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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. The full coding regions of ABO gene and the erythroid cell-specific regulatory elements in intron 1 of them were amplified with polymerase chain reaction and then directly sequenced. The novel alleles were confirmed by cloning and sequencing. Phylogenetic tree was made using CLUSTAL W software. 3D structural analyses of the glycosyltransferases (GTs) with some typical mutations were performed by PyMOL software. RESULTS: Forty-eight distinctly rare ABO alleles were identified in 211 Chinese variant individuals, including 16 novel ABO alleles. All of the alleles were categorized as 5 groups: 16 ABO*A alleles, 23 ABO*B alleles, 4 ABO*BA alleles, 4 ABO*cisAB alleles, and 1 ABO*O alleles. ABO*A2.08 and ABO*BA.02 were the relatively predominant A and B subgroup alleles, respectively. According to the phylogenetic tree, 28 alleles (5 common alleles and 23 alleles identified in our laboratory) were classified into 3 major allelic lineages. The structural analysis of 3D homology modeling predicted reduced protein stability of the mutant GTs and may explain the reduced ABO antigen expression. CONCLUSIONS: The molecular basis of ABO variants was analyzed, and 16 novel ABO alleles were identified. The results extended the information of ABO variants and provided a basis for better transfusion strategies and helped to improve blood transfusion safety.

The uneven bars are among one of the advantage projects of Chinese women gymnastics team, and Jaeger Salto stretched to hang on HB belongs to difficult E group action. We made kinematic analysis of Jaeger Salto stretched to hang on HB of the elite uneven bars athlete Huidan Huang on the 2013 National Games of the women's gymnastics qualifying, The conclusive kinematic characteristics of this high skilled element can provide useful information for the technical training of coaches and athletes, and enrich gymnastic theories.

To detect platelet anti-HPA-1a and -1b antibodies using recombinant GPIIIa fragments coupled to Luminex beads.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.The sensitivity of MAIPA and Luminex bead assay to detect anti-HPA-1a was dilution 1/64 (i.e. 1.56 IU/ml) and far more than dilution 1/2048 (i.e. 0.049 IU/mL), respectively. The Luminex bead assay could specifically identify negative and positive controls of anti-HPA-1a and -1b. All results of 36 blinded samples by Luminex assay were accordant to reference results except one sample which contained high concentration antithetical antibody and resulted in false positive of anti-HPA-1b. Cross-reactivity was also not observed with the samples containing HLA, ABO or other platelet antibodies.The Luminex beads coupled with recombinant GPIIIa fragments can be used to detect HPA-1 system antibodies with sufficient sensitivity and specificity, that is suitable for the detection of platelet alloantibodies in clinical alloimmune thrombocytopenia.

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). The platelets were incubated with FITC-labeled CD36 and PE-labeled CD41 monoclonal antibodies, then the expression level of CD36 was detected by flow cytometry. The CD36 expression on monocytes for the samples of CD36-deficiency on the platelets was further analyzed. The results showed that 7 samples with CD36 antigen deficiency were found in 192 apheresis platelet donors. The frequency of CD36 deficiency was 3.6% and all of them were typeII deficiency. The significant difference of CD36 antigen expression was observed in the platelet donors of Hangzhou population, among them 59 individuals with low expressed CD36 antigen and 126 individuals with highly expressed CD36 antigen were found according to the geometric mean fluorescence intensity. It is concluded that the CD36 antigen deficient phenotype existed in the population, these data will provide the information for research of the CD36 antigen distribution and help to solve the platelet transfusion refractoriness.

To explore the molecular basis of an individual featuring an ABx variant of ABO blood group system.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). And the products were sequenced bidirectionally following enzyme digestion. Exons 6 and 7 were also subcloned and analyzed for haplotypes of the ABO gene.Erythrocytes of the proband have expressed a strong A antigen and a weak B antigen, which was identified as a rare ABx variant in addition with other serological features. Nine heterozygous sites in exon 6 (297A/G) and exon 7 (467C/T, 526C/G, 657C/T, 703G/A, 796C/A, 803G/C, 808T/A, 930G/A) of the coding region of the ABO gene were identified. Based on haplotype analysis, one allele was determined as common A102, whilst another was consistent with B101 except for an 808TA mutation which has resulted in replacement of phenylalanine with isoleucine at position 270 of glycosyltransferase B.The 808TA mutation of the glycosyltransferase B gene may decrease the enzymatic activity and result in the Bx variant.

