Flow cytometric DNA analysis.

Flow cytometric DNA analysis, including principles, techniques, and applications, is reviewed. Flow cytometry, a relatively recently developed technology, is being increasingly used in the diagnosis and treatment of a wide variety of benign and malignant diseases. Current major applications include phenotypic analysis of cells, cell sorting, and DNA analysis. DNA analysis by flow cytometry is rapid, reliable, and reproducible. Flow cytometric DNA analysis offers a quick, reliable method of analyzing cellular DNA content capable of identifying cell populations with abnormalities in total DNA content.     

Flow cytometric DNA analysis of lung cancer cell lines.

Tumor DNA content (ploidy) was analyzed by use of flow cytometry (FCM) in 17 lung cancer cell lines which were subcultured in our laboratory. The study included 6 adenocarcinomas, 2 squamous cell carcinomas, 1 adenosquamous cell carcinoma, 5 large cell carcinomas, and 3 small cell carcinomas. Of the 17 lung carcinoma cell lines, 15 revealed aneuploid patterns with DNA index above 1.1, whereas one had diploid. The mean DNA index (DI) in adenocarcinoma, was 1.34 +/- 0.09, DI 1.6, in squamous cell carcinoma, DI 1.0 in adenosquamous cell carcinoma, DI 1.70 +/- 0.66 in large cell carcinoma, and DI 1.29 in small cell carcinoma. Of the 17 cell lines, three lines showed multiploid patterns with clinically poor prognosis and indicated heterogeneity. Flow cytometric DNA analysis using lung cancer cell lines could provide further basic study of lung cancer cells and give a useful information on the degree of the malignancy clinically.     

Flow cytometric analysis in platelet crossmatching using a platelet suspension immunofluorescence test

BACKGROUND: The sensitivity of flow cytometric measurement of platelet antibodies in a crossmatch technique was investigated. STUDY DESIGN AND METHODS: The corrected count increment after platelet transfusion was compared with the fluorescence ratio determined by flow cytometric measurement. RESULTS: When crossmatching was performed before transfusion(s) in alloimmunized patients, a fluorescence ratio < or = 1.7 was associated with satisfactory responses (corrected count increment > or = 7.5), and the predictive values for negative and positive crossmatch results were 94 and 87 percent, respectively. Analysis of antigen preservation during platelet storage with antibodies to HLA alpha-chain, HLA-B27, HPA-1a, and HPA-3a showed that platelets can be stored, refrigerated, for up to 4 weeks without significant loss of HLA class I and HPA-1a. There was a slight but continuous loss of HPA-3a upon storage. CONCLUSION: Flow cytometric measurement of fluorescence in the platelet suspension immunofluorescence test can be used for platelet crossmatching, with sensitivity and predictive values comparable to those of previously described techniques and with the advantage of automation.     

Flow cytometric DNA analysis of human endometrial carcinoma

Analysis of endometrial carcinoma of 15 cases and the normal endometrium from 12 control cases was made using flow cytometry. The proliferative activity and the ploidy level of the tumors were determined and a comparative study of the tumors classified by the nuclear grade of the tumor was made. In comparison with normal endometrium, endometrial carcinoma tissue had a significantly larger S-phase fraction of the cell cycle. Moreover, among the endometrial carcinoma cases, the percentage of S-phase cells was found to increase with increases in abnormalities of nuclear morphology. Endometrial carcinoma cases had higher levels of G2+M phase fractions than normal endometrium, but not significantly so. The proliferation index of endometrial carcinomas was significantly higher than that in normal endometrium, and it was found that proliferative activity became more robust the more irregular the nuclear morphology. With regard to the ploidy level, all of the normal endometria and Nuclear Grade 1 or 2 endometrial carcinoma cases showed a DNA distribution between diploid and tetraploid. In contrast, Nuclear Grade 3 cases could be subdivided into two groups, one which had a DNA distribution between diploid and tetraploid with an extremely high proliferation index and the other which had an aneuploid DNA stem line.     

Flow cytometric analysis of Ki-67 reactive antigen]

Monoclonal antibody Ki-67 is a useful and easy means for evaluating cell proliferative activity. Furthermore, it appears to provide valuable prognostic informations in some neoplastic diseases. However, almost all studies on solid tumors were performed only by microscopic observation using immunohistochemistry, and not by flow cytometry. Therefore, the information deduced from the findings is limited. The method of DNA/Ki-67 double staining of solid tissue and cultured cells for flow cytometry is shown, and the significance and the usefulness of flow cytometric study of Ki-67 in clinical use are discussed.     

