Xianguo Xu

Molecular basis of D variants in Chinese persons

BACKGROUND: Most studies of the molecular basis of D variants have been conducted in Caucasian and African populations. There are limited data on the molecular basis for D variants in Chinese populations.

Molecular basis for para-Bombay phenotypes in Chinese persons, including a novel nonfunctional FUT1 allele

BACKGROUND: The para‐Bombay phenotype is characterized by H‐deficient or H‐partially deficient red blood cells (RBCs) in persons who secrete ABH antigens in their saliva. The studies that determined the genotypes for two Chinese individuals with the para‐Bombay phenotype are described.

A dispermic chimera was identified in a healthy man with mixed field agglutination reaction in ABO blood grouping and mosaic 46, XY/46, XX karyotype

BACKGROUND Chimerism is the presence of two or more genetically distinct cell populations in one organism. Here, we reported the identification of dispermic chimerism in a 25-year-old male. METHODS Blood grouping was performed with standard gel centrifugation test cards. ABO and HLA-A,-B,-C,-DRB1 and -DQB1 loci genotyping was determined with PCR sequence-based typing. A quantitative analysis of dual red cells populations was measured by flow cytometer. The karyotype was analyzed by G-banded chromosomes. Short tandem repeat (STR) analysis was performed on blood, buccal mucosal and hair shafts samples. RESULTS A mixed-field agglutination with anti-B antibody was observed with gel centrifugation tests, which showed a double populations of O and B groups RBCs. Two groups RBCs were also observed by flow cytometer with nearly 90% O group cells and 10% B group cells. The normal O01,O02,B101 alleles were identified in DNA sample of the proband. STR analysis revealed three alleles for D8S1179,D3S1358,TH01,D13S317,D16S539,D2S1338,D19S433,TPOX and D18S51 loci. HLA-DRB1 and -DQB1 loci had three alleles and a karyotypic mosaic was found with 60% 46, XY and 40% 46, XX karyotype in the proband. In all studies, the third allele was attributable to a dual paternal contribution. CONCLUSION A individual with dispermic chimerism was identified, which would generate by fertilization of an oocyte and the corresponding second polar body by two different sperms.

Serological characteristic and molecular basis of A2 subgroup in the Chinese population

BACKGROUND A2 phenotype is a common subgroup of blood group A, but the serological characteristic and genetics basis of A2 phenotype currently was rare reported in the Chinese Han population. Here, a large scale study of the serology and genetics of A2 and A2B phenotypes was performed. METHODS/MATERIALS 11263 Chinese individuals with group A and AB phenotypes were determined for A2 antigen with the standard serological method. The full coding region of the ABO gene was sequenced in the individuals with A2 and A2B phenotypes. Some samples including each ABO genotypes were chosen for determining the activity of glycosyltransferase A (GTA) in plasma. RESULTS 134 individuals were assigned as A2 and A2B phenotypes in 11263 individuals. There was imbalance in A2 and A2B phenotypes and the proportion of A2B among AB samples was significantly higher than that of A2 in group A samples. All samples of the A2 and A2B phenotypes were classified into A2-related allele group, A1-related allele group and the other group based on kind of the ABO genotype. Four novel A2-related alleles (A217, A218, A219, A220) were identified. The individuals with same genotype showed different agglutination strength with anti-A1 and anti-H on their RBCs. The plasma from individuals with A2-related allele had almost no GTA activity, while plasma from individuals with A1-related allele had some GTA activity. CONCLUSION A2 and A2B phenotypes could derive from different genotypes and the serological characteristic may be heterogeneity in the Chinese Han population.

Molecular basis and zygosity determination of D variants including identification of four novel alleles in Chinese individuals

The frequency and molecular basis of the D variants have been reported in the Caucasian and African populations, but relatively little information was known in the Chinese population. Here, a study was investigated in Chinese persons with weak or discrepant D serologic typing.

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. Ultrasound is employed here to enhance the loading of trehalose into human platelets. Two frequencies were used, 25 kHz and 800 kHz. The estimated intensity of ultrasound in the sample was varied from 0 to 1.5 W/cm(2). The trehalose concentration in the platelets was 11.27+/-2.53 mmol/L when Wolkers et al.s method was used without ultrasound. The application of 0.8 W/cm(2), 800 kHz ultrasound for 1h increased the concentration of trehalose loaded by 54%. The application of 0.8 W/cm(2), 25 kHz ultrasound for 30 min increased the trehalose concentration that was loaded by 172%. The number and mean volume of the platelets following ultrasonic radiation in these two cases remained normal as compared with fresh untreated platelets. Morphological examination of the radiated platelets showed slight changes. Although further work is needed, ultrasound has been shown to be efficient for the loading of trehalose into platelets.

A 425T>C mutation in the B allele for the ABO transferase is associated with the B3 phenotype in Han Chinese persons.

