Advances in the field of xenotransplantation raise the intriguing possibility of using porcine red blood cells (pRBCs) as an alternative source for blood transfusion. Serologically, pRBCs share a number of characteristics with human red blood cells (RBCs), so pRBCs are considered the most likely donor for xenotransfusion. However, xenoantigens on porcine erythrocytes play major roles in antibody-mediated RBC destruction. Although the alphaGal epitope (Galalpha1, 3Galbeta1, 4GalNAc-R) is the major xenoantigen on porcine erythrocytes and is responsible for the binding of the majority of human natural antibodies, other non-alphaGal xenoantigens have been identified. The importance of these non-alphaGal xenoantigens in binding human natural antibodies and subsequently triggering immunological responses cannot be underestimated.
Animals ; Blood Group Antigens ; immunology ; Erythrocyte Transfusion ; methods ; Erythrocytes ; cytology ; immunology ; Humans ; Swine ; Transplantation, Heterologous ; immunology

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

α-Gal, the main xenotransplantation antigen, can lead to hyperacute rejection (HAR) in xenotransplantation. This study was purposed to investigate the effect of recombinant α-galactosidase (α-Gal antigen) on the Holstein-Friesian(H-F) red blood cells (RBC). The enzymelysis method was used to digest the α-Gal antigen on H-F RBC; the saline and anti-human globulin methods were used to perform the agglutination test of H-F RBC and human plasma; the flow cytometry was used to detect the α-Gal antigen on surface of H-F RBC, fluorescence intensity of FITC-IB4 and FITC-IgG labeled RBC. The results indicated that the saline and anti-human globulin method showed α-galactosidase-treated H-F RBC fail to agglutinate with human pooled plasma; the flow cytometry showed the fluorescence intensity of FITC-IB4 and FITC-IgG labeled RBC decrease 99.0% and 87.8%, respectively. It is concluded that the novel α-galactosidase can be used to cleared the α-Gal antigen on the surface of H-F RBC and α-galactosidase-treated H-F RBC may be considered as human blood substitute.
Animals ; Blood Substitutes ; Cattle ; Erythrocytes ; immunology ; Female ; Humans ; Transplantation, Heterologous

We evaluated the clinical usefulness of simultaneous LISS/Coombs and NaCl/Enzyme testing using the gel method for screening and identification of unexpected antibodies in 15,014 samples. When unexpected antibodies were detected by either screening test, those antibodies were identified using both the LISS/Coombs and the NaCl/Enzyme gel test. The positive screening rates of the LISS/Coombs, NaCl/Enzyme, and combined tests (excluding 25 autoantibody cases) were 0.48%, 1.29%, and 1.39%, respectively. Among the 57 samples positive by both screening methods, the antibodies in 19.3% could be identified only by the NaCl/Enzyme method. Among the 137 samples positive only by NaCl/Enzyme screening, 74.5% showed positive results in antibody identification only by the NaCl/Enzyme test, although 7.3% were also positive in the LISS/Coombs test. The NaCl/Enzyme method thus showed about threefold higher detection rates than the LISS/Coombs method, especially in screening for Rh antibodies, and higher exact identification rates and discriminatory power for identifying mixed antibodies. Addition of the NaCl/Enzyme method to routine laboratory procedures may detect and identify considerable numbers of significant antibodies that might be missed if only the LISS/Coombs method is used.
Antibodies/*analysis/immunology ; *Coombs' Test ; Erythrocytes/*immunology ; Hemagglutination Tests/*methods ; Humans ; Isoantibodies/analysis ; Reagent Kits, Diagnostic

