OBJECTIVETo analyze related factors which affect GPA mutation frequency of workers exposed to benzene, with the Glycophorin A (GPA) mutation assay and explore the possibility of GPA mutation frequency as an index of predicting the risk of benzene poisoning.

METHODSThe erythrocytes were bound with fluorescent-labeled monoclonal antibody after isolated and fixed from the peripheral blood, and then the GPA mutation assay was performed using the flow cytometry (FCM). The related factors of GPA mutation frequency were analyzed by statistical methods.

RESULTSThe GPA mutation frequency of chronic benzene poisonings was significantly higher than that of their controls (P < 0.05). Significant direct correlation was found between age, length of service, accumulative exposure score and the GPA mutation frequency of workers exposed to benzene (P < 0.01). However, there was significantly inverse correlation between the 3AB index and the GPA mutation frequency (GPAN0: r(s) = -0.589, P < 0.01, GPANN: r(s) = -0.615, P < 0.01). In the multiple factor regression analysis on GPA mutation frequency, benzene exposure and individual susceptibility both entered model of multiple factors analysis, the coefficient of determination of benzene-exposed workers was 0.819.

CONCLUSIONExposure to benzene and individual susceptibility are the most important factors that affect GPA mutation frequency. GPA mutation frequency increases with the benzene exposure and individual susceptibility.

Benzene ; poisoning ; Glycophorin ; genetics ; Humans ; Mutation ; Mutation Rate ; Occupational Exposure

OBJECTIVES: To assess the availability of the glycophorin A (GPA) assay to detect the biological effect of ionizing radiation in workers exposed to low-doses of radiation. METHODS: Information on confounding factors, such as age and cigarette smoking was obtained on 144 nuclear power plant workers and 32 hospital workers, by a self-administered questionnaire. Information on physical exposure levels was obtained from the registries of radiation exposure monitoring and control at each facility. The GPA mutant assay was performed using the BR6 method with modification by using a FACScan flow cytometer. RESULTS: As confounders, age and cigarette smoking habits showed increasing trends with GPA variants, but these were of no statistical significance. Hospital workers showed a higher frequency of the GPA variant than nuclear power plant workers in terms of the NO variant. Significant dose-response relationships were obtained from in simple and multiple linear regression models. The slope of the regression equation for nuclear power plant workers was much smaller than that of hospital workers. These findings suggest that there may be apparent dose-rate effects. CONCLUSION: In population exposed to chronic low-dose radiation, the GPA assay has a potential to be used as an effective biologic marker for assessing the bone marrow cumulative exposure dose.
Biomarkers* ; Bone Marrow ; Glycophorin* ; Linear Models ; Nuclear Power Plants ; Surveys and Questionnaires ; Radiation, Ionizing ; Registries ; Smoking

BACKGROUND: The Polycomb-group gene Bmi-1 is known to be a molecular regulator of self-renewal of normal and leukemic stem cells and be involved in various aspects of cellular proliferation, differentiation, and survival. METHODS: This study evaluated the effects of overexpression of Bmi-1 on human cord blood CD34+ cells. Bmi-1 was introduced into CD34+ cells through lentivirus transduction. Bmi-1 expressing CD34+ cells were applied to colony forming assay, stromal co-culture, and cytokine-stimulatied culture. RESULTS: Ectopic expression of Bmi-1 resulted in the increased number of erythroid colonies in primary and secondary colony forming assay in an erythropoietin dependent manner. In stromal co-culture, Bmi-1-expressing postnatal hematopoietic stem cells seemed to lose the ability of self-renewal, as determined by week 5 cobblestone area-forming cell assay and by week 5 secondary colony assay. In cytokine-stimulated suspension culture of Bmi-1-transduced CD34+ cells, we observed increased erythropoiesis marked by Glycophorin A expression. CONCLUSION: Our data suggest that ectopic expression of Bmi-1 in human hematopoietic stem/progenitor cells may result in the differentiation to the erythroid lineage rather than promoting self-renewal.
Cell Proliferation ; Coculture Techniques ; Erythropoiesis ; Erythropoietin ; Fetal Blood ; Glycophorin ; Hematopoietic Stem Cells ; Humans* ; Lentivirus ; Stem Cells