This study was aimed to establish the real-time fluorescent quantitative PCR (RT-qPCR) with erythrocyte Kidd blood group gene for detecting the hematopoietic chimera and to investigate the feasibility of this method. The TaqMan MGB probes and special primers were designed on basis of difference of erythrocyte Kidd blood group alleles, the hematopoietic chimerism was detected by RT-qPCR, the DNA chimerism was simulated by means of dilution of multiple proportions, and the sensitivity analysis was performed. The results showed that the RT-qPCR with erythrocyte Kidd blood group gene could effectively distinguish JK*A and JK*B alleles. There was no significant difference between the theoretic value and the practical measured value by this method (P0.05). As 156 donor's cells could be discriminated from 10(4) chimeric cells, this method may effectively detect donor's cells with correlation coefficient 0.998. It is concluded that the established RT-qPCR with erythrocyte Kidd blood group gene shows the feasibility for quantitative detection of hematopoietic chimera, and may be used to quantitatively detect chimera in a certain range.

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. Intact RhD cDNA, DEL9 and DEL89 transcripts were obtained by one-step and two-step RT-PCR, respectively. The obtained products were cloned into pCR4 TOPO sequencing vector for choosing the right transcript. RHD gene was amplified again from the sequencing plasmid DNA, and then subcloned into pcDNA3.1/V5-His TOPO expression vector; DEL9 and DEL89 were cloned into the expression vector directly. Gene sequence and direction were identified by sequencing. The results showed that the sequence and direction of target genes were right, thus these 3 different expression vectors were correctly constructed. It is concluded that expression vector is constructed from different transcripts of RHD gene, which lays a foundation for further exploring the membranous protein expression of Rh(D) antigen.

To analyze the molecular basis for an individual with ABx09 phenotype of ABO subtype.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. The amplified products for exons 5 to 7 were also cloned by TOPO TA cloning sequencing kit to split the two alleles apart, selected colonies were sequenced bidirectionally for exons 5 to 7 of the ABO gene. The samples of the proband's parents were collected, then serological test of the blood group and sequence analysis for exons 6 and 7 of ABO gene were preformed.Both A and B antigens were detected on red blood cells of the proband and there was anti-B antibody in the serum. There was no G deletion at position 261, while 297AG in exon 6, 467CT, 526CG, 657CT, 703GA, 796CA, 803GC, 889GA and 930GA heterozygote in exon 7 were detected by direct DNA sequencing, which can be assigned for A102Bx09 genotype. After cloning and sequencing, two alleles A102 and Bx09 were obtained. The sequence of Bx09 had one nucleotide changes (G to A) at position 889 compared with that of B101, which resulted in an amino acid change of Glu to Lys at 297 position. The Bx09 in the proband was inherited from her mother by family investigation.G to A at nt889 of alpha-1,3 galactosyltransferasegene can result in Bx09 phenotype, with the presence of anti-B antibody in serum.

To elucidate the serological characteristics and molecular mechanism of a blood donor with weaken B antigen.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. The alternative splicing isoforms of ABO cDNA were obtained by reverse transcription-PCR(RT-PCR) and analyzed with cloning and sequencing techniques. The level of methylation of the CpG island in ABO gene promoter was analyzed by bisulfite sequencing method.The serological characteristic of the donor showed that the B antigen was decreased obviously without anti-B antibodies in serum and the B glycosyltransferase activity was decreased as well. The genotype of the donor was B101/O01 without any other mutations in the full-length coding sequences and splice receptor sites. The nucleotide characteristics of the 5'-UTR was consistent with B101/O01 and no any abnormity was identified in the promoter, enhancer and the negative regulatory sequence regions. The integrative cDNA transcript of ABO gene was obtained and no new splicing isoform was found. Compared with the normal B phenotype, a number of methylated CpG sites were found near the promoter of ABO gene in this sample.The methylation in the CpG island of ABO gene promoter region may cause weak expression of the B antigen.

