Background: The reduction of erythrocytes from cord blood is very need for long - term storage of C034 cells for transplantation. Reduced erythrocyte will reduces preservative blood volume, preservatives and freely HST when defrosting, so stem cells are better protected. Objectives: To study selection of the best centrifugal procedure to reduce maximal erythrocytes and lose minimal C034 cells from cord blood. Subjects and methods: 20 blood samples selected from 60 cord blood units was used for this study. The study was carried out through two steps. In the first step, the centrifugal speed was fixed and the centrifugal time was changed.In the second step, the centrifugal time was fixed, the centrifugal speed was changed. From collected results the best appropriate procedure to reduce erythrocytes from cord blood have been selected. Results: The procedure of gradient centrifuge with speed of 500g in 6 minutes isolated> 50% of erythrocytes, kept > 84% of CD34 cells and then centrifuge of 1000 g in 10 minutes reduced about 40% of volume of nuclear cell - suspension. Conclusion: The procedure can use for preparation of stem cell suspension from cord blood to storage in nitrogen liquid. \r\n', u'\r\n', u'
Erythrocytes/ pathology ; Fetal Blood/ chemistry ; drug effects ; immunology

This study was purposed to evaluate the effect of trehalose-loading on physiological and biochemistry properties of red blood cell (RBC) membrane. The samples were divided into the control group (RBC without trehalose loading) and the test group (RBC with trehalose loading). Osmotic fragility reaction was used to determine the osmotic fragility change of loaded RBC membrane in NaCl solution of different osmotic concentration. Flow cytometry and deformeter were used to assay the integrality and deformability of the RBC, respectively. The results showed that the NaCl solution osmotic concentrations were 160 mOsm and 121.4 mOsm, respectively when the haemolysis rate was 50% of the control group and the test group. Flow cytometry data demonstrated that incubation of RBC in a hypertonic trehalose solution resulted in a fraction of cells with different complexity that attached to little Annexin V-FITC, and that it could be removed by washing and resuspending the RBC in an iso-osmotic (300 mOsm PBS) medium. The deformability of the loaded RBC descend, the statistical difference was significant between control and test groups (P < 0.01). It is concluded that the membrane physiological and biochemistry stability and membrane integrality of RBC in a hyper osmotic pressure can be retained after trehalose loading.
Blood Preservation ; methods ; Cryopreservation ; methods ; Erythrocyte Membrane ; drug effects ; Erythrocytes ; drug effects ; Humans ; Osmotic Fragility ; drug effects ; Trehalose ; pharmacology

OBJECTIVETo study comparatively the cytotoxicity induced by acid bentonite and organic bentonite.

METHODSThe cytotoxicity of two kinds of bentonite was detected using CCK8 assay, neutral red uptake (NRU) assay, lactate dehydrogenase (LDH) leakage assay, apoptosis assay and hemolysis assay. In hemolysis assay human erythrocytes served as target cells and were exposed to the two kinds of bentonite at the doses of 0, 0.3125, 0.6250, 1.2500 and 2.5000 mg/ml for ten min. In other four assays, human B lymphoblast cells (HMy2.CIR) served as target cells and were exposed to the two kinds of bentonite at the doses of 0, 10, 20, 30, 60, 120 and 180 microg/ml for four h.

RESULTSIn hemolysis assay, the hemolysis rates induced by two kinds of bentonite at all doses were significantly higher than that of control (P<0.05); in CCK-8 assay, the cellular activities in acid bentonite group at the doses > or =30 microg/ml and in organic bentonite group at the doses > or =20 microg/ml were significantly lower than that of control (P<0.01); the similar results appeared in NRU assay and LDH assay, and the dose-effect relationship was observed in above 4 assays. In apoptosis assay, the early apoptosis cell rates in acid bentonite group at the dose of 180 microg/ml and in organic bentonite group at the doses of 120,180 microg/ml were significantly higher than that of control (P<0.05). Moreover, the results of five in vitro assays indicated the cytotoxicity induced by organic bentonite was higher than that induced by acid bentonite.

CONCLUSIONTwo kinds of bentonite could induce cytotoxicity, such as apoptosis and damage of cell membrane. The cytotoxicity of organic bentonite is higher than that of acid bentonite due to the different industrial treatment and characteristics of two kinds of bentonite particles.

