Objective To explore a molecular method for the determination of RHD zygosity. Methods Two pairs of primers were designed specific for downstream rhesus box and hybrid rhesus box according to the sequences in GenBank. Together with a pair of internal control primer, a dual-tube polymerase chain reaction (PCR) method was established for determination of the RHD zygostity. Thirty Rh D-positive, D-negative and weak D phenotype samples were evaluated by taking a recent restriction fragment length polymorphism (RFLP) method as reference. Results The results of the dual-tube PCR and RFLP are identical in 29 samples, which are also in concordance with the Rh phenotype, respectively. In one weak D sample, however, both methods tested as RHD -/RHD -, which showed that there was one RHD - haplotype in this individual, but a false negative result in testing downstream rhesus box. Conclusions The dual-tube PCR is a less-labored method for RHD zygosity detection. It can be used clinically in prenatal test to determine RHD zygosity of pregnant women′s partner for prediction of fetus RhD phenotype.

Objective To analyze the genomic structure of a novel RHD allele. Methods Through polymerase chain reaction (PCR), sequence specific primer-PCR(SSP-PCR) and genomic DNA sequencing,the RHD gene in a D-negative individual was detected. Results In SSP-PCR tests, the sample was tested negative for exons 3~7, 9~10 and intron 2(Din 2), but was positive for the Rh downstream Box3. The RHCE genotype was Ccee. Three PCR methods were used to detect intron 10(Din10) of RHD gene;all gave negative results. Through a RHD full length coding region sequencing method, the sample was found to have sequences identical to normal RHD in exons 1 and 2, while exons 3~10 were negative. Conclusion The sample is D antigen negative, RHD gene positive, with amalgamative allele of RHD-CE(2~10).

Objective Study on the relations between Rh hemolytic disease of the newborn (HDN) and the influencing factors of producing anti-D. Methods D antigens of 32 RhD-negative pregnant women and their newborns are determined by indirect antiglobulin test (IAT) and absorption/elution test. With polymerase chain reaction (PCR) and direct genomic DNA sequencing, we detect the RHD gene in weak D pregnant women identified serologically, and we analyzed the situation of fetomaternal hemorrhage (FMH) of the D-negative women with more than 2 gestations with flow cytometry. Results Among 32 pregnant women of RhD-negative detected by first test, there are 18 pregnant women with two and more pregnancy. In these 18 pregnant women, 3 cases are identified as D el phenotype, 1 case is designated as D category VI type III, the rest 14 cases are truly D-negative pregnant women. Among the truly D-negative multi-pregnant women, 2 produce anti-D in sera and 13 are detected fetal erythrocytes in their peripheral blood by flow cytometry. However there are no anti-D detected in sera of D-negative first-pregnant women. Conclusion No anti-D allo-immune response were observed in all first-time pregnant women. In multi-pregnant women, however, 14.3% produce anti-D and result in HDN of Rh.

Objective To investigate the clinical use of blood in Shenzhen,in order to make the blood transfusion more appropriate and safer.Methods The medical records of 2 597 blood transfusion receipts from 3 hospitals in 2006 were reviewed.The use of blood components in each department,unreasonable prescriptions of very small amount of blood transfusion,and the combined transfusion of RBC and plasma were analyzed.Results The proportion of reasonable transfusion of RBC,plasma,platelet concentrates and cryoprecipitate were 66.44%,24.65%,97.97% and 61.29%,respectively.These proportion were 91.54%,51.85%,98.86%,and 100%,respectively,in internal medicine department,and 50.22%,19.86%,89.58%,42.86%,respectively,in surgery department.Unreasonable prescription of low-volume blood transfusion and the combined transfusion of RBC and plasma were 47.05% and 51.17%.Conclusion Except for plasma transfusion,the internal medicine departments did fairly well in blood component transfusion.Since the unreasonable transfusion still exists,the clinical doctors should be further trained in the appropriate use of blood,and get rid of the old practice of whole-blood-dependence.

