Objective:To analyze the polymorphism and haplotypes of HLA-A,B,DRB1 loci in Guangzhou blood donors.Methods:PCR-SSP methods were applied to determine HLA-A,B,DRB1 genotypes of 1 691 unrelated healthy individuals from Guangzhou,and the frenquencies of alleles and haplotypes were calculated with software of Pypop.Results:37 HLA-A alleles,76 HLA-B alleles and 43 HLA-DRB1 alleles were detected,and A02:07-B46:01-DRB109:01 and A33:03-B58:01-DRB103:01 were the most common haplotypes in Guangzhou blood donors.Conclusion:The distribution of alleles and haplotypes of Guangzhou blood donors are close to those of southern Chinese population and provide more genetic information in anthropology,transplantation and studies involoing disease association.

Objective:To characterize the allele frequencies and its polymorphisms of human platelet antigen (HPA) in Guangzhou population.Methods:A total of 500 samples from healthy voluntary platelet donors in Guangzhou were genotyped for HPA-1 to-17 by PCR-SSP.Results:HPA-1a to-17a alleles were found in each of the samples;The gene frequencies of HPA-1a to-17a were 99.8%,99.85%,56.3%,99.9%,98.8%,98.6%,100%,100%,100%,100%,100%,100%,100%,100%,55.1%,and 100%,100% respectively.The gene frequencies of HPA-1b to-6b and-15b alleles were 0.2%,0.15%,43.7%,0.1%,1.2%,1.4% and 44.9% respectively;HPA-7b to-14b and-16b-17b were not detected.In summary Guangzhou population displayed higher frequency of HPA-1a to-17a and HPA-3b,-15b.Compared with those of other Han populations in China,HPA frequency of Guangzhou people was significantly different from that of Beijing;Compared to that of the European,American,English and Egyptians,HPA frequency was different significantly.While HPA frequency was different from those of Japanese and Thais.This study for the first time investigated the assortment of HPA genes and its frequency,there were 40 assortments in Guangzhou population,only 5 assortment of HPA gene frequencies more than 10%,35 assortment of HPA gene frequencies less than 9%.Conclusion:HPA distribution in Guangzhou population appears to have local characteristics.This study confirms the ethnic and original difference of HPA.The allele frequencies and its polymorphisms of HPA in Han population are shown North-South differences.Races and countries outside Asia are also shown differences.The basic information provided by this study of the HPA system polymorphisms is useful to guide the design of the local HPA genotype database of volunteer platelet donors.It's also useful to avoid the PTR,and the HPA related clinical research.

Objective To establish a cell line stably expressing the human CD36 by using TA clo-ning and cell transfection technology and to analyze its application to the detection of anti-CD36 antibodies. Methods Total RNA was isolated from human platelets and then used to synthesize complementary DNA ( cDNA) . Sequence of the gene encoding CD36 on human platelets was obtained by PCR amplification. The recombinant vector was transformed into TOP10 E. coli after TA cloning. The positive recombinant pcDNA3. 1/V5-CD36 plasmid was screened out by blue-white selection and then sequenced. The correctly constructed plasmid coated with Effectene? Transfection Reagent was transferred into HEK293T cells. Fluo-rescence-activated cell sorting was performed to screen out the cell line that could stably express the CD36 on human platelets. The transfected cell line-based flow cytometry analysis and antibody capture assay ( ACA) were established and used for antibody detection in nine serum samples positive for anti-CD36 antibodies. Results The HEK293T cell line stably expressing the recombinant CD36 was successfully established. Compare with the monoclonal antibody immobilization of platelet antigens assay ( MAIPA) , anti-CD36 anti-bodies could be easily identified in nine serum samples by using the transfected cell line-based flow cytome-try analysis and ACA. Conclusion This study suggests that the HEK293T cells stably expressing the re-combinant CD36 could be used in flow cytometry analysis and ACA for the detection of anti-CD36 antibod-ies. It also paves the way for further researches on the mechanism of CD36 in other diseases.

OBJECTIVETo analyze the sequence of a novel human leukocyte antigen (HLA)-A*33:44 allele.

METHODSA novel HLA-A allele was found by double-stranded sequencing combined with single-stranded sequencing. The frequency of the novel allele was determined by population survey.

RESULTSGenomic sequence of this novel HLA-A*33:44 allele (accession No. HQ873871) has differed from HLA-B*33:03:01 by one nucleotide in exon 4, which resulted in nt 866 G→ A substitution, which results in an amino acid substitution from Gly(GGT) to Asp(GAT) at codon 265. This alternation is a new single nucleotide polymorphism compared with other HLA-A alleles. The frequency of this new allele is less than 0.0003 in Chinese Han population.

CONCLUSIONA mutation has been found in exon 4 of the novel HLA-A*33:44 allele, which may provide more information for HLA gene study.

Adult ; Alleles ; Amino Acid Substitution ; Asian Continental Ancestry Group ; genetics ; Female ; HLA-A Antigens ; genetics ; Humans ; Male ; Polymorphism, Single Nucleotide ; Sequence Analysis, DNA ; methods

Antibodies against human CD36 are responsible for several immune-mediated disorders. The detection of anti-CD