The application of SPR technology in prenatal testing of fetal RHD blood type gene / 国际检验医学杂志

Objective To study the feasibility of detecting fetus RhD type gene by Surface Plasmon Reso-nance(SPR)technology,and to establish a new rapid diagnosis method for fetus RhD type gene.Methods The different types of DNA corresponding RNA probes were fixed on the surface of SPR chip by using amino cou-pling methods,and optimize the chip analysis condition,and then using the RNase H enzyme hydrolysis,signal amplification detection,at last the detection conditions were determined.We use the RhD type gene exon 5,7 of RNA probe to test its corresponding DNA molecules,and analyse the specificity and sensitivity of SPR chip detection signal.Results The SPR technique for detecting the exon 5,7 of RhD blood type gene shows good sensitivity and specificity in all,SPR technology can specifically detect the Exon 5,7 of RhD blood type gene, and the sensitivity of for detecting RhD gene exon 5,7 is 100 pmol/L by SPR.Conclusion The SPR technolo-gy can quickly detect RhD gene accordingly,SPR technology is simple,rapid,reliable and label-free,w hich can provide a new way predicting fetal RhD type for RhD negative prenatal.

Analysis and identification of a novel CD36 allele, 1142 T>G / 中国组织工程研究

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.

Detection of platelet antibody by surface plasmon resonance technique and its preliminary application / 重庆医学

Objective To study the platelet antibody screening and crossing match by surface plasmon resonance(SPR) ,and to find a new way for platelet compatibility testing .Methods The corresponding universal platelet antigen was fixed on the SPR chip surface by the amino coupling method .Platelet antibody positive and negative control serum were analysed by SPR micro-ar-ray ,the stability ,sensitivity and specificity of this technique were discussed ,and compared with MAIPA assay .Finally we used the SPR technology to cross match for ten cases of the platelet antibody positive patients before infusion ,and to evaluate the effect of platelet infusion .Results For the SPR technology ,the stability ,sensitivity and specificity of platelet antibody detection were all better ,106 cases of the repeated platelet transfusion samples were tested by SPR assay and MAIPA method ,there was no signifi-cant difference between them(chi-square=0 .333 ,P>0 .05) ,the total consistency was 97 .2% .The 10 cases of platelet antibody positive patients were crossed match before platelet transfusion by SPR technology ,the good results of 8 cases of them were found by the clinical tracking evaluation ,1 h CCI>7 .5 ,24 h CCI>4 .5 .Conclusion SPR technology for screening platelet antibody mat-ches with MAIPA method in basic quality ,but SPR assay is simple ,rapid ,reliable and intuitive ,label free ,which can satisfy the re-quirements for clinical rapid detection of platelet antibody screening and crossing match .

Correlation study of regulary B cell and effective Th cell in children with chronic idiopathic thrombocytopenic purpura / 国际检验医学杂志

Objective To study the changes of the peripheral blood regulatory B cells and related cytokines in children patients with chronic idiopathic thrombocytopenic purpura (CITP),and to analyze their correlations with effective Th cells,and to discuss their roles in the pathogenesis of CITP.Methods The peripheral blood mononuclear cells (PBMC)were separated in 30 cases of CITP,then percentages of Th1 ,Th17,Th22 and Breg cells were detected by the flow cytometry(FCM).IFN-γ,IL-17,IL-22 and IL-10 levels in culture supernatant were measured by the ELISA method.Results Compared with the control group,the proportions of the peripheral blood Th1 ,Th17,Th22 subsets in CITP children were increased[(18.4±4.7)% vs.(10.82±4.4)%;(2.42±1.02 )% vs.(1 .23±0.42)%;(0.79±0.24)% vs.(0.26±0.11)%],the proportion of Breg cells was reduced significantly,the differ-ences had statistical significance[(0.83±0.21)% vs.(1 .89±0.12)%,P <0.05].In CITP children,the IFN-γ,IL-17,IL-22 levels in culture supernatant were higher than those in the control group[(278.2±121.2)pg/mL vs.(181.8 ±82.2)pg/mL;(214.8 ± 100.5)pg/mL vs.(161 .4±67.8)pg/mL;(122.16±22.2)pg/mL vs.(90.8±31.1)pg/mL],but the IL-10 level was lower than that in the healthy control group[(27.4±12.6)pg/mL vs.(46.1±16.2)pg/mL),differences between them had statistical signifi-cance(P <0.05).Besides,there was a positive correlation between the proportion of Breg cells and cytokine IL-10 level(r=0.617, P <0.05 ),however,there was a negative correlation between the proportion of Breg cells with Th1 ,Th17 and Th22 cells (P <0.05),between IL-10 level with IFN-γ,IL-17 and IL-22 levels(P <0.05).Conclusion The downregulation of Breg cells proportion may participate in the immune disorder mechanism of effective Th cells in CITP children,which can provide a new idea for the im-munoregulation therapy in CITP children.