Fetal nucleated red blood cells (nRBCs) are rare in maternal circulation, but their presence constitutes a potential source of non-invasive prenatal genetic diagnosis. This study was undertaken to establish a non-invasive prenatal genetic diagnosis method using isolated fetal nRBCs. A multi-step method including triple density gradient and magnetic activated cell sorting (MACS) using CD45 and CD71, cytospin centrifugation, K-B staining, and glycophorin A-immuno fluorescence in situ hybridization (GPA-immuno FISH) was performed. The study population included 65 patients from 8 to 41 weeks of gestation, and fetal nRBC was separated from all cases. The number of fetal nRBCs retrieved was 12.8 +/- 2.7 in 8 to 11 gestational weeks, 15.2 +/- 6.5 in 12 to 18 gestational weeks, 16.4 +/- 6.5 in 19 to 23 gestational weeks, 10.6 +/- 3.2 in 24 to 28 gestational weeks, and 5.5 +/- 1.9 in 35 to 41 gestational weeks: the mean number of nRBCs collected from 20 ml of maternal peripheral blood was 13.7 +/- 6.2. The highest value of yield was 45.6% from 12 to 18 weeks gestation. The fetal sex determination confirmed by amniocentesis or chorionic villus sampling showed 100% sensitivity and 91.7% specificity for males; 91.7% sensitivity and 100% specificity for females. We showed that fetal cells can be reliably enriched from maternal blood and that they can be used for detecting specific chromosomes by FISH with a specificity superior to current non-invasive methods.
Erythrocytes/immunology* ; Female ; Fetal Blood/immunology* ; Gestational Age ; Glycophorin ; Human ; Immunomagnetic Separation ; Immunophenotyping ; In Situ Hybridization, Fluorescence* ; Pregnancy ; Prenatal Diagnosis*

OBJECTIVETo detect the base sequences of all exons and part of introns in the GYPA gene of the glycophorin GPA and to investigate the polymorphism of M, N alleles in Chinese population.

METHODSA total of 225 blood sample were randomly colleeted from unrelated Chinese volunteers and were detected by serology techniques. The primers were designed by self, the seguencing of GYPA gene related with sample exon 1-7 full length sequences of bases and intron-1-7 partial sequence was performed, the polymorphism of M, N gene mutation in mucleotide sequence was analysed.

RESULTSThe results of M and N genotyping were in agreement with the results of serological detection. The 23rd base of intron-2, the 55th base of intron-3, the 63rd base of intron-4, the 55th, 189th and 190th base of intron-6, the 712th base variation of exon-7 in the gene M and N were used to subdivide the gene M and N into the mutant M103, M201, M202, N101, N102, N103, N104, and N201. At the same time, it was found that 42th and 54th base were mutated, the base T was inserted between 59th and 60th base in the intron-2, the new mutations occurred in the alleles 28, 29, 65 and 102 in intron-3 in this study.

CONCLUSIONThe polymorphism of the the Chinese population's GYPA gene occurs in all the exons and partly in the introns. The gene polymorphism of M and N blood group in Chinese population might provide the theoretical basis for the studies of clinical blood transfusion, human population genetics and molecular biology.

Alleles ; Asian Continental Ancestry Group ; Blood Group Antigens ; Blood Grouping and Crossmatching ; Exons ; Genotype ; Glycophorin ; Humans ; Introns ; Polymorphism, Genetic

UNLABELLEDObjective： To study the effect of PD98059, a specific inhibitor of Ras/Raf/MEK/ERK signaling pathway, on the proliferation and apoptisis of bone marrow CD71(+), CD235a(+) nucleated erythrocytes in patients with high altitude polycythemia (HAPC) and the pathogenesis of HAPC.

METHODSThe CD71(+) and CD235a(+) nucleated erythrocytes in HAPC patients and controls (patients with simple obsolete stracture) were sorted by using the immunemagnetic beads, then were added with 5, 10, 20 µmol/L of PD98059 and DMSO (as control) and were cultured for 72 h under hypoxia. The cell apoptosis was detected by flow cytometry with Annexin V and PI double staining, the cell proliferation was detected by CCK8 method, at same time the erythroid colong-formation ability of bone marrow mononuclear cells (BMMNC) treated with 5, 10, 20 µmol/L of PD98059 and DMSO was observed.

