Molecular Basis of ABO Variants Including Identification of 16 Novel Subgroup Alleles in Chinese Han Population.

Introduction: The characteristic of ABO blood subgroup is crucial for elucidating the mechanisms of such variant phenotypes and offering useful information in blood transfusion.

Methods: In total, 211 ABO variants including part of available family members were investigated in this study. The phenotypes of these individuals were typed with serologic methods.

[Analysis of Human Platelet Antigen-1 System Alloantibodies Using Recombinant GPIIIa Fragments Coupled to Luminex Beads].

Objective: To detect platelet anti-HPA-1a and -1b antibodies using recombinant GPIIIa fragments coupled to Luminex beads.

Methods: The sensitivity of 2 techniques, monoclonal antibody specific immobilization of platelet antigen (MAIPA) and Luminex bead assay, was compared using 12 twofold-serial dilutions (from neat to 1 in 2048) of an anti-HPA-1a WHO international standard. The specificity of Luminex assay to identify anti-HPA-1a and -1b antibodies was assessed using 8 negative or positive controls and 36 blinded samples provided by WHO Platelet Workshop.

[Establishment of method detecting CD36 expression on human platelet and its application].

The individual with the deficiency of CD36 antigen on platelet displayed the risk of anti-CD36 immune reaction induced by transfusion, which is one of the reasons for platelet transfusion refractoriness (PTR). This study was purposed to detect the expression level of CD36 antigen on platelet by flow cytometry among apheresis platelet donors of Hangzhou area, and the frequency of CD36 deficiency was analyzed. Platelet-rich plasma (PRP) was separated from fresh anticoagulant whole blood by centrifugation, then the platelets were washed and adjusted to 1×10(6).

[Study on a novel mutation of B glycosyltransferase gene related with an ABx variant].

Objective: To explore the molecular basis of an individual featuring an ABx variant of ABO blood group system.

Methods: Serological assays were used to characterize the erythrocyte phenotypes and salivary ABH secretors. All of the seven exons and flanking introns of ABO glycosyltransferase gene were amplified with polymerase chain reaction (PCR).

[Construction of expression vector from different transcripts of RHD gene].

RHD gene has different alternative transcripts. This study was aimed to construct expression vector of normal mRNA, DEL9 and DEL89 transcripts from RHD gene. Total RNA was extracted from Rh(D) positive umbilical blood cells of newborn.

[Identification of an ABx09 phenotype of ABO subtype].

Objective: To analyze the molecular basis for an individual with ABx09 phenotype of ABO subtype.

Methods: The ABO group antigens on red blood cells of the proband were identified by monoclonal antibodies, and the ABO antibody in serum was detected by standard A, B, O cells. The exons 1 to 7 of ABO gene were amplified by polymerase chain reaction (PCR) respectively and the PCR products were sequenced directly.

[Molecular mechanism of an individual with weaken B phenotype in ABO blood group].

Objective: To elucidate the serological characteristics and molecular mechanism of a blood donor with weaken B antigen.

Methods: The ABO blood group antigens on red blood cells were identified by monoclonal antibodies, the ABO antibodies in serum were detected by standard A, B, O cells and the activity of the B glycosyltransferase was analyzed. The full-length sequence and 5'-untranslated region (5'-UTR) sequence of ABO gene were amplified by polymerase chain reaction (PCR) respectively and direct sequencing.

[C742T mutation of α1, 3 N-acetyl-D-galactosaminyltransferase gene is responsible for A2 subgroup].

The objective of this study was to analyze the molecular genetic basis for 2 individuals with A2B phenotype of ABO subtype. The ABO group antigens on red blood cells were identified by monoclonal antibodies and the ABO antibodies in serum were detected by the standard A, B, O cells. The exon 5 to exon 7 coding region of ABO gene was amplified by polymerase chain reaction (PCR) and the PCR product was sequenced directly after the enzymes digested.

Moisture sorption characteristics of freeze-dried human platelets.

Freeze-drying is a promising method for a long-term storage of human platelets. The moisture sorption characteristics of freeze-dried human platelets (FDHPs) were studied in this paper. The moisture sorption isotherms of FDHPs and freeze-dried lyophilization buffer (FDLB) were measured at 4, 25, and 37 °C.

[Para-Bombay phenotype caused by combined heterozygote of two bases deletion on fut1 alleles].

This study was purposed to investigate the molecular basis of a para-Bombay phenotype for screening and identification of rare blood group. ABO and H phenotypes of the proband were identified by serological techniques. The exon 6 to exon 7 of ABO gene and full coding region of α-1,2-fucosyltransferase (fut1) gene of the proband were analyzed by polymerase chain reaction and direct sequencing of the amplified fragments.

[Cloning and sequence analysis of 5'untranslated region of human ABO gene].

Objective: To clone and investigate the polymorphism of the 5'-untranslated region (5'-UTR) of the human ABO gene, in order to provide the basis for exploring the transcriptional regulation of the human ABO histo-blood group genes.

Methods: ABO phenotypes of 30 unrelated healthy blood donors were determined by serological technique, their genotypes were analyzed by sequencing the exons 6 and 7 of the ABO gene. Nearly 5 kb of the 5'-UTR of ABO gene was obtained by PCR amplification and sequencing was performed bidirectionally.

[Study on polymorphism of membrane glycoprotein genes related to human platelet alloantigens].

Objective: To investigate the polymorphisms of platelet membrane glycoprotein genes related to human platelet alloantigen (HPA)-1 to 17w.

Methods: The DNA segments of platelet membrane glycoprotein genes related to HPA-1 to 17w were amplified using author's designed primers. The amplification products were purified and directly sequenced to identify the HPA genotype and glycoprotein gene polymorphisms.