Analysis of frequency of a RHD1227A allele in Chinese Hans / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine the frequency of RHD1227A allele in Chinese Hans.</p><p><b>METHODS</b>For a total of 890403 ethnic Han blood donors, the D antigen was determined with a saline method and indirect antiglobulin test. The RHD1227A allele and number and type of zygosity of RHD gene were determined with PCR sequence specific primer (PCR-SSP). Allelic frequency was calculated through statistics.</p><p><b>RESULTS</b>In total 2385 donors were found to be Rh-negative, 108 individuals were found to be weakly positive for D antigen (including weak D and partial D phenotypes). The remaining 887 910 individuals were Rh-positive. Among the Rh-negative individuals, 516 were found with RHD1227A. Among these, 467 were RHD1227A/d and 49 were RHD1227A/RHD1227A. Two of 108 D antigen weak-positive individuals were found as RHD1227A/RHD+. In addition, 8 of 1073 random Rh-positive samples were found to be RHD1227A/RHD+. The allele frequency of RHD1227A in the population was calculated as 0.004 036. The figure should be 0.006 682 if calculated based on the detected rate of the allele in Rh-negative individuals, and 0.007 884 if calculated based on the reported average phenotype rate of DEL in Rh-negative individuals.</p><p><b>CONCLUSION</b>By taking main influencing factors such as the RHD zygosity, the rate of RHD1227A and DEL phenotype may be determined. The allele frequency of RHD1227A in Chinese Hans is between 0.004 036 and 0.007 884.</p>

Expression of apoptosis protein in stored apheresis platelets / 吉林大学学报(医学版)

Objective To analyze the change of phosphotidyl serine(PS) in apheresis platelets(plts) during storage process,and to evaluate the quality of the plts during the storage period.Methods The apoptosis ligand Fas(CD95) was used to induce plts as apoptosis model,and by which the method to detect plts PS by flow cytometry(FCM) was established.Subsequently the resting PS expressive rate and the induced PS expressive rate of 30 apheresis plts,which induced by CD95,were determined using the FCM method established in storage process.Results At the 1st,5th,7th,8th day of storage process,the PS expression of plts kept on increasing.The PS expressive rate of plts were 2.03%?0.91%,3.26%?1.86%,8.98%?4.60%,26.68%?21.28%,and 38.85%?26.43%,respectively,and there were significant differences among groups respectively(P0.05).Conclusion The PS expression of apheresis plts increase significantly with the prolongation of storage time;Fresh plts has the ability of anti-apoptosis;Apheresis plts could keep normal function until storage 7 d.