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. The amplified product was also cloned by TOPO TA cloning sequencing kit to split the 2 alleles apart and chosen colonies were sequencing bidirectionally for exon 6 to 7 of ABO gene. The results showed that both A and B antigen were identified on red blood cells of the individuals and there was anti-A1 antibody in their serum. There was no 261G deletion and showed 297A/G, 467C/T, 526C/G, 657C/T, 703G/A, 742C/T, 796C/A, 803G/C, 930G/A heterozygotes by direct DNA sequencing. After cloning and sequencing, it was obtained the B101 and one novel A allele. The novel allele has one nucleotide change at 742 position C to T compared with A102, which results in an amino acid Arg to Cys at 248 position and was nominated as A213. It is concluded that C742T mutation of the α1, 3 N-acetyl-D-galactosaminyl-transferase gene can lead to A2 phenotype and with anti-A1 antibody in serum.

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. The haplotype of compound heterozygote of fut1 was also identified by cloning sequencing. The results indicated that a rare para-Bombay phenotype was confirmed by serological techniques. Two deletion or insertion variant sites near nucleotide 547 and 880 were detected in fut1 gene. The results of cloning sequence showed that one haplotype of fut1 gene was two bases deletion at 547-552 (AGAGAG→AGAG), and another one was two bases deletion at position 880-882 (TTT→T). Both two variants caused a reading frame shift and a premature stop codon. It is concluded that a rare para-Bombay phenotype is found and confirmed in blood donor population. The molecular basis of this individual is compound heterozygote of two bases deletion on fut1 gene which weaken the activity of α-1, 2-fucosyltransferase.

To investigate the polymorphisms of platelet membrane glycoprotein genes related to human platelet alloantigen (HPA)-1 to 17w.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.Thirteen new single nucleotide polymorphisms (SNPs) and a micro-satellite sequence were found in the glycoprotein genes from the 112 random samples, in which two SNPs (1333G/A and 1960G/A) in ITGB3 gene result in two amino acid change (V419M and E628K) on glycoprotein GPIIIa.New variants in platelet membrane glycoprotein genes were identified, which may lead to structure change of platelet membrane glycoprotein and affect the accuracy of partial HPA genotyping method.

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.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. Haplotypes of samples with heterozygous sites in the 5'-UTR of ABO gene were separated and analyzed after cloning.Twenty polymorphic sites were identified in these samples where ABO genotypes were consistent with serological phenotypes. It included sixteen nucleotide sequence variations, one 8 bp deletion, one 6 bp deletion/insertion, one 36 bp insertion and one 43 bp repeats. Among them, 11 were novel polymorphic sites. Seven different haplotypes of 5'-UTR of ABO gene were defined to the cis/trans linkage of those mutations.There were polymorphisms in the 5'-UTR of ABO gene and the nucleotide sequences near the proximal promoter were conservative.

In order to explore the effects of 35CT and 682AG 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. After screening by using G418, H antigen expression on the COS-7 was tested by flow cytometry and fut1 mRNA was detected by real-time PCR. The results indicated that three kinds of recombinant plasmids pcDNA3.1/V5-His-wild (35C + 682A), pcDNA3.1/V5-His-35T and pcDNA3.1/V5-His-35T-682G were successfully constructed. After transfection, the H antigen expressed on membrane of COS-7 cells at the second day, with the maximum level of expression at the fourth day. When compared with pcDNA3.1/V5-His-wild transfected cells, the H antigen expression level of the 35T and 682G + 35T recombinant plasmids in the transfected cells was 52.7% and 13.3% on the fourth day, respectively. Although the level of fut1 mRNA decreased with prolonging of time, the mRNA expressed on the pcDNA3.1/V5-His-35T-682G transfected cells reached to 14% of the wild plasmids on the first day. It is concluded that 682AG mutation obviously reduces the activity of alpha-(1,2) fucosyltransferase, while 35CT mutation leads to partial reduction of H antigen in vitro expression.

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. The results showed that the DNA amount extracted from sorted Treg cells was fit for STR detection. All STR alleles specific for recipient or donor could be detected and the quantitative results were consistent with theoretic values in over 10% recipient chimeras. But only partial recipient alleles could be detected and the quantitative results were different from theoretic values in less then 1% recipient chimeras. It is concluded that a quantitative chimerism analysis of Treg cell based on immune sorting is established. The sensitivity and accuracy for chimera detection are 1% to 10%, and this method can be used to monitoring hematopoietic chimerism following allogeneic hematopoietic stem cell transplantation.