Flow cytometric analysis of cytologic specimens in hematologic disease

Cytologic evaluation of body fluids and fine needle aspirations (FNA) is frequently required in patients with hematologic diseases. In this study we have correlated immunophenotyping and DNA analysis by flow cytometry with cytologic findings and tissue biopsies from 20 patients with body fluid specimens and 5 with FNA. Nineteen of 25 cases, including all FNA cases, had an immunophenotype consistent with malignancy: 12 monoclonal B-cell lymphomas, 2 T-cell lymphomas, 2 T-ALL and 3 non-T-ALL. By cytologic examination, 14 of these 19 cases were positive for malignant cells, 2 suspicious and 3 negative; the latter 5 cases, including 2 FNA cases, had small monoclonal B-cell populations detected by flow cytometry. Six cases had a benign immunophenotype; cytologic examination was benign in 4 of these and suspicious for lymphoma in 2. Our results show the feasibility of using flow cytometry to evaluate body fluids or FNA and demonstrate that small malignant populations that may be missed by routine cytology can be detected by flow cytometry.     

Flow cytometric measurement of NK cell cytotoxicity.

A simple and fast method to measure the natural killer (NK) cell cytotoxicity has been established. In our novel assay, following the incubation of K562 target and effector cells, the cells were stained with FITC-conjugated anti-transferrin receptor (CD71) mAb. The K562 targets alone were labeled with anti-CD71 mAb, since CD71 is highly expressed on K562 cells but not on mononuclear cells. The fluorescence intensity of CD71 on K562 cells were analyzed using flow cytometry, and the cytotoxicity was evaluated. In our assay, designated as a "CD71 assay", no spontaneous release of anti-CD71 mAb was observed from labeled K562 cells and living K562 cells were able to be divided from dead K562 cells. The data obtained by this assay was well correlated with that by the standard 51Cr-release assay as well as the C-FDA assay. In addition, the CD71 assay is a safe and simple analytic procedure since no isotope is required.     

Flow cytometric analysis of chromosome aberration on tumor cells]

Chromosomal changes plays an important role in malignant transformation. Generally, the process of karyotype instability with grows aneuploidy occurs in tumor. But it is very difficult to obtained Flow karyotype from tumor cells. There are many technical problem in the analysis of chromosomes aberration in tumor cells. Problem is technical procedure of isolation from metaphase chromosomes. It is important to choice of swelling buffer and treatment times. Such technic affect for Flow karyotype pattern. We try to obtained Flow karyotype from chinese hamster cell and V-79 cells and we reported a recent new techniques of cell preparation and chromosomes suspension.     

Automated analysis of reticulocytes

An evaluation of the performance of the GEN-S analyzer in examining the parameters of reticulocytes is described. The automated and manual methods of counting the relative quantity of reticulocytes were compared. It was established that the precision of the analyzer significantly surpasses the manual method results. The variation coefficient for the parameters of examination of reticulocytes is lower at a higher content of reticulocytes and the accuracy of the method is better if the content of reticulocytes in blood is higher. The parameters, characterizing the fraction of immature reticulocytes, are least accurate. The study demonstrated that the automated method of analyzing the reticulocytes by using the GEN-S analyzer can be used to ensure a more accurate monitoring of changes of the function of erythropoesis at different anemic conditions and to assess the conducted therapy.     

Near-infrared fluorescence imaging of murine atherosclerosis using an oxidized low density lipoprotein-targeted fluorochrome

