reticulocyte maturation can be used in clinical practice.

Automation↗

PubMed1985

Pattern of DNA synthesis and its effect on the classification of cells by flow cytometry.

A flow cytometer produces a DNA distribution which contains information about the proportions of cells in various phases of the cell cycle. In this report we show that the form assumed for the rate of DNA synthesis in a cell plays an important part in the estimation of those proportions. We compare three forms for the rate of DNA synthesis, one of which is derived from knowledge of the mechanism of DNA replication, and find that they give substantially different estimates of the proportions of cells in the G1 and G2 + M phases.

Cell Cycle↗

PubMed1993

Routine monitoring of Cryptosporidium oocysts in water using flow cytometry.

A flow cytometric method for the routine analysis of environmental water samples for the presence of Cryptosporidium oocysts has been developed. It uses a Coulter Epics Elite flow cytometer to examine water samples and to separate oocysts from contaminating debris by cell sorting. The sorted particles are then rapidly screened by microscopy. The method has been evaluated and compared with direct epifluorescence microscopy on 325 river, reservoir and drinking water samples. The technique was found to be more sensitive, faster and easier to perform than conventional epifluorescent microscopy for the routine examination of water samples for Cryptosporidium.

Animals↗

PubMed1986

Detection of minimal residual disease in acute leukemia by flow cytometry.

Commercial flow cytometers can detect and enumerate rare cells at the level of 1 per 10(5) other cells within a reasonable measuring time, provided that the rare cells can be uniquely labeled with fluorescent marker. This detection level is sufficient for the enumeration of normal hematopoietic stem cells and committed progenitor cells. Detection at this level is useful for the quantitation of residual leukemic cells in remission bone marrow, for the analysis of the proliferative state of these cells as well as of normal stem cells, which are of importance in choosing the optimal chemotherapy regimen, and for monitoring the efficacy of maintenance chemotherapy. A further improvement in the speed of flow cytometers would be required, however, to make full use of the bone marrow samples.

Animals↗

PubMed2000

Opsonization and phagocytosis of Plasmodium falciparum merozoites measured by flow cytometry.

A flow cytometric phagocytosis assay was established to investigate the role of anti-merozoite antibody, complement, and cytokines on the phagocytosis of Plasmodium falciparum merozoites by human neutrophils. This assay involved allowing fluorescein isothiocyanate-labeled merozoites to interact with phagocytes and analysis of the cells on a FACScan with Lysis II software. To differentiate the proportion of neutrophil surface-bound merozoites from the merozoites ingested by neutrophils, the fluorescence of bound merozoites was quenched by adding trypan blue. The data showed that sera from malaria-immune individuals in the Solomon Islands and Papua New Guinea promoted merozoite engulfment by neutrophils. The cytokines tumor necrosis factor alpha, gamma interferon, granulocyte-macrophage colony-stimulating factor, and interleukin-1beta significantly increased the amount and the rate of merozoite phagocytosis by neutrophils. Optimum merozoite phagocytosis occurred when both cytokines and anti-malarial antibody were present.

Animals↗

PubMed1998

Simultaneous identification of antibodies to Brucella abortus and Staphylococcus aureus in milk samples by flow cytometry.

Two flow cytometric assays are described herein. The single cytometric test (SCT) detects antibodies to either Brucella abortus or Staphylococcus aureus in the serum or milk of a cow or water buffalo. The double cytometric test (DCT) detects both anti-B. abortus and anti-S. aureus antibodies concurrently. In the SCT, the sample to be tested is incubated in succession with the antigen (either B. abortus or S. aureus) and the proper secondary antiserum (fluorescein isothiocyanate-labelled rabbit anti-cow immunoglobulin antiserum or rabbit anti-water buffalo immunoglobulin antiserum). In the DCT, the sample to be tested is incubated first with B. abortus and S. aureus antigens and then with the secondary antiserum. The B. abortus antigen used in the DCT is covalently bound to 3-microm-diameter latex particles. The difference in size between B. abortus and S. aureus permits the establishment of whether the antibodies are directed against one, the other, or both antigens. When compared to the complement fixation test, the SCT and DCT each show a specificity and a sensitivity of 100%. The SCT has been used previously to detect anti-S. aureus antibodies. Here its use is extended to the detection of anti-B. abortus antibodies. The DCT is described here for the first time. The DCT appears to be useful for large-scale brucellosis eradication programs. It offers the possibility of using one test to identify animals that are serologically positive for both B. abortus and S. aureus.