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. The diploid of the individual with B phenotype was separated into its haploid components with a haplotype specific extraction method. The exons 6 to 7 of the two single ABO haplotypes were then amplified and sequenced separately. The results indicated that 3 samples had mutation at 743 site in total 417 individuals, in which 2 individuals were with O phenotype and 1 individual was with B phenotype. The DNA sequencing of exon 6 and 7 in 2 samples with O phenotype showed 261G deletion and 743G/C heterozygotes. The DNA sequencing of exon 6 and 7 in the sample with B phenotype showed 261G/deletion and 297A/G, 526C/G, 743G/C, 657C/T, 703G/A, 796C/A, 803G/C, 930G/A heterozygotes. After separating of the 2 single strands in the B sample with haplotype specific extraction, an O and B101 allele were identified after sequencing. The novel allele was submitted to the Blood Group Antigen Gene Mutation database and is named as O61. It is concluded that 743GC is a novel mutation in exon 7 of ABO and a novel O61 allele with 743GC has been identified.

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. Ten exons of RHD gene in these samples were amplified by PCR-SSP, and positive exons were DNA sequenced. The results indicated that 30 real RhD negative and 8 RhDel phenotypes were identified in 38 initial RhD negative samples. Ten exons were complete negative in 28 real RhD negative samples and only exon 1, 2 and 10 were positive in 2 real RhD negative samples amplified by PCR. All 10 exons in 8 RbDel samples were positive and a DNA variant (1227GA) was found in 8 RhDel samples. It is concluded that all exons are absence in most real RhD negative phenotypes, and the partial exons absence is also found in some real negative phenotypes among Yiwu population in Zhejiang province of China. The G to A mutation at position 1227 is found in all RhDel phenotypes.

To analyze the molecular genetic basis for a new B112 allele of ABO blood group and the pedigree of the proband.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. The exons 5-7 of ABO gene for the proband was amplified by polymerase chain reaction and the amplified products were digested with double enzymes and sequenced for exons 6 and 7. A magnetic bead-based, haplotype specific extraction was used to separate the diploid sample of the proband into its haploid components. The exons 6 and 7 of the two single ABO haplotypes were then amplified and sequenced separately. The samples of the parents of the proband were collected, and the blood group serological test and sequence analysis for exons 6 and 7 of ABO gene were performed.The serum characteristic of the proband was consisted with the normal B phenotype. The DNA sequencing of exons 6 and 7 showed 261G/del, 297A/G, 526C/G, 559C/T, 657C/T, 703G/A, 796C/A, 803G/C and 930G/A heterozygotes and was assigned for B/O genotype. After separation of the two single strands of the proband with haplotype specific extraction, a B112 and an O01 allele were identified after sequencing. The B112 allele had one nucleotide change (C to T) at position 559 compared with B101, which resulted in an amino acid change at position 187 (Arg to Cys). The B112 in the proband was identified to inherit from his mother after pedigree analysis, the ABO blood group serological characteristics and sequences of exons 6 and 7 of the mother were identical to that of the proband.A novel B112 allele of ABO group system with 559CT was identified. It had normal B antigen expression, suggesting that Arg118Cys of alpha-1, 3 galactosyltransferase did not affect its enzyme activity.

To investigate the molecular genetic basis of para-Bombay phenotype in a Chinese family.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. The haplotypes of compound heterozygote of the FUT1 gene were also analyzed by cloning sequencing.Three para-Bombay phenotypes were identified in nine family members by serological technology. Three heterozygous variants (35C/T, 235G/C and 682A/G) were found in FUT1 gene of the proband, and the hapotype of FUT1 gene was h(235C)/h(35T+628G)according to the cloning sequencing. The alleles h(235C)and h(35T+628G) caused G79R, A12V and M228V amino acid substitutions in alpha-1,2-fucosyltransferase, respectively.A novel 235GC mutation of FUT1 gene which was associated with para-Bombay phenotype was found in the Chinese pedigree.