The aim of this study was to explore the feasibility of detecting plaques using an NIR797 fluorochrome-labeled, anti-oxLDL antibody (anti-oxLDL-NIR797) and near-infrared fluorescence (NIRF) imaging in a murine model of atherosclerosis. Anti-mouse oxLDL polyclonal antibodies were conjugated to NIR797 dyes to synthesis oxLDL-targeted NIRF probe. In situ and ex vivo NIRF imaging of the high-cholesterol diet-induced atherosclerotic lesions of apoE?/? mice (baseline) as well as ex vivo NIRF imaging in the progression and regression group (without or with atorvastatin treatment for another 8 weeks) were performed 24 h after an intravenous injection of 1 mg/kg of anti-oxLDL-NIR797, while phosphate-buffered saline (PBS) was used for the controls. The plaque areas were investigated using Oil Red O (ORO) staining. Aortas isolated from the apoE?/? mice 24 h post-injection exhibited a selective, strong, heterogeneous NIRF signal enhancement in the aortic root, arch, and bifurcation, whereas the PBS and competitive inhibition groups had limited NIRF signal changes (p < 0.05). There was a significant correlation between ORO staining and NIRF in the atherosclerotic aortas that received anti-oxLDL-NIR797. Immunofluorescence studies confirmed the colocalization of the oxLDL/macrophages and NIR797 fluorochromes. Furthermore, the atherosclerotic lesions of atorvastatin-treated mice showed reduced anti-oxLDL-NIR797 uptake and oxLDL expression. These results indicate that NIRF plaque imaging is feasible with an oxLDL-targeted NIRF probe. Thus, oxLDL-based molecular imaging of atherosclerotic plaques is feasible and may provide important methods for characterizing vulnerable plaques and monitoring the response to therapeutic interventions for atherosclerosis.     

Flow cytometric bromodeoxyuridine (BrdU)/DNA analysis using fresh solid tumors and its clinical significance]

It is very important to determine the biological characteristics of individual cancers, not only in order to evaluate malignant potentials but also to decide on the most effective anticancer therapy. A method which combines BrdU labeling and flow cytometric bromodeoxyuridine (BrdU)/DNA analysis makes it possible to plot the DNA synthetic rate of cells against their DNA content. Hence, this method yields more useful information than the previous method of flow cytometric DNA analysis. When this method is performed correctly, it can be also applied to fresh solid tumors. In this paper, we will describe the results of our preliminary investigation and discuss its clinical significance.     

流式细胞术诊断血小板无力症

建立血小板膜糖蛋白（ＧＰ）的流式细胞术（ＦＣＭ）检测方法并初步用于诊断血小板无力症（ＧＴ）。结果表明：ＦＣＭ不但能灵敏、快速、准确、特异地检测单个或亚群血小板膜ＧＰ的变化，而且很容易鉴别ＧＴ及其杂合子携带者，为ＧＴ的诊断提供了一种新方法。     

Flow cytometric analysis of platelet membrane antigens during and after continuous-flow plateletpheresis

BACKGROUND: The influence, extent, and duration of changes in platelet antigen expression caused by blood-biomaterial interaction in plateletpheresis were assessed. STUDY DESIGN AND METHODS: Twenty-two apheresis donors were studied by using two automated continuous-flow apheresis devices. Blood samples were taken before, during, and for 4 days after extracorporeal circulation. The platelet surface expression of glycoproteins CD41a, CD42b, CD62p, and CD63 was analyzed by flow cytometry. RESULTS: Over the course of plateletpheresis, there was a significant increase in mean channel fluorescence intensity (MCFI) of CD62p, from 25.1 +/− 7.9 (mean +/− SD) to 50.4 +/− 28.9, and of CD63, from 22.3 +/− 6.5 to 33.3 +/− 13.2. There was a significant decrease in CD41a expression as measured by the MCFI, from 1129.8 +/− 125.0 to 1066.6 +/− 102.2, and in CD42b MCFI, from 329.6 +/− 49.4 to 321.4 +/− 52.0. The two apheresis devices showed different platelet activation kinetics, but the overall MCFI of CD62p and CD63 did not significantly diverge after 60 minutes of apheresis. CD62p and CD63 expression as measured by the MCFI returned to preapheresis levels during the follow- up period in 25 and 25 of 44 procedures, respectively, within 24 hours; in 10 and 13 of 44 procedures after 48 hours; in 7 and 3 of 44 procedures after 72 hours; and in 2 and 3 of 44 procedures on Day 5. CONCLUSION: The varying kinetics of expression, as measured by the MCFI, of platelet antigens CD62p, CD63, CD41a, and CD42b during extracorporeal circulation may be useful for biocompatibility testing. Activated platelets continue to circulate in donors for several days after cytapheresis, which suggests that a sufficient interval between apheresis procedures is necessary to avoid the collection of activated platelets.     