This study was purposed to establish the method of quantifying RhD antigen on red blood cells (RBC) by flow cytometry (FCM) and to explore the expression of D antigen on RBC of different RhD serotype. RhD(+) RBCs and RhD(-) RBCs were mixed in 1:1 ratio. Cells were stained by the indirect method (IgG anti-D as the first antibody, FITC-anti-IgG F(ab')2 as the second antibody), and the ratio of RhD(+) on RBCs was quantified by FCM. The optimal dosage of IgG anti-D was defined. Expression of RhD antigen on RBC of RhD(+), weak D, RhDel and RhD(-) type were detected by FCM. The results showed that optimal dilution of IgG anti-D monoclonal antibody was 1:4, 1x10(6) cells/50 microl. The percentage of D(+) RBC of RhD(+), weak D, RhDel and RhD(-) type were 96.8+/-2.97%, 79.5+/-9.88%, 47.8+/-11.43%, 3.7+/-2.96%, respectively. The mean fluorescence intensity (MFI) of RhD antigen expression of RhD(+), weak D, RhDel and RhD(-) type were 33.3+/-6.21 Dal, 18.6+/-5.39 Dal, 7.10+/-1.17 Dal, 0.79+/-0.55 Dal, respectively. In conclusion, there are significant differences of RhD antigen expressions among RBC of different RhD serotypes. The level of antigen on RhD(+) RBC is the highest and then weak D the next, while the level of antigen on RhDel RBC is the lowest level.
Blood Donors ; Erythrocytes ; immunology ; metabolism ; Flow Cytometry ; methods ; Humans ; Rh-Hr Blood-Group System ; immunology ; metabolism

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

The objective of this study was to investigate the serologic characters of enzyme-only red cell antibody and its clinical significance, and to provide basis for the safety of blood transfusion. The patient serum containing enzyme-only antibody was used to react with the red cells of donors, panel cells and auto-cells in various medium. Absorption and elution test were also per formed. The results showed that this blood sample was found to contain an antibody that reacted with donor red cells and panel cells only in papain medium, but was not demonstrable by indirect antiglobulin test and other method s. Decline of antibody titers was observed after absorption test, but antibody activity was not detected in the elute. The patient underwent transfusion with 600 ml of Rh type identical RBCs, without any hemolytic transfusion reaction. In conclusion, enzyme-only antibody usually doe s not lead to hemolytic transfusion reaction.
Aged ; Aged, 80 and over ; Erythrocytes ; immunology ; Hemolysis ; Humans ; Isoantibodies ; immunology ; Male ; Papain ; pharmacology ; Transfusion Reaction

OBJECTIVETo explore the immune response of silicotic rats to sheep red blood cells(SRBC).

METHODSSilicotic rats were immunized with SRBC by tracheal instillation(Group 1) or intraperitoneal injection (Group 2), and non-silicotic rats were immunized by tracheal instillation as normal control(Group 3). The levels of serum hemolytic index(HC50) were measured on 7, 12, 20, 25, and 32 days after primary immunization and 5, 12, 15 days after the second immunization. Special anti-SRBC IgG was measured with ELISA(A490 nm) on 12, 20, 25, 32 days and 5, 12, 15, 27 days respectively. Delayed-type hypersensitivity(DTH) to SRBC was measured 20 days after second immunization and DTH reaction was determined at 24, 48, 72, and 96 h after administration. Total cell count and cell populations in the bronchoalveolar lavage fluid(BALF), lung associated lymph node(LALN) and spleen weight, special IgG secreted from spleen cells were measured at the end of the experiment.

RESULTSThe HC50 of Group 1(47.4 +/- 1.0, 52.2 +/- 4.6, 31.1 +/- 11.9, 43.8 +/- 3.5, 33.6 +/- 16.8, 49.0 +/- 2.3, 92.9 +/- 20.2, 87.7 +/- 5.2) were statistically higher than those of Group 3(40.4 +/- 10.6, 2.8 +/- 2.5, 0.8 +/- 0.6, 6.6 +/- 5.8, 1.4 +/- 0.1, 36.5 +/- 16.5, 53.0 +/- 33.2, 2.6 +/- 2.2). The special anti-SRBC IgG response in Group 1(1.67 +/- 0.19, 1.98 +/- 0.36, 1.12 +/- 0.50, 1.38 +/- 0.30, 2.75 +/- 0.15, 2.60 +/- 0.28, 2.86 +/- 0.10, 2.50 +/- 0.20) were much stronger than those in Group 3 (0.59 +/- 0.30, 0.56 +/- 0.21, 0.21 +/- 0.16, 0.22 +/- 0.01, 0.81 +/- 0.25, 0.74 +/- 0.25, 0.69 +/- 0.26, 1.38 +/- 0.41). Furthermore, the results of DTH showed positive response and the ratios for diameter of skin rash > 5 mm at 24, 48, 72, 96 h were 16/16, 16/16, 16/16, 15/16 respectively in Group 1, while those in Group 3 were 8/15, 1/15, 1/15, 1/15 respectively. Total cell count in the BALF, LALN and spleen weight, and special IgG secreted from spleen cells in Group 1 were higher too. Group 2 expressed almost of the same but with mild immunologic responses as Group 1.