To investigate the molecular genetic basis of the B3 variant of ABO blood group system with mixed-field hemagglutination in Chinese.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. Mutations in exons 6 and 7, including partial intron of the ABO gene, were screened by polymerase chain reaction and DNA sequencing. Haplotypes of the two individuals were also analyzed by sequencing.A mixed-field hemagglutination of RBCs with anti-B and anti-AB antibodies was detected in the two unrelated individuals. Exogenous ABO-incompatible RBC transfusion and endogenous genetic chimera were excluded by sequential agglutination method and STR. The ABO phenotypes of the two individuals were classified as A1B3 according to the ABO subgroup definition. The sequence region from intron 5 to 3'-UTR of the B allele was identical to that of ABO*B101 allele, except for a T to C substitution at nucleotide position 425 in exon 7. This substitution resulted in an amino acid change of M142T in the B glycosyltransferase.A novel B allele with 425TC substitution resulting in B3 subgroup was identified in two Chinese individuals.

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. The haplotypes of diverse genotypes were also analyzed by cloning sequencing. Twelve samples were identified as AweakB or ABweak phenotype by serological technology, and were also identified as cisAB variants including four genotypes by directly sequencing. Two cisAB alleles were found by haplotype sequencing. One allele was cisAB01, in which four nucleotide acid alterations were observed (467CT and 803GC in exon 7, 163TC and 179CT in intron 6); the other allele maintained a nucleotide acid of A101 allele (803G) compared with B101 allele, which resulted in a polypep-tide containing 176G, 235S, 266M, and 268G four amino acids. This is a novel allele, which has been named cisAB05 by Blood Group Antigen Gene Mutation Database. According to systematically investigation of the molecular genetic basis of the cisAB variants in Chinese population, we found a novel cisAB05 allele and presumed that the cisAB01 allele is derived from homologues exchange of A101-B101 combination.

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. In conclusion, anti-Le(b) antibody can result in hemolytic transfusion reaction, FUT3 gene is homozygous for le(59, 508) allele resulting in Le (a-b-) phenotype.

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% - 53.5% at different conditions of lyophilization, the size and shape of ice crystals in lyophilized platelets of different test groups were not similar, the expression and distribution of platelet activation markers (PAC-1 and CD62p) after rehydration were relatively similar to fresh platelets, expression rate of PAC-1 was lower (0.03% - 0.22%), meanwhile there were differences of CD62p expression levels between different groups. The optimal theoretic composition of prefreezing conditions obtained on basis of platelet recovery rate was A(2)B(1)C(1)D(3), i.e, first, suspensions of platelets on the program control cooling apparatus were lyophilized with a rate of 20 degrees C/min and maintained at -40 degrees C for 2 hours, then the annealing was conducted at -30 degrees C for 0.5 hours with a heating rate of 1.5 degrees C/min; finally, the freeze-drying program was carried out to the end. It is concluded that the freezing rate, annealing rate, annealing temperature and holding time of annealing all impact on the recovery of lyophilized platelets. The preservation qualities of lyophilized platelets were affected by the various combinations of prefreezing parameters.

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. All individuals contained KIR3DL3, KIR3DP1, KIR2DL4 and KIR3DL2.; 20 different KIR genotypes and 12 haplotypes have been found, the most common KIR genotypes was 2,2 with frequency 29.6% and KIR haplotype was 2 with frequency 53.0%. The A KIR haplotype was the most prevalent with frequency 67.2%; 12 pairs (44.4%) HLA identical sibling donor-recipients showed KIR match in genotype and haplotype, 13 pairs (48.1%) with one KIR haplotype mismatch and 2 pairs (7.4%) with two KIR haplotype mismatch; 1 pair was matched between donor KIR2DL1 and patient HLA-Cw (Lys80) ligand, 17 pairs were matched between KIR2DL2/KIR2DL3 and HLA-Cw (Asn80) ligand, 5 pairs were matched between KIR3DL1 and HLA-Bw4 ligand. It is suggested that the probability of KIR mismatch is high in HLA-identical sibling.