[In vitro expression activity of α-(1,2) fucosyltransferase with 35C > T and 682A > G mutations].

In order to explore the effects of 35C > T and 682A > G mutations on the activity of alpha-(1,2) fucosyltransferase, the coding region of fut1 gene was amplified by polymerase chain reaction (PCR) from genomic DNA. PCR product was ligated into expression vector using TOPO TA cloning kit to obtain the recombinant plasmids. The recombinant plasmids were transfected into COS-7 cells by liposome method.

[Quantitative chimerism analysis of regulatory T cell subsets based on immunomagnetic sorting].

The aim of study was to explore the feasibility of quantitative chimerism analysis of regulatory T (Treg) cells using immune sorting coupling short tandem repeat (STR) method. 14 sets of artificial chimera samples were prepared by mixed lymphocytes according to different proportion. The CD4(+)CD25(+) Treg cells were harvested by negative and positive selection of immunomagnetic beads, then the STR polymorphisms of 16 loci in sorted Treg cells was analyzed.

[Molecular basis of a new O61 allele in ABO blood group].

Objective of this study was to explore the molecular basis of a new O61 allele in ABO blood group. The ABO group antigens on red cells of the blood samples were identified by monoclonal antibodies and the ABO antibody in serum was detected by the standard A, B, O red cells. The coding region sequences of exon 5 to exon 7 in ABO gene were amplified by polymerase chain reaction (PCR) and the amplification products were purified with double enzyme digestion and directly sequenced for exon 6 and 7.

[Molecular basis for real RhD negative and RhDel phenotypes in Yiwu population of Zhejiang Province in China].

This study was purposed to investigate the molecular basis for RhD negative phenotype in Yiwu population in Zhejiang Province of China. The RhD negative samples were screened by saline agglutination test in blood donors. Some real RhD negative and RhDel phenotypes were identified using anti-human globulin test and absorbtion elution test.

[Molecular genetic analysis of a new B112 allele of ABO blood group].

Objective: To analyze the molecular genetic basis for a new B112 allele of ABO blood group and the pedigree of the proband.

Methods: The ABO group antigens on red cells of the proband were identified by monoclonal antibodies. The ABO antibody in serum was detected by using standard A, B and O cells.

[Analysis of alpha-1,2-fucosyltransferase gene mutations in a Chinese family with para-Bombay phenotype].

Objective: To investigate the molecular genetic basis of para-Bombay phenotype in a Chinese family.

Methods: ABO and H phenotypes of the proband and his pedigree were characterized by serological techniques. The exons 6 and 7 of the ABO gene and full coding region of alpha-1,2-fucosyltransferase (FUT1) gene of the pedigree were analyzed by polymerase chain reaction and direct sequencing of the amplified fragments.

An experimental study of the use of ultrasound to facilitate the loading of trehalose into platelets.

Trehalose is widely used as a freeze-drying protectant in biomaterial preservation. For this purpose, trehalose has to be loaded into the cells but this is difficult and many methods have been tried. The application of ultrasound can temporarily permeabilize cell membranes, which offers a non-chemical, non-viral, and non-invasive method of cellular drug delivery.

[A novel M142T mutation in the B glycosyltransferase gene associated with B3 variant in Chinese].

Objective: To investigate the molecular genetic basis of the B3 variant of ABO blood group system with mixed-field hemagglutination in Chinese.

Methods: Serological techniques were performed to characterize the erythrocyte phenotype of two discrepant samples. A sequential agglutination method and 13 short tandem repeat (STR) loci were tested to exclude the possibility of exogenous or endogenous DNA chimera.

[Molecular genetic analysis of rare cisAB variants in Chinese population.].

We investigated the molecular genetic basis of rare cisAB variants at the ABO locus in Chinese population. Serological techniques were performed to characterize erythrocyte phenotype of 12 discrepant samples and 116 randomly selected samples. Mutations of complete exon 6 and 7 including flanking intron in the ABO genes were screened by PCR and directly sequencing.

[Effect of prefreezing parameters on human platelet cryopreservation].

This study was aimed to investigate the effect of prefreezing parameters such as freezing rate, annealing, rate, annealing temperature, holding time in annealing before lyophilization on lyophilized platelets by orthogonal tests. The recovery rate, the morphological and ultrastructural changes, the activation and aggregation of lyophilized platelet after rehydratation were detected by cytometer, scan electron microscopy, flow cytometry and aggregation reaction on thrombin, respectively, and complex evaluation was carried out. The results showed that the recovery rate of lyophilized platelets was 91% - 53.

[Identification of the hemolytic transfusion reaction caused by lewis antibody using serological and molecular biological methods].

To analyse the reason for one case of hemolytic transfusion reaction, antibodies in a patient's serum were identified using panel cells and Le (a-b-) phenotype cells, patient phenotype was identified by using anti-Le(a) and anti-Le(b) blood grouping reagents and the entire coding region of FUT3 gene was amplified by PCR and sequenced directly. The results showed that both IgM anti-Le(a) and anti-Le(b) antibodies were detected in patient's serum. Red cells was typed as Le (a-b-) phenotype and the FUT3 genotype was homozygote for non-functional le(59, 508) alleles.

[Analysis of natural killer cell immunoglobulin-like receptor gene family in HLA-identical sibling].

The aim of study was to analyze natural killer immunoglobulin (Ig)-like receptor (KIR) gene content in HLA-identical sibling and to investigate the possibility of their KIR match. Samples were genotyped for HLA by Luminex method and polymerase chain reaction sequence based typing, the KIR gene was detected by polymerase chain reaction sequence-specific primers. The results showed that 17 KIR genes could be observed in the 27 pairs HLA-A, -B, -Cw and -DRB1 locus identical sibling samples.