Apoptosis ; drug effects ; Bentonite ; toxicity ; Cell Line ; Cytotoxicity Tests, Immunologic ; Erythrocytes ; drug effects ; pathology ; Hemolysis ; drug effects ; Humans ; Lymphocytes ; drug effects ; pathology

The effects of amphotericin B, an antifungal antibiotic, on erythrocyte volume and cation permeability were investigated by measuring the hematocrit, cell volume, cation content, fragility and osmotic behavior in rat erythrocytes, in vitro. 1. When erythrocytes were incubated in a Ringer solution containing amphotericin B (5-25 microgram/ml) the hematocrit and the cell volume increased, the effect being proportional to the concentration of the drug and the incubation time period. 2. Amphotericin B increased the Na content and decreased the K content of the erythrocyte. In normal Ringer solution (NaCl-Ringer)containing amphotericin B the magnitude of cellular Na gain was greater than that of K loss. Therefore, the total cellular cation content increased. On the other hand, when cells were incubated in the amphotericin B containing Ringer solution in which NaCl was replaced by Na2SO4 (Na2SO4-Ringer) the magnitude of cellular K loss exceeded that of cellular Na gain. Consequently, the total cellular cation content was reduced. 3. Amphotericin B increased cell volume (hematocrit) when erythrocytes were incubated in the Na2SO4-Ringer solution. 4. The fragility of erythrocytes increased when cells were preincubated in the amphotericin B containing normal Ringer solution, whereas it decreased in tile cells preincubated in the amphotericin B containing Na2SO4-Ringer solution. 5. The cell volume was linearly related to the reciprocal of medium osmolality(200 to 900 mOsm/kg H2O) in both NaCl-and Na2SO4-Ringer solutions, and the linearity was not altered by amphotericin B. The antibiotic did not change the slope of the correlation line (V vs. 1/OSM). It, however, increased the intercept of the line with the ordinate in normal Ringer solution and decreased that in the Na2SO4-Ringer solution. These results indicate that amphctericin B alters the cell volume by changing the permeability of Na and K across the membrane.
Amphotericin B/pharmacology* ; Animal ; Erythrocyte Volume/drug effects* ; Erythrocytes/analysis* ; In Vitro ; Osmotic Fragility/drug effects ; Potassium/blood* ; Rats ; Sodium/blood*

OBJECTIVETo observe the effect of Coptidis Rhizoma (CR) on hemolysis and antioxidant system of normal mice and its impact on the functions, while evaluating the oxidation reduction property of CR and berberine.

METHODIn the whole animal experiment, normal mice were orally administered with CR at the dose of 1.2 g x kg(-1) for three days. Their blood were collected to detect the hemoglobin in plasma, the content of serum bilirubin, the number of peripheral blood reticulocytes, the T-AOC in whole blood, measure the contents of glucose-6-phosphate-dehydrogenase (G6PD), superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde (MDA) of RBC membrane, determine the activity of Na(+)-K(+)-ATPase, Ca(2+)-Mg(2+)-ATPase, fluidity, and observe its impact on the liquidity and deformability of RBCs. According to the electrical and biochemical experiment, the voltammetric behaviors of CR and berberine on glassy carbon electrode were evaluated using cyclic voltammetry. In the RBC in vitro experiment, the impact of Coptidis Rhizoma on autoxidation hemolysis rate of RBCs of normal mice was observed.

RESULTThere was no significant effect on hemoglobin, serum bilirubin, and reticulocyte count in normal mice administrated with CR at the dose of 1.2 g x kg(-1), and so is on RBC membrane SOD, G6PD, MDA, GSH and whole blood T-AOC activity. In addition, CR had also no significant effect on Na(+)-K(+)-ATPase, Ca(2+)-Mg(2+)-ATPase activity, and no notable impact on the fluidity and deformability of RBCs. There were two oxidation peaks at -0.27 V and 0.60 V induced by CR and one oxidation peak induced by berberine at 0.56 V, with no reduction peak at fly-back. CR could significantly inhibit oxidative hemolysis in RBCs at the dose of 0.125-2 g x L(-1) in vitro.

CONCLUSIONThe normal dose of Coptidis Rhizoma can not cause hemolysis of RBC, and also can not change antioxidant system and functions of RBC, CR and berberine show antioxidant (reducing) properties.