CONCLUSIONSilicosis-induced extremely strong DTH and over-response of humoral immunity to some antigens may contribute to the likelihood of silicosis complicated with tuberculosis.

Animals ; Erythrocytes ; immunology ; Hypersensitivity, Delayed ; etiology ; Immunization ; Immunoglobulin G ; blood ; Rats ; Sheep ; Silicosis ; immunology

The present study evaluated the effects of infected culture supernatant of erythrocytes, fractionation of culture supernatant and serum from dogs infected with Babesia gibsoni (B. gibsoni) on the maturation of canine reticulocytes in vitro. The SDS-PAGE demonstrated that significantly broader bands were generated by both the infected culture supernatant of erythrocytes and the serum from dogs chronically infected with B. gibsoni. The culture supernatant of erythrocytes infected with B. gibsoni strongly suppressed the maturation of reticulocytes. Prior studies showed that chronically infected serum had inhibitory effects on both the maturation of reticulocytes and the canine pyrimidine 5'-nucleotidase subclass I and purine-specific 5'-nucleotidase activity. In addition, serum free infected culture supernatant of erythrocytes had an inhibitory effect on the morphological maturation of reticulocytes. These results suggest that infected serum and culture supernatant of erythrocytes might accumulate excess proteins and/or metabolites as a result of the inhibited maturation of reticulocytes and decreased activity of erythrocyte 5'-nucleotidase. Furthermore, the fractions observed at >150 kDa- and 150-70 kDa- in the infected culture supernatant and serum retarded the maturation of canine reticulocytes in vitro. The results obtained from the in vitro examinations, in the present study, suggested that B. gibsoni itself and/or its metabolites might release certain proteins in the infected culture supernatant and serum from infected dogs and as a result delay morphological maturation of canine reticulocytes.
Animals ; Babesia/*immunology ; Babesiosis/blood/immunology/parasitology/*veterinary ; Cell Differentiation/immunology ; Dog Diseases/*blood/immunology/*parasitology ; Dogs ; Electrophoresis, Polyacrylamide Gel ; Erythrocytes/*immunology ; Reticulocytes/*immunology

This study was aimed to explore impact of removal of cell membrane G alalpha1-3Gal beta1-4Glc NAc epitopes (called alpha-Gal) and chemical modification of other xenoantigen on bovine red blood cell (bRBC) and porcine red blood cell (pRBC) antigenicity and to compare their modified erythrocytes, in order to provide basis for development of human blood substitute with rich source, high safety and efficacy. bRBC and pRBC were subjected to both enzymatic removal of membrane alpha-Gal with recombinant coffee bean alpha-galactosidase (rC alpha-GalE) and covalent attachment of benzotriazole carbonate-linked methoxypolyethylene glycol (mPEG-BTC, MW = 20 kD). The effects of treatment were measured by hemagglutination, flow cytometric assay of IgG binding and clinical cross-match testing to human sera. The results showed that although alpha-galactosidase treatment reduced hemagglutination titers to levels similar to negative control, the combination of the treatments was most effective. Clinically used cross-match tests between bRBC, pRBC and human sera demonstrated increased compatibility. Bovine RBC were more robust than pRBC, and had less xenoantigens, and had longer half life than pRBC in vivo. These characteristics suggested that bRBCs were more suitable to investigation as an alternatives to hRBC in clinical transfusion than pRBC. These data suggested that strategies to remove or mask xenoantigens on bRBC reduce antigenicity sufficiently to allow in vitro cross-match compatibility to human sera, and therefore bRBC following modification may be considered as human blood substitute.
Animals ; Antigens, Heterophile ; immunology ; Blood Substitutes ; Cattle ; Disaccharides ; immunology ; Epitopes ; immunology ; Erythrocyte Membrane ; immunology ; Erythrocyte Transfusion ; methods ; Erythrocytes ; immunology ; metabolism ; Humans ; Swine ; alpha-Galactosidase ; immunology