RESULTSWith the increase of PD98059 concentration, the apoptosis rate of bone marrow CD71(+) and CD235a(+) nucleated erythrocytes in HAPC patients was enhanced (r=0.807,P<0.01), while the proliferation rate of CD71(+) and CD235a(+) nucleated erythrocytes in HAPC patients dereased (r=0.502,P<0.01). The erythroid colong-formation ability of BMMNC in HAPC patients decreased with the increase of PD98059 concentration (r=0.504,P<0.01). There were statistic differences among different groups at 7 and 14 d.

CONCLUSIONThe MEK specific inhibitor PD98059 can inhibit the proliferation and promote the apoptosis of CD71(+) and CD235a(+) nucleated erythrocytes in HAPC patients, then inhibit the excessive accumulation of erythrocytes.

Altitude Sickness ; Apoptosis ; Bone Marrow ; Bone Marrow Cells ; Cell Proliferation ; Erythroblasts ; Erythrocyte Count ; Erythrocytes ; Flavonoids ; Flow Cytometry ; Glycophorin ; Humans

Red blood cells that agglutinate with most normal adult sera but never with own sera are termed polyagglutinable and can be separated by patterns of lectin reactivity into various types. Among these polyagglutination, activation of the T cryptantigen occurs when carbohydrate structures on glycophorins A and B lose sialic acid and express the disaccharide Gal beta-l-3 GalNac which reacts with the peanut agglutinin, a lectin from Arachis hypogaea. T activation is a temporary condition due to exposure of the membrane antigen to the action of microbial neuraminidase. In T activated red cells, the following hazards, which are theoretically possible, are spontaneous polyagglutination of red cells in vitro, in vivo and severe blood transfusion reactions. We experienced a case of T activation in 6 month old girl with bacterial meningitis caused by Streptococcus pneumoniae. The reactivity to lectins indicated the patient's red cells were T activated. We report a case of T activation in an infant with the review of literature.
Adult ; Arachis ; Blood Transfusion ; Erythrocytes ; Female ; Glycophorin ; Humans ; Infant ; Lectins ; Membranes ; Meningitis, Bacterial ; N-Acetylneuraminic Acid ; Neuraminidase ; Peanut Agglutinin ; Streptococcus pneumoniae

PURPOSE: This study was performed to induce proliferation and differentiation of erythroid progenitors from cord blood CD34 cells using extracellular matrix (ECM) and stromal cells. METHODS: Cord blood mononuclear cells were separated using Ficoll-Hypaque and CD34 cells were purified by Mini-MACS column. Cells were cultured in IMDM medium including 10% FBS and several cytokines (EPO, flt3-ligand, SCF and TPO) upto 14 days. ECM like laminin, collagen IV, fibrinogen and fibronectin were used alone or in combination. Stromal cells were derived from BM mononuclear cells for 4 weeks of culture and irradiated before use. On day 14 of stromal coculture of cord blood CD34 cells, flow cytometric analysis were done with fluorescence isothiocyanate-conjugated (FITC) antihuman glycophorin A antibody for erythroid cells. RESULTS: ECM-treated group showed 16.3~31.3 fold increase of the cell numbers on day 14 and there were no difference from cytokines-treated group. Stromal cells induced great amount of fold increase compared with ECM-treated group on day 7 (25.4 fold vs. 2.2~5.4 fold). The cell numbers increased upto 16.4 fold on day 7, 92.8 fold on day 10, and 198.4 fold on day 14 with stromal cells. Erythroid progenitors expressing glycophorin A increased from 2.78% on day 0 to 21.57% on day 7 and 43.87% on day 14. CONCLUSION: Stromal coculture of cord blood CD34 cells induced marked proliferation and differentiation of erythroid cells compared with cytokines or ECM-treated group. Efficient in vitro erythroid culture might have implications for gene therapy in RBC defects or developing blood substitutes for transfusions.
Blood Substitutes ; Cell Count ; Coculture Techniques ; Collagen ; Cytokines ; Erythroid Cells ; Extracellular Matrix* ; Fetal Blood* ; Fibrinogen ; Fibronectins ; Fluorescence ; Genetic Therapy ; Glycophorin ; Laminin ; Stromal Cells*