Flow cytometric analysis of cellular DNA content in epithelial ovarian cancer]

Tumor DNA content (ploidy) was determined by flow-cytometry (FCM) on tissue from 32 epithelial ovarian cancer patients. Staining for DNA analysis was achieved with Propidium Iodide. Peripheral blood lymphocytes were used as reference diploid cell population. Of the 32 patients, 26 (81.3%) had tumors which were aneuploid, whereas 6 (18.7%) were diploid. DNA aneuploid cell lines were found in 100% of serous adenocarcinoma, in 57% of mucinous adenocarcinoma, in 67% of endometrial adenocarcinoma, in 88% of clear cell carcinoma and in 75% of undifferentiated carcinoma. The DNA ploidy abnormalities differed in each histologic characteristic of epithelial ovarian cancer.     

Flow cytometric monitoring of antibiotic-induced injury in Escherichia coli using cell-impermeant fluorescent probes

Three fluorescent nucleic acid binding dyes-propidium iodide, TO-PRO-1, and SYTOX green-were evaluated, and their abilities to distinguish between bacterial cells with and without an intact cytoplasmic membrane were compared. Each dye was readily able to discriminate between healthy and permeabilized cells of Escherichia coli, although SYTOX green showed a greater enhancement in fluorescence intensity on staining-compromised, as opposed to healthy, cells in log-phase growth, than either PI or TO-PRO-1. Flow cytometric analysis of E. coli stained with these dyes after exposing them to several antimicrobial agents showed that all three dyes were able to detect antimicrobial action. Notably, however, the intensity of the cell-associated fluorescence was related to the mechanism of action of the antimicrobial agent. Large changes in fluorescence intensity were observed for all the dyes subsequent to beta-lactam antibiotic action, but smaller changes (or no change) were seen subsequent to exposure to antimicrobials acting directly or indirectly on nucleic acid synthesis. Furthermore, cell-associated fluorescence did not relate to loss of viability as determined by plate counts. Despite offering much insight into antimicrobial mechanisms of action, these fundamental problems become relevant to the development of rapid antimicrobial susceptibility tests if colony formation is used as the standard.     

急性白血病复发外周血白血病细胞的流式细胞术检验分析

目的分析急性白血病(AL)患者外周血白血病细胞流式细胞术检测情况,为分析AL的临床疗效和预后提供参考。方法采用流式细胞术检测87例AL患者(急性髓细胞白血病53例、急性淋巴细胞白血病34例)外周血标本,同时检测骨髓细胞形态学变化。结果急性髓细胞白血病检测的灵敏度为95.6%、特异度为34.5%、阳性预测值为81.3%;急性淋巴细胞白血病检测的灵敏度为87.3%、特异度为45.6%、阳性预测值为68.9%,差异均有统计学意义(P0.05);检出微小残留病变阴性19例,24个月后复发率为26.31%,微小残留病变阳性68例,24个月后复发率为86.76%,两者复发率比较差异有统计学意义(P0.05);在所有微小残留病变阳性患者中,高表达者复发率(88.23%)高于低表达者(47.09%),差异有统计学意义(P0.05)。结论流式细胞术检测外周血白血病细胞对诊断AL复发和指导临床用药有重要意义。     

Flow cytometric features of angioimmunoblastic T-cell lymphoma

BACKGROUND: The immunophenotypic features of angioimmunoblastic T-cell lymphoma (AILT) have not been well described. METHODS: We retrospectively reviewed our institutional experience with the flow cytometric features of 16 cases of AILT. RESULTS: Multiparameter flow cytometry was able to identify a distinct population of immunophenotypically aberrant T cells in 15 of 16 cases. In 13 lymph node specimens, the neoplastic cells ranged from 1.9 to 87% (median 23%) of cells. The ratio of reactive to neoplastic T cells ranged from 0.01 to 20 (median 1.5); reactive T cells outnumbered neoplastic in 9/13 (69%) cases. The neoplastic populations expressed CD2, CD4, CD5, and CD45RO in all cases, lacked expression of CD8 and CD56 in all cases, and showed negative or dim surface CD3 in most cases. CD10 was expressed by the neoplastic populations in 11 of 14 cases at diagnosis; in 3 of these 11 only a subpopulation of the neoplastic cells was CD10(+). CD10 tended to be absent on neoplastic cells in staging bone marrows. The neoplastic population in all but one of the 15 positive cases possessed multiple immunophenotypic abnormalities and these were generally retained during the follow-up analyses of several cases. CONCLUSIONS: These results indicate the potential utility of flow cytometry in the diagnosis and follow-up of AILT.