The B3 phenotype is characterized by mixed-field hemagglutination of red blood cells (RBCs) with anti-B and antiA,B. The frequencies of B3 and of A1B3 among group AB Chinese persons are approximately 1 in 900 and about 1 in 1800, respectively. Although B3 is the most common B subtype in the Chinese population, only two articles have been published addressing its molecular genetic basis in the population. This study presents the molecular genetic analysis of two unrelated Chinese individuals with the A1B3 phenotype. Both probands denied receiving a stem cell transplant or a transfusion. The RBCs of the two probands showed strongly positive reactions with anti-A, anti-A,B, and antiA1; weakly positive reaction with anti-H; and mixed-field agglutination with anti-B by microscopy. Reverse grouping showed no reactivity with groups A, B, or O reagent RBCs. No free RBCs were observed after a sequential agglutination method to observe for rosettes microscopically, indicating that the probands’ RBCs expressed the same antigen in varying degrees but that there was not a dual population of RBCs. This excluded exogenous ABOincompatible RBCs from transfusion or contamination as a cause of the findings. The possibility of an endogenous genetic chimera as an explanation was also excluded by testing for 13 short-tandem-repeat (STR) loci and a sexual locus on the probands’ peripheral blood DNA using two STR amplification kits (data not shown). Based on the serologic characteristics, the two probands’ phenotypes were classified as A1B3 according to the ABO subgroup definitions. As the majority of the coding sequences lie in Exons 6 and 7 of the ABO gene, we used a polymerase chain reaction (PCR) technique to analyze these two exons’ sequences of the two probands and 113 randomly selected individuals as in our previous study. The blood groups A, B, O, and AB of 113 individuals were 35 (31%), 29 (26%), 42 (37%), and 7 (6%), respectively. A 2749-bp fragment spanning a region from intron 5′ to 3′-UTR was amplified by the following primer pairs: E67F, 5′-CTCAAGGGGCTG TTCTGAAG-3′, and E67R, 5′-GCGATTGCGTGTCTGTG TAT-3′. Amplification products were then purified and bidirectionally sequenced with the following five forward or reverse primers: F1, 5′-CCTGTCCCTTTGTTCTCCAA-3′; R2, 5′-GCTCTGCCAGCATCCTCTTTCT-3′; F3, 5′-GGGCT CGCCAGGTCGGTAA-3′; F4, 5′-GAGGACTCGCTCAGGACA GGG-3′; and R5, 5′-AGGACGGACAAAGGAAACAGA-3′. The purified products were also ligated into pCR-TOPO vector and cloned to analyze the haplotypes of the ABO gene. According to the results of direct sequencing of the PCR products, the genotype of the probands could be assigned as A1B with position 425 in the cDNA nucleotide sequence heterozygous for nucleotides T and C. The cloning sequencing confirmed further that the sequence of the B allele of the two probands was identical to that of ABO*B101 allele, except for a 425T>C nucleotide substitution in Exon 7 (Fig. 1). This substitution results in an amino acid change at Met142Thr in the B glycosyltransferase. The allele is now designated as ABO*B305 on the Blood Group Antigen Gene Mutation Database (http://www.ncbi.nlm.nih.gov/gv/mhc/xslcgi. cgi?cmd=bgmut/home) and has been submitted in GenBank with Accession Number EU363231. The sequences of A1 allele in the probands showed known nucleotide polymorphisms specific for the common ABO allele A101 (Individual 1) and A102 (467C>T, Individual 2), respectively. The 425T>C mutation was not found in 113 randomly selected individuals with the common ABO phenotypes as analyzed by DNA direct sequencing. The population frequency estimate of this allele is less than 0.0044 (1/226). The same mutation at the 425T of the ABO gene has also been identified in another subgroup allele. One individual with Ael phenotype was found to heterozygote with a common O01 allele and an A allele with a mutation of 425T>C. There may be different reasons to explain why the 425T>C mutation on the A allele causes the Ael phenotype but causes B3 phenotype on B allele. The first possible reason is that attempts to classify or distinguish phenotypes based on agglutination characteristics are subjective and often depend on the characteristics of the antibody used for testing. The second important reason is that an antigen expression may also depend on the other ABO allele inherited. Here the cells appear to have reactivity characteristic of B3 (when inherited with the A1 allele), but the possibility remains that this B305 allele may express a phenotype different from B3 when inherited with an O or A2 allele. In conclusion, 425T>C in the B allele is associated with two unrelated A1B3 individuals. As fewer B antigenic determinants were present on the B3 RBCs, we postulate that the predicted amino acid change M142T derived from 425T>C affected the B glycosyltransferase activity rather than the specificity of the enzyme. Further investigation into glycosyltransferase activity in an in vitro *These authors contributed equally to the manuscript.

Two novel mutations in KLF1 were identified in Chinese individuals with In(Lu) phenotype: NOVEL MUTATIONS IN KLF1

I n the In(Lu) phenotype, Lutheran antigens are barely detectable and there is also weakened expression of some other blood group antigens outside of the Lutheran system. The frequency of the In(Lu) phenotype was approximately 1 in 3000 blood donors in South-East England and 1 in 5000 of South Wales blood donors. It was reported that the In(Lu) phenotype may result from heterozygosity for inactivating mutations in KLF1. Here, we report that two novel mutations in KLF1 were responsible for the In(Lu) phenotype.

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. The experimental data were fitted to Brunauer-Emmett-Teller (BET) and Guggenheim-Anderson-de Boer (GAB) equations. There were no significant statistical differences (P>0.05) between the sorption characteristics of FDHPs and FDLB at 4 and 25 °C, while FDHPs absorbed more water at 37 °C. The net isosteric heat of sorption was derived. The heat for FDHPs showed an abnormal negative value at low moisture contents when 25 and 37 °C data were used. Dynamic sorption experiments were carried out at 25 °C with environmental water activity controlled at 0.75, 0.85, and 0.90. The moisture diffusion coefficient was fitted to be 8.24×10−12 m2/s when experimental data at initial time were used. These results would be helpful in choosing prehydration and storage condition for FDHPs.