Animals ; Antioxidants ; pharmacology ; Berberine ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Erythrocytes ; drug effects ; metabolism ; Female ; Hemolysis ; drug effects ; Male ; Mice

To study the hemolytic effect of polyphyllin II (PP II) mediated by anion channel protein and glucose transporter 1 (GLUT1), in order to initially reveal its hemolytic mechanism in vitro. In the experiment, the spectrophotometric method was adopted to detect the hemolysis of PP II in vitro and the effect of anion channel-related solution and blocker, glucose channel-related inhibitor and multi-target drugs dehydroepiandrosterone (DHEA) and diazepam on the hemolysis of PP II. The scanning electron microscope and transmission electron microscope were used to observe the effect of PP II on erythrocyte (RBC) morphology. The results showed that PP II -processed blood cells were severely deformed into spherocytes, acanthocyturia and vesicae. According to the results of the PP II hemolysis experiment in vitro, the anion hypertonic solution LiCl, NaHCO3, Na2SO4 and PBS significantly inhibited the hemolysis induced by PP II (P < 0.05), while blockers NPPB and DIDS remarkably promoted it (P < 0.01). Hyperosmotic sodium chloride, fructose and glucose at specific concentrations notably antagonized the hemolysis induced by PP II (P < 0.05). The glucose channel inhibitor Cytochalasin B and verapamil remarkably antagonized the hemolysis induced by PP II (P < 0.01). The hemolysis induced by PP II could also be antagonized by 1 gmol x L(1) diazepam and 100 μmol x L(-1) DHEA pretreated for 1 min (P < 0.01). In conclusion, the hemolytic mechanism of PP II in vitro may be related to the increase in intracellular osmotic pressure and rupture of erythrocytes by changing the anion channel transport activity, with GLUT1 as the major competitive interaction site.
Animals ; Diosgenin ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Erythrocytes ; cytology ; drug effects ; Hemolysis ; drug effects ; Hemolytic Agents ; pharmacology ; Sheep

BACKGROUNDCisplatin based chemotherapy is a well recognized risk factor for coagulation disorders and thrombosis. The pathophysiological mechanisms by which cisplatin promote thrombosis are not well understood.

METHODSRed blood cells (RBCs) were separated from peripheral blood of patients with breast cancer (n = 10) and healthy adults (n = 6) and treated with cisplatin. Coagulation time of RBCs was assessed by one step recalcification time and the productions of thrombin, intrinsic and extrinsic factor Xa were measured in the presence or absence of various concentrations of lactadherin. Exposed phosphatidylserine was stained with lactadherin and observed by confocal microscopy and flow cytometry.

RESULTSNeither fresh RBCs nor RBCs treated without cisplatin had potent procoagulant activity. Cisplatin treatment increased procoagulant activity of RBCs in a cell number- and concentration-dependent manner. Exposed phosphatidylserine was stained with lactadherin and after cisplatin treatment, strong fluorescence was revealed by confocal microscopy. Lactadherin bound RBCs from patients with breast cancer increased from (1.9 +/- 0.5)% on control RBCs to (68.0 +/- 3.5)% on RBCs treated with 10 micromol/L cisplatin for 24 hours.

CONCLUSIONSCisplatin treatment increases procoagulant activity of RBCs, which have a strong association with exposure of phosphatidylserine. The increased procoagulant activity may contribute to the pathogenesis of thrombophilia during cisplatin based chemotherapy in breast cancer patients.

Antineoplastic Agents ; pharmacology ; Blood Coagulation ; drug effects ; physiology ; Cisplatin ; pharmacology ; Erythrocytes ; drug effects ; physiology ; Humans ; In Vitro Techniques

OBJECTIVETo summarize the results of general survey, primary structure analysis and related functional studies of abnormal hemoglobins (Hbs) found in Hunan Province.

DATA SOURCESInternational Hb journals, Chinese biochemical and biomedical journals and other articles relevant to hematology.

STUDY SELECTIONAll Hb variants found in Hunan and identified by primary structure analysis during 1980 - 1991 were included.