Animals↗

PubMed1990

Reticulocyte counting using flow cytometry.

A flow cytometric method for the quantitation of reticulocytes was refined for routine laboratory use. Blood (2 microliters) is added to 2 ml of 0.4 microM thiazole orange in phosphate buffered saline, incubated at room temperature for 90 minutes, and analysed on a Coulter EPICS Profile flow cytometer, with gating for red cells on the basis of forward and right angled light scatter. Blood (2 microliters) is also incubated with phosphate buffered saline alone as an unstained control. The adult reference range (mean +/- 2 SD), established from 30 laboratory personnel, is 19.4-59.2 x 10(9)/l (0.2-1.6%). Comparison of this technique was made on 39 selected patient samples with visual counting of cells stained with brilliant cresyl blue. The correlation between the two methods was 0.99 with slope 0.96 and intercept 0.02. The precision of the automated technique in three subjects with reticulocyte counts of 0.12%, 1.84%, and 14.3% was 33.3%, 7.3%, and 1.4%, respectively (coefficient of variations). In three patients studied serially after intensive chemotherapy, in whom the reticulocyte count quantitated by routine visual methods approached zero (0-0.1%) for eight to 18 days, the automated counts varied between 0 and 0.5%. Flow cytometric reticulocyte counting is thus a simple and highly reliable methodology for the quantitation of normal and raised reticulocyte counts but cannot be reliably used to quantitate a subnormal level.

Benzothiazoles↗

PubMed1993

Zinc-silicon interactions influencing sperm chromatin integrity and testicular cell development in the rat as measured by flow cytometry.

Flow-cytometric procedures were used to determine effects of dietary Zn and Si variations on rat testicular cell development, including integrity of caudal epididymal sperm chromatin structure defined as the susceptibility of DNA to denaturation in situ. Concentrations of 4 (deficient), 12 (adequate), and 500 (excessive) mg of Zn/kg of diet were used with Si concentrations of 0 (low), 540 (medium), and 2,700 (high) mg/kg of diet in a 3 x 3 factorial arrangement. Three-week-old Sprague-Dawley male rats were fed the experimental diets for 8 wk. Rats fed the Zn-deficient/Si-low diet demonstrated significant deviations in the ratio of testicular cell types present, including a reduction of S phase and total haploid cells. Furthermore, approximately 50% of epididymal sperm had a significant decrease in resistance to DNA denaturation in situ. In the Zn-deficient/Si-medium treatment, the effects of Si on animal and testicular growth, distribution of testicular cell types, and sperm chromatin structure integrity were quite similar to the effects of the Zn-adequate diets. A toxic effect of Zn on sperm chromatin structure integrity observed in the Zn-excess/Si-medium treatment seemed to be counteracted by Si in the Zn-excess/Si-high treatment. Silicon at medium and high levels seems to affect Zn metabolism through potentiation and antagonistic reactions, respectively. Zinc deficiency likely disrupts the normal sperm chromatin quaternary structure in which Zn plays a role by providing stability and resistance to DNA denaturation in situ.

Animals↗

PubMed1995

Application of biotin, digoxigenin or fluorescein conjugated deoxynucleotides to label DNA strand breaks for analysis of cell proliferation and apoptosis using flow cytometry.