This study was purposed to investigate the molecular genetics basis for para-Bombay phenotype. The para-Bombay phenotype of two probands was identified by routine serological techniques. The full coding region of alpha (1, 2) fucosyltransferase gene (FUT1 and FUT2) in the probands was amplified by polymerase chain reaction and the amplified fragments were directly sequenced, meanwhile the mutations of FUT1 were also identified by TOPO TA cloning sequence method. The results indicated that two heterozygous mutations were detected by directly sequencing in two probands: AG deletion at position 547 - 552 and C to T mutation at position 658. Two different mutations were confirmed to be true compound heterozygotes with each mutation on a separate homologous chromosome by TOPO TA cloning sequence method. AG deletion at position 547 - 552 caused a reading frame shift and a premature stop codon. C658T mutation resulted in Arg--Cys at amino acid position 220. It is suggested that the FUT1 mutation of two probands are compound heterozygous mutation with different chromosomes, which are named h1h3 and may be the genetics basis of para-Bombay phenotype.

To analyze the allelic polymorphism of killer cell immunoglobulin-like receptor 2DL3 (KIR2DL3) gene in Zhejiang Han population.Allelic of KIR2DL3 was detected by PCR sequence based typing (PCR-SBT) method. Two specific fragments of KIR2DL3 were amplified using different primers and sequenced for exons 4,5,7,8,9 of KIR2DL3. All the samples would be assigned to KIR2DL3 allele according to the polymorphism site patterns.Two KIR2DL3 alleles were observed, with KIR2DL3*001 having higher frequency, 0.77.The PCR-SBT method is reliable and there are distinctive frequencies of KIR2DL3 alleles in Zhejiang Han population.

To investigate the frequency of RHD 1227A allele in Rh negative population and random population, an AS-PCR (allele specific-polymerase chain reaction) method was employed to detect RHD 1227A allele. RHD gene copy was determined by D zygosity test and RHD exon 9 nucleotide sequence analysis. The results showed that among 143 Rh negative donors, forty-one RHD 1227A allele carriers were detected, and 8 (19.51%) out of which were RhCCdee, 32 (78.05%) were RhCcdee, and 1 (2.44%) was RhCcdEe. Thirty-five Rh negative RHD 1227A carriers had RHD gene deletion, and the remaining carriers were RHD 1227A homozygous. Seven (1.43%) individuals were detected with RHD 1227A allele among 489 random donors. They were all G/A heterozygous at RHD 1227 site. Serological test indicated that they were normal Rh positive phenotype. It is concluded that the frequency of RHD 1227A allele is 16.43% among Rh negative population and 0.72% among the random population.

To investigate the molecular genetic basis of the Bw variant and identify novel alleles at ABO locus in Chinese Han population.Serological techniques were performed to characterize erythrocyte phenotype of a proband. Mutations of the ABO gene were screened by polymerase chain reaction, reverse transcription-polymerase chain reaction and DNA sequencing.The proband was identified as Bw phenotype by serological technology and family study. A novel Bw variant allele was identified in the gDNA and cDNA. The novel allele was observed a missense mutation (278 C to T) at the exon 6 which resulted in an amino acid substitution (P93L) compared with B101 allele. The 278 C to T was the first report mutation position in exon 6 among Bw alleles, so the P93L amino acid substitution was different from others Bw variants which had amino acid substitutions in a conserved functional domain reported previously.A novel Bw allele (278 C to T) responsible for Bw variant is reported in Chinese population.

In order to establish a genotyping method for DEL phenotype in Zhejiang Han population, an AS-PCR method was developed according to the RHD 1227A allele sequence. Its specificity and sensitivity were assessed in two Rh negative populations whose RHD 1227A or DEL phenotype status was known. The results showed that in evaluation of the method by detecting 50 RHD 1227A positive and 50 RHD 1227A negative individuals, the genotyping method displayed a sensitivity of 100% and a specificity of 100%; in evaluation of the method by detecting 33 DEL positive and 89 DEL negative individuals, the sensitivity was 100%, however, there were two serologically negative samples which were confirmed as positive using genotyping method. After re-testing these two samples with serological method and sequence analysis, it was found that original serological method gave false negative results and genotyping method still showed 100% specificity. The minimal target DNA concentration of this genotyping method is 8.13 ng/microl. In conclusion, designed genotyping method can be used to identify DEL phenotype efficiently in Zhejiang Han Rh negative population.