BACKGROUND: Arsenic trioxide (As2O3) has been identified as an effective drug for the treatment of acute promyelocytic leukemia (APL). However, the role of As2O3 during the erythroid differentiation of human leukemic cells remains unknown. In this study, we investigated the in vitro effects of As2O3 on the erythroid differentiation of the K562 cell line and also on the expression and regulation of the apoptotic modulators of this process. METHODS: The K562 cells were cultured in the presence of 0.1, 0.5 and 1.0micrometer As2O3, or they were cultured in the presence of 1.0 and 10micrometer all trans retinoic acid (ATRA). The expression of glycophorin A before and after treatment with As2O3 or with ATRA in the K562 cells was assessed by flow cytometry and western blotting. The expressions of Bcl-2 and caspase-3 were determined by western blotting. RESULTS: The viability of the K562 cells was not decreased after treating with 0.1 and 0.5micrometer of As2O3, but the viability was significantly reduced at a dose of 1.0micrometer Caspase 3 activation was not observed at 0.1 and 0.5micrometer of As2O3 until 12 days, but Caspase 3 was activated by 1.0micrometer of As2O3 from day 3. The expression of glycophorin A was increased in dose dependent manner by As2O3 treatment, but this was not changed in the ATRA treated K562 cells. The expression of Bcl-2 was increased by 0.1 and 0.5micrometer of As2O3, but it was abruptly reduced by 1.0micrometer of As2O3. CONCLUSION: These results suggest that As2O3 induces the erythroid differentiation of K562 cells and that 1.0micrometer of As2O3 induces apoptosis through the down-regulation of Bcl-2.
Apoptosis* ; Arsenic* ; Blotting, Western ; Caspase 3 ; Cell Line ; Down-Regulation* ; Flow Cytometry ; Glycophorin ; Humans* ; K562 Cells ; Leukemia* ; Leukemia, Promyelocytic, Acute ; Tretinoin

BACKGROUND: Hemopoiesis and erythropoiesis have been studied mainly in immortalized cell lines and semisolid medium. But cell lines do not represent normal erythropoiesis, besides, in semisolid medium the cells are immobilized that it is difficult to do additional immunologic, biochemical, and molecular biologic experiments. In the present study we used a two-phase liquid culture system to isolate and quantify erythroid progenitors from peripheral blood and cord blood. METHODS: Peripheral and cord blood were obtained from three healthy donors and three full-term deliveries, respectively. Mononuclear cells were separated by density gradient centrifugation and were cultured in the first phase at a density of 5x106/mL in alpha- minimal essential medium ( -MEM). After 5~7 days of incubation at 37degrees C in an atmosphere of 5% CO2 with extra humidity, the nonadherent cells were harvested and recultured in the original volume of -MEM containing 10ng/mL stem cell factor and 1U/mL erythropoietin (EPO). Cellular morphology was observed by preparing cytocentrifuge slides stained with Wright Giemsa. On days 8, 10, 12, and 16 of the second phase, hemoglobin (Hb)- containing cells were counted on hemocytometer after staining with acid benzidine and glycophorin A-positive erythroid cells were scored by a flow cytometer. RESULTS: Pronormoblasts first started to appear on days 4~5 in the secondary culture. On day 10 basophilic normoblasts could be seen and on days 12~14 orthochromatic normoblasts were present. Both from peripheral and cord blood the maximum number of benzidine and glycophorin A-positive cells were achieved after 10 days and the total erythroid cell yield was approximately 1x106/mL. CONCLUSION: Using two-phase liquid culture, erythroid cell yield reached 1x106/mL both from peripheral and cord blood. In addition, this culture system permits the study of the effect of various culture conditions and components without terminating the culture, therefore it might provide us a useful experimental tool for studying pathogenesis and therapeutic modalities in genetic erythroid disorders as well as erythroid cell development.
Atmosphere ; Basophils ; Cell Line ; Centrifugation, Density Gradient ; Erythroblasts ; Erythroid Cells ; Erythropoiesis ; Erythropoietin ; Fetal Blood ; Glycophorin ; Humans ; Humidity ; Stem Cell Factor ; Tissue Donors