A novel frequent BRCA1 allele in Chinese patients with breast cancer / 华中科技大学学报（医学）（英德文版）

The whole length of exon 11 of BRCA1 was sequenced (total 3427 bp) in 59 patients and 10 healthy female blood donors. To allow a rapid determination of the different BRCA1 alleles, a sequence-specific primer PCR method (PCR-SSP) was established and was applied to 57 additional female donors. Finally, the full-length coding region of BRCA1 was analyzed through reversed-transcriptase PCR (RT-PCR) and cDNA sequencing (total 5554 bp) in one donor with wild-type allele and 2 patients with one or two mutated alleles. By genomic DNA sequencing, 5 homozygous polymorphisms were observed in 18 patients: 2201C>T, 2430T>C, 2731C>T, 3232A>G and 3667A>G All of them were previously observed in Caucasians, Malay and Chinese, but for the first time the mutations were found in one allele (GenBank AY304547). Twenty-six patients and 4 donors were heterozygous at these 5 nucleotide positions. The remaining 15 patients and 6 donors showed a sequence identical with the standard BRCA1 gene. Combined the PCR-SSP results and in a summary, 6 of 67 (9.0 %) healthy individuals were homozygous for the mutated allele, whereas 18 of 59 (30.5 %) breast cancer patients were homozygous. A Chi-square test showed a significant correlation between homozygous mutated BRCA1 allele and breast cancer. The cDNA sequencing showed that 2 additional mutations, 4427T>C in exon 13 and 4956A>G in exon 16, were found. A new BRCA1 allele, which is BRCAI-2201T/2430C/2731T/3232G/3667G/4427C/4956G (GenBank AY751490), was found in Chinese. And the homozygote of this mutated allele may implicate a disease-association in Chinese.

A Novel Frequent BRCA1 Allele in Chinese Patients with Breast Cancer / 华中科技大学学报（医学）（英德文版）

The whole length of exon 11 of BRCA1 was sequenced (total 3427 bp) in 59 patients and 10 healthy female blood donors. To allow a rapid determination of the different BRCA1 alleles, a sequence-specific primer PCR method (PCR-SSP) was established and was applied to 57 additional female donors. Finally, the full-length coding region of BRCA1 was analyzed through reversed-transcriptase PCR (RT-PCR) and cDNA sequencing (total 5554 bp) in one donor with wild-type allele and 2 patients with one or two mutated alleles. By genomic DNA sequencing, 5 homozygous polymorphisms were observed in 18 patients: 2201C＞T, 2430T＞C, 2731C＞T, 3232A＞G and 3667A＞G. All of them were previously observed in Caucasians, Malay and Chinese, but for the first time the mutations were found in one allele (GenBank AY304547). Twenty-six patients and 4donors were heterozygous at these 5 nucleotide positions. The remaining 15 patients and 6 donors showed a sequence identical with the standard BRCA1 gene. Combined the PCR-SSP results and in a summary, 6 of 67 (9.0 %) healthy individuals were homozygous for the mutated allele, whereas 18 of 59 (30.5 %) breast cancer patients were homozygous. A Chi-square test showed a significant correlation between homozygous mutated BRCA1 allele and breast cancer. The cDNA sequencing showed that 2 additional mutations, 4427T＞C in exon 13 and 4956A＞G in exon 16, were found. A new BRCA1 allele, which is BRCA1-2201T/2430C/2731T/3232G/3667G/4427C/4956G (GenBank AY751490), was found in Chinese. And the homozygote of this mutated allele may implicate a disease-association in Chinese.

Analysis on Prenantal Fetomaternal Immuno-state of 32 RhD-negative Pregnant Women / 中华围产医学杂志

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.

Multiple RhD isolations produced by alternative splicing / 中国输血杂志

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.

Applications of RHD zygosity test through polymerase chain reaction for prediction of fetus Rh D-positive phenotype / 中华妇产科杂志

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.

Review of 2597 cases of blood transfusion and evaluation of the appropriateness / 中国输血杂志

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.

Analysis of genomic structure of a novel RHD allele / 中国输血杂志

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).

RHD genotyping for the Chinese / 中国输血杂志

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.

A study on immuno-state and RHD gene of a rare D~(VI) type Ⅲ pregnant woman / 中国输血杂志

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.