The purpose of this study was to investigate the molecular genetic basis of A2 subgroup and identify the novel alleles at ABO locus in Chinese Han population. All seven exons and their flanking sequences, enhancer and promoter in the ABO gene of five samples from individuals with serological discrepancies were amplified by polymerase chain reaction (PCR); the PCR products were screened by directly sequencing; the haplotypes of exon 6 and 7 were analyzed by TOPO cloning sequencing. The results showed that five samples were identified as A2 or A2B subgroup by serological technology. The A201 and A205 alleles were confirmed in one A2B individual and one A2 individual, respectively. A novel A2 variant allele was identified in three A2B individuals. The two nucleotide acid alterations (467CT and 539GC) at the exon 7 resulting in two amino acid substitutions (P156L and R180P) in this novel allele were observed, when compared with A101 allele. It is concluded that the polymorphism of A2 allele is found to be relatively variable in Chinese population, and a novel A208 allele responsible for A2 subgroup is firstly reported.

To investigate the alpha-1, 3/4-fucosyltransferase gene (FUT3) polymorphism associated with Lewis blood group in Zhejiang population, the Lewis phenotypes of 183 random samples from Chinese blood donors in Zhejiang province were identified by standard serological techniques. The entire coding region of FUT3 gene were amplified by PCR from genomic DNA of 39 Lewis negative and 9 Lewis positive phenotype samples and sequenced directly. The haplotypes of FUT3 allele were identified by TOPO cloning sequencing method. The results showed that the frequency of true Le (a-b-) phenotype in Zhejiang population was 10.4% according to serological and molecular biological methods. Five nucleotide acid variant sites (59TG, 202TC, 314CT, 508GA and 1067TA) were detected in all 48 sequencing samples. Besides the wild type Le allele, 2 common (le(59, 1067) and le(59, 508) and 3 rare non-functional le alleles (le(59), le(1067) and le(202, 314) were found in this population. In conclusion, the polymorphism of non-functional FUT3 allele was found to be relatively variable in Chinese Zhejiang population.

To investigate the molecular basis of partial D phenotypes in Chinese, D variants with weak D expression was screened by using indirect anti-human globulin test (IAT) method, the polymerase chain reaction-sequence specific primer (PCR-SSP) method was employed to amplify RHD specific exons and their flanking regions. The amplification products were sequenced directly to determine the molecular basis of D variants. The results showed that ten cases of partial D phenotypes, including one case of D Va (Kou.), one case of D Va (Hus.), one case of D Va-like (YH.), and seven cases of D VI type III, were detected from 22 cases of weak D phenotype respectively. All ten cases of partial D phenotypes had one RHD allele deleted. In conclusion, the molecular basis of ten cases of partial D phenotype was confirmed, including D Va (Kou.) and D Va-like (YH.) phenotypes reported firstly in Chinese population.

To investigate the molecular genetics basis for one para-Bombay phenotype, the red blood cell phenotype of the proband was characterized by standard serological techniques. Exon 6 and 7 of ABO gene, the entire coding region of FUT1 gene and FUT2 gene were amplified by polymerase chain reaction from genomic DNA of the proband respectively. The PCR products were purified by agarose gels and directly sequenced. The PCR-SSP and genescan were performed to confirm the mutations detected by sequencing. The results showed that the proband ABO genotype was A(102)A(102). Two heterozygous mutations of FUT1 gene, an A to G transition at position 682 and AG deletion at position 547-552 were detected in the proband. A682G could cause transition of Met--Val at amino acid position 228, AG deletion at position 547-552 caused a reading frame shift and a premature stop codon. The FUT2 genotype was heterozygous for a functional allele Se(357) and a weakly functional allele Se(357), 385 (T/T homozygous at position 357 and A/T heterozygous at 385 position). It is concluded that the compound heterozygous mutation--a novel A682G missense mutation and a 547-552 del AG is the molecular mechanism of this para-Bombay phenotype.