A flow cytometric method has recently been developed using biotinylated dUTP (b-dUTP) in a reaction catalyzed by terminal deoxynucleotidyl transferase (TdT) to identify the endonuclease-induced DNA strand breaks occurring during apoptosis. Counterstaining of DNA makes it possible to relate apoptosis to cell cycle position or DNA index. In the present study, we compared this method with one using digoxigenin-conjugated dUTP (d-dUTP) to label apoptotic cells. The discrimination of apoptotic from nonapoptotic cells was similar when incorporation of d-dUTP was compared with b-dUTP. Both techniques resulted in a 20-30 fold increase in staining of apoptotic over nonapoptotic cells although somewhat less background fluorescence was observed with the d-dUTP. Direct labeling with fluoresceinated dUTP (f-dUTP) was less sensitive in detecting DNA strand breaks, but had the advantage of simplicity. The principle of labeling DNA strand breaks using TdT was also employed to identify DNA replicating cells. To this end, the cells were incubated in the presence of BrdUrd, then exposed to UV light to selectively photolyse DNA containing the incorporated BrdUrd. DNA strand breaks resulting from the photolysis were then labeled with b-dUTP or d-dUTP. This approach is an alternative to immunocytochemical detection of BrdUrd incorporation, but unlike the latter does not require prior DNA denaturation, thus can be applied when the denaturation step must be avoided. The method was sensitive enough to recognize DNA synthesizing cells that were incubated with BrdUrd for only 5 min, the equivalent of replication of less than 1% of the cell's genome. The discrimination between apoptotic vs. BrdUrd incorporating-cells is based on different extractability of DNA following cell fixation. This method can be applied to analyze both cell proliferation (DNA replication) and death (by apoptosis) in a single measurement.

Apoptosis↗

PubMed1993

Rapid determination of sperm cell concentration in bovine semen by flow cytometry.

A flow cytometric technique is described for determining sperm concentration in fresh or extended semen with improved accuracy, precision, repeatability, ease of conduct, and rapidity. The technique is designed to measure the ratio of a known number of fluorescent beads admixed with sperm stained with either acridine orange or propidium iodide. A significant advantage of the technique is the distinct resolution between sperm and other particles (e.g., somatic cells, fat droplets, and bacteria in the semen or extender) that interfere in other counting protocols. Field testing of this protocol over the past 3 yr has demonstrated its superiority over the Coulter counter, hemacytometer, and spectrophotometer for accuracy in counting sperm in extended semen and the accuracy of counting sperm in straws based on preextension spectrophotometric determination of sperm concentration. Sperm chromatin quality can be determined simultaneously with this sperm counting procedure. This approach to counting sperm provides an excellent procedure for quality control of sperm numbers in processed semen.

Acridine Orange↗

PubMed1998

Analysis of megakaryocytes by flow cytometry.

A flow cytofluorometric measurement of megakaryocyte ploidy has been adapted from Tomer's method. Briefly, bone marrow is aspirated through a medium containing theophyllin, prostaglandin-E1 and other antiaggregant agents. Megakaryocytes-enriched buffy coats are recovered. Megakaryocytes are then stained for GP IIIa coupled with FITC and for DNA (propidium iodide). A Becton Dickinson FACScan flow cytometer is used for measuring the ploidy distribution of GP IIIa positive cells. The method we developed presents several advantages. Firstly, the time required is greatly decreased in comparison with other studies. Secondly, the washing steps are limited in number allowing a diminution of the cell loss. Thirdly, the method ensures a better megakaryocyte preservation. Finally, selection of megakaryocytes by ploidy and expression of GP IIIa can be made easily because of the simultaneity of the two stainings as well as by the use of a precise gating on the flow cytometer. Based on these results, we conclude that the present method provides a better means for the isolation and analysis of human normal megakaryocytes. This technique has been applied to the analysis of megakaryocyte populations from patients with abnormal platelet counts. In chronic myeloid leukemia patients, analysis of ploidy distribution shows a shift toward the low ploidy while in patients with immune thrombocytopenic purpura, polycythemia vera and essential thrombocythemia the ploidy distributions are shifted toward the high ploidy.