DATA EXTRACTIONData concerning 11 types of Hb variants found in 3 districts and 7 counties in Hunan Province were briefly documented. Their frequencies of occurrence were calculated and their distributions among Han, Yao, Tujia and Dong ethnic groups were listed.

RESULTSThirty-six cases with abnormal Hb were identified out of 7412 individuals screened in Hunan. 11 different types of Hb variants were recognized by primary structure analysis in 19 propositi along with their family members, including 5 alpha-chain variants, 4 beta-chain variants, 1 delta-chain variant and 1 delta-beta chain fusion variant. Oxygen equilibrium characteristics, reaction dynamics, the rate of globin chain synthesis (RGCS), morphology observation by electron microscopy and DNA analysis were all used in the functional studies of hemoglobinopathies.

CONCLUSIONSThe average incidence of abnormal Hbs in Hunan is 0.486%. In Jianghua County, whose inhabitants are mostly of the Yao ethnic group, the incidence is significantly higher (1.09%). Hb Jianghua [beta120(GH3) Lys-->lle] and Hb Shuangfeng (SF) [alpha27(B8) Glu-->Lys] were two new variants first reported in international literature; whereas Hb Lille [alpha74(EF3) Asp-->Ala], HbA(2) Flatbush [delta22(B4) Ala-->Glu] and Hb Lepore-Boston [delta87(F3)-beta116(G18)] were the first three instances to be found in China. Hb SF displayed an oxygen affinity 1.5-fold higher than that of HbA at pH 7.4 and 25 degrees C with its oxygen equilibrium curve shifted to the left. Reticulocytes of Hb SF heterozygote showed unbalanced RGCS, quite similar to that found in beta-thalassemia minor. Erythrocytes of Hb SF heterozygote were changed to spherocytes and began to lyse after incubation with sodium salicylate or sulfadiazine (pH 7.4, 37 degrees C) for 2 - 4 h. These findings explained the sudden attack of hemolytic anemia provoked by two drugs in Hb SF propositus. The genotype of a patient with Hb Q-H disease is identified as -,-/-,alpha(Q) by DNA restriction mapping.

Erythrocytes ; drug effects ; Globins ; biosynthesis ; Hemoglobins, Abnormal ; chemistry ; genetics ; physiology ; Humans ; Mutation

Rh is a very important blood group like ABO blood system in transfusion medicine. It causes severe transfusion reaction and hemolytic disease of the newborn (HDN) if RhD blood group does not match between the donor and the recipient. The population of RhD negative is only about 0.2% - 0.5% in Chinese. Conversion of RhD positive RBCs to RhD negative is very important in clinical transfusion. This study was to try to modify RhD antigen located on the surface of A, B, O and AB red blood cells in order to convert RhD positive to RhD negative by the modification of four kinds of methoxypolyethylene glycol (mPEG) derivatives and to observe the effect of mPEG modification on cell morphology, structure and function. The result demonstrated that modification efficiency of mPEG-BTC (mPEG-benzotriazole carbonate) was better than other three kinds of mPEG derivatives. It could camouflage RhD antigen efficiently when the concentration reached to 1 mmol/L. The result also showed that there were no harmful effects of mPEG modification on cell morphology, osmotic fragility, hemolysis, AchE, cholesterol, ATP, 2,3-DPG and deformability. It is suggested that success in converting RhD positive RBCs to RhD negative was preliminarily achieved.
Erythrocytes ; drug effects ; immunology ; physiology ; Humans ; Polyethylene Glycols ; pharmacology ; Rh-Hr Blood-Group System ; immunology

In order to optimize the preservation of blood, 3 kinds of antioxidant were selected and each of them can be injected directly into vein, then the optimal dose of these antioxidants was chosen using statistical method; ISMC (injectio salvia miltiorrhizae composita), ginaton and the combination of ISMC and ginaton were added into blood as optimal dose, some references as ATP, EI and so on were observed during blood preservation. The results showed that all of the three kinds of antioxidants increased ATP, EI and decreased FHb during blood preservation. It is concluded that both of ISMC and ginaton can effectively optimize the preservation of blood and combination of ISMC and ginaton can produce additive effect.
Adenosine Triphosphate ; metabolism ; Antioxidants ; pharmacology ; Blood Preservation ; Erythrocytes ; drug effects ; physiology ; Humans ; Salvia miltiorrhiza