To investigate the h4 allele (C35T) frequency of alpha-1,2-fucosyltransferase gene in Chinese population.The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for identifying C35T variant was established by using PCR to amplify a 125 bp FUT1 gene fragment, including C35T variant sequence, and the PCR product was digested by Hae III restriction enzyme. One hundred and fifty-eight random samples from Chinese blood donor were screened by PCR-RFLP.Among 158 Chinese individuals with normal ABO and H blood group phenotypes, 8 and 83 were homozygous with 35T/T and 35C/C, respectively, while 67 were heterozygous with 35C/T. The allele frequencies were compatible with Hardy-Weinberg equilibrium.The C35T substitution of FUT1 gene is not a mutation which gives rise to a non-functional h allele responsible for para-Bombay phenotype but a single nucleotide polymorphism in Chinese population.

The aim of this study was to clone human RHD gene and to investigate its expression in transduced K562 cells. Total RNA was extracted from reticulocyte of cord blood. RHD and RHCE genes were amplified using RT-PCR method. The amplified products were cloned into pGEM-T plasmid by TA ligation and several clones were screened by direct sequencing method in order to obtain the RHD gene. RHD gene was subcloned into pcDNA3.1(-) expression vector, then the recombined plasmids were transduced into K562 cells with superfect transfection reagent kit. Finally transcription and expression of RHD gene in K562 cells were detected. The result showed that RHD gene has been cloned sucessfully, the inserted sequence and direction of RHD cDNA in its recombined pcDNA3.1(-) vector were identified using enzyme cutting and sequencing method. After transduced with recombined pcDNA3.1(-) vector, K562 cells could transcribe RHD mRNA in its cytoplasm and express RhD antigen on its membrane surface. In conclusion, RhD antigen can expressed in K562 cells with RHD cDNA transduction, and the expression system in vitro may be helpful to further investigate the molecular basis of RhD variants.

To probe into the molecular genetics basis for para-Bombay phenotype.Red blood cell phenotype of the proband was characterized by serological techniques. Exons 6 and 7 of ABO gene, the entire coding region of alpha(1,2) fucosyltransferase (FUT1) gene and FUT2 gene were amplified by polymerase chain reaction (PCR) from genomic DNA of the proband respectively. The PCR products were excised and purified from agarose gels and were directly sequenced.AG at 547-552 deletion homozygous allele was found in the proband, which caused a reading frame shift and a premature stop codon. Parents of proband were heterozygous carriers.Two base deletion at position 547-552 of alpha (1,2) fucosyltransferase gene may cause para-Bombay phenotype.

To study the molecular genetic basis for p phenotype.Red blood cell phenotype of the proband was characterized by standard serological technique. Exon 3 of the alpha (1,4) galactosyltransferase gene was amplified by polymerase chain reaction from genomic DNA of the proband. The amplified PCR products were excised and purified from agarose gels and direct sequenced.Homozygous single nucleotide G deletion at position 300 or 301 was found in the proband, which caused a reading frame shift at codon 101,resulting in a premature stop at codon 113. Parents of the proband were heterozygous carriers.A novel single guanosine deletion at position 300 or 301 of alpha (1,4) galactosyltransferase gene was determined, which may be one facet of the molecular basis for p phenotype.

To investigate genetic polymorphism of six short tandem repeat (STR) loci (D3S1358,D16S539, TH01,TPOX, CSF1PO,D7S820) in Zhejiang She ethnic population and Han population.By use of AmpFlSTR Cofiler kit, 6 STR loci in 108 She samples and 102 Han samples were amplified. The PCR products were electrophoresed by ABI Prism 377 sequencer; the data were analyzed by Genescan software.All genotype frequencies of the 6 STR loci in She and Han ethnic groups met Hardy-Weinberg equilibrium. In the She population, the heterozygosities (H) in D3S1358,D16S539,TH01,TPOX, CSF1PO and D7S820 were 0.8028, 0.9148, 0.7522, 0.6728, 0.9123,0.8338, the exclusion of paternity(EP) were 0.4067, 0.6057, 0.4437, 0.3200, 0.5250, 0.5358, discrimination power (DP) were 0.6690, 0.7841, 0.6447, 0.5382, 0.7298, 0.7296.The combined DP, PE and polymorphism information content were 0.9991,0.9805,0.9988 respectively. There were significant differences at D3S1358, D16S539 and TPOX loci, compared with Hans.She population has its own STR allele distribution characteristic. The above data obtained from She population can be used not only in genetic researches and population investigation, but also in human identity and paternity testing.