Objective Study on fetomaternal immuno state and RHD type of a pregnant woman of weak D phenotype.MethodsThrough polymerase chain reaction (PCR)、direct genomic DNA sequencing and flow cytometry.ResultsIn both sequence specific promer (SSP) PCR and the sequencing PCR tests, the sample was detected negative in exons 3 6 of the RHD gene, whereas all other exons (exons 1 2,7 10) were tested positive. And the sequence of detected exons (exons 1 2,7 10) are the same with normal RHD in GenBank (accession no. AJ299020 1 and AJ299026 9). Serologically and genetically, the sample can be designated as D category VI type Ⅲ. Through a duce tube PCR method, the RhD zygosity of this individual was typed CD VI e/cde。In flow cytometry, a few fetal erythrocytes were detected in peripheral blood of the mother. However there were no anti D detected in sera and hemolytic disease of the newborn(HDN) observed at all.ConclusionSevere cases of HDN have occurred in D positive babies born to partial D mother with anti D, although HDN don't take place in this case. We may still consider D VI phenotype individuals as D positive donors and D negative receiptions in our transfusion practice and in clinical anti D allo immune prophylaxis and monitoring.

Objective To study whether RHD gene exists multiple mRNA produced by alternative splicing. Method RhD mRNA was analyzed through reverse transcriptase PCR (RT-PCR) and cDNA sequencing in 4 individuals with different Rh phenotypes (CcDDEe、CCDDee、CcDDee and ccDdee). Results The individuals with different Rh phenotypes had identical and intricate RhD mRNA isolations, which included normal mRNA, and 4 other transcripts with deletions of exon 7, exon 9, exon 7 and 9, and exon 7 to 9. Those transcripts had the same sequences as normal RhD mRNA except exon(s) deletion, which showed RHD gene existed alternative splicing, and it happened in exons or introns 7-9 regions. Conclusion There are intricate and different cassette-exons RhD transcripts produced through alternatively spliced exons 7 to 9. And those RhD mRNA isolations might code proteins with different C-terminus.

Objective To establish a RHD genotyping method specific for the Chinese. Methods Six pairs of primers specific for most alleles found in the Chinese according to the records in NCBI GenBank, were designed, and a multi-tube sequence-specific primer PCR (PCR-SSP) method was established with a pair of internal control primer in each reaction. The method was evaluated with samples serologically determined and full length RHD sequenced from 89 Rh-negative, 28 D el, and 13 Rh-positive, weak D and partial D phenotype of Chinese Hans. Furthermore, 318 random samples from blood donors were genotyped and the results were compared with serological results of those samples. Results The PCR-SSP results were in concordance with serological results (100%) in all samples, and all RHD positive, D antigen negative alleles (or nonfunctional alleles) observed in the Chinese up to now could be detected or implicated, including D el phenotype especially D el allele existing in Rh-positive individuals (RHD/RHD1227A). This genotype was detected with a rate of 8/318, and allele frequency should be 0.012579 Conclusion Our method is rapid and easy, with high accuracy in the testing of the Chinese.

G mutation.

BACKGROUND:As a main antigen of platelet, CD36 antigen is also known as platelet glycoprotein IV (GPIV). The mutation of CD36 gene may result in deficiency of the antigen.
OBJECTIVE:To identify a novel CD36 alele.
METHODS: DNA was isolated from peripheral blood sample, and 12 coding regions of CD36 gene were amplified by PCR. Sequencing-based typing was used to analyze the sequence of the target regions. The derived sequences were aligned with the standard sequence of NG_008192 in GenBank to identify the novel alele.
RESULTS AND CONCLUSION: 1142 T>G mutation was detected in exon 12 of CD36 gene of the proband, and the other regions were consistent with the standard sequence. No data or report about 1142 T>G was found in GenBank or National Center for Biotechnology Information (NCBI), and thus it was reported to GenBank and received by number KM275213. 1142 T>G results in amino acid 381 Leu>Ser of the CD36 protein. There is a big difference in hydrophilia and polarity of the two amino acids. Also the 381 amino acid locates in highly conserved region. Thus it is speculated that 1142 T>G may reduce or vanish the activity of the protein.