Adolescent↗

PubMedDate not supplied

[Determination of the radiosensitivity of interphase-dying cells in the thymus, spleen and bone marrow of rats by flow cytometry].

The flow cytofluorometry of cells stained with a DNA-specific probe was used to determine the share of dying cells (containing less than 2C DNA) in thymus, spleen and bone marrow cells of irradiated rats. The cell death curves for spleen and bone marrow had a plateau by the 6th h, and for thymus, by the 10th h following irradiation with different doses. On the basis of the dose-response relationship the share of cells dying in the interphase was determined in each organ under study, and dose-response curves shaped. All the curves had no shoulder. Do was 3.0, 3.0 and 3.7 Gy for thymus, spleen, and bone marrow cells, respectively.

Animals↗

PubMed2005

Comparison of Microcapillary Cytometry Technology and Flow Cytometry for CD4+ and CD8+ T-Cell Estimation.

An alternative technology for the estimation of T cells based on a microcapillary technique (Guava Technologies, Hayward, CA) was compared to FACSCount (Becton Dickinson, San Jose, CA). Samples from 51 human immunodeficiency virus-infected and 21 healthy individuals were tested. The correlation (r) of the two systems for CD4(+) T cells was 0.994, and the coefficient of variation was 6.5%, establishing equable performance between the two technologies.

CD4-CD8 Ratio↗

PubMed2000

Evaluation of platelet function by flow cytometry.

Platelet function in whole blood can be comprehensively evaluated by flow cytometry. Flow cytometry can be used to measure platelet reactivity, circulating activated platelets, platelet-platelet aggregates, leukocyte-platelet aggregates, procoagulant platelet-derived microparticles, and calcium flux. Clinical applications of whole blood flow cytometric assays of platelet function in disease states (e.g., acute coronary syndromes, angioplasty, and stroke) may include identification of patients who would benefit from additional antiplatelet therapy and prediction of ischemic events. Circulating monocyte-platelet aggregates appear to be a more sensitive marker of in vivo platelet activation than circulating P-selectin-positive platelets. Flow cytometry can also be used in the following clinical settings: monitoring of GPIIb-IIIa antagonist therapy, diagnosis of inherited deficiencies of platelet surface glycoproteins, diagnosis of storage pool disease, diagnosis of heparin-induced thrombocytopenia, and measurement of the rate of thrombopoiesis.

Antibodies, Monoclonal↗

PubMed1998

Bone marrow staging in patients with non-Hodgkin's lymphoma: is flow cytometry a useful test?

BACKGROUND: Flow cytometric analysis of bone marrow often is used as an adjunct to morphologic evaluation in the staging of patients with non-Hodgkin's lymphoma (NHL). The goal of this study was to define objectively the benefit of flow cytometry in this setting. METHODS: The authors reviewed retrospectively all bone marrow specimens submitted between January 1992 and December 1994 to the Washington University Department of Pathology for flow cytometric immunophenotyping to rule out NHL. Results of morphologic examination and flow cytometry were reviewed independently and the ability to detect bone marrow involvement compared. RESULTS: Two hundred and seventy-three bone marrow specimens from 190 patients with an established diagnosis of NHL were submitted for flow cytometric analysis at initial presentation, restaging, and/or recurrence. Morphologic evaluation was negative in 69%, positive in 23%, and equivocal in 8%. Flow cytometry was negative in all but 1 morphologically negative bone marrow specimens and 40% of morphologically involved bone marrow specimens. Two of 23 morphologically equivocal bone marrow specimens were positive by flow cytometry. An additional 86 specimens were obtained to rule out NHL in patients without an established diagnosis of NHL. The majority of patients had a history of human immunodeficiency virus infection, cytopenia, or unexplained fevers. Morphologically, one specimen was involved with NHL, 5 were equivocal, and 80 were negative. All specimens were negative by flow cytometry. CONCLUSIONS: In this study, flow cytometric analysis improved the detection of NHL in bone marrow in only 3 of 273 samples, 2 of which were suspicious morphologically. Flow cytometry of bone marrow aspirates has a limited role in the routine staging and follow-up of patients with an established diagnosis of NHL.

Bone Marrow Examination↗

PubMed1997

Image cytometry for quantitative analysis of DNA in the testes of infertile men with varicocele: comparison with flow cytometry.

PURPOSE: The first aim of this study is to evaluate the usefulness of image cytometry comparing flow cytometry to analyze the spermatogenesis using testicular biopsy specimens obtained from infertile male. The second is to investigate the spermatogenesis of infertile men with varicocele by image cytometry. MATERIALS AND METHODS: A total of 76 biopsy specimens of testicular tissue were obtained from 38 infertile men with varicocele and 6 specimens of normal testicular tissue controls were obtained from 6 volunteers at vasectomy. They were evaluated by image cytometry and pathohistological examination. Out of 76 specimens, 21 biopsy tissues were also analyzed by flow cytometry, and the results by both image cytometry and flow cytometry were compared. Two specimens from Sertoli cell only testes were also examined by image and flow cytometry. RESULTS: In comparative study, image cytometry revealed a decrease in the proportion of haploid and diploid cells (18.8 +/- 6.6% and 32.4 +/- 7.4%), and an increase in that of tetraploid cells (21.8 +/- 5.6%) vs. the flow cytometry findings (haploid, 25.9 +/- 9.2%; diploid, 36.8 +/- 6.5% and tetraploid, 15.1 +/- 5.9%). Image cytometric analysis of the testes of varicocele patients showed 15.9 +/- 5.6% haploid, 31.7 +/- 7.9% diploid and 16.2 +/- 8.9% tetraploid cells in the left testis of infertile men, while their right testis showed 16.6 +/- 5.4% haploid, 30.0 +/- 7.6% diploid and 17.2 +/- 6.4% tetraploid cells. The testes of normal men showed 37.6 +/- 8.3% haploid, 19.4 +/- 4.8% diploid and 17.4 +/- 5.8% tetraploid cells. The left and the right testes of the patients with varicocele showed a lower proportion of haploid cells and a higher proportion of diploid cells vs. the testes from normal men. The proportions of the haploid and diploid cells are correlated with sperm concentration. CONCLUSION: Image cytometry could exclude interstitial cells, Sertoli cells, and sperm on the static image, so produced an accurate assessment of spermatogenesis. The alteration in the germ cell population in the testes of infertile men with varicocele suggested that the impairment mostly affected the cells in the meiotic stage.

Adult↗

PubMed2026

Standardizing stem cell enumeration: A methodological comparison of single and dual flow cytometry platforms.

Two flow cytometry methods are used for stem cell (CD34+) enumeration; single platform (SP) and dual platform (DP). While several studies reported comparable results, others suggested superiority of the SP method. This study evaluated variations between both methods using a modified workflow. A total of 54 fresh and thawed specimens, including mobilized peripheral blood, apheresis products, and umbilical cord blood, were analyzed using both methods. High concordance between SP and DP methods was observed for absolute viable CD34+ counts in fresh and thawed specimens (p = 0.088 and 0.427, respectively), as well as for CD34+ viability (p = 0.085 and 0.801). Absolute viable WBC counts were comparable between methods in thawed specimens (p = 0.124), whereas a modest statistical variation was observed in fresh specimen group (p = 0.039), largely influenced by umbilical cord blood samples. Variation in absolute viable CD34+ counts remained within clinically acceptable limits, with median variations of 2.4 for fresh and 1.4 for thawed samples. SP and DP methods demonstrated high concordance for absolute viable CD34+ enumeration and CD34+ viability in fresh and thawed specimens. Although a modest variation in viable WBC counts was observed in fresh samples, this did not affect CD34+ enumeration and remained clinically acceptable. While SP provides a standardized approach, the DP method offered greater gating flexibility, with fewer technical resources required, and was approximately 70% more cost-effective, supporting its use as a practical alternative in appropriate laboratory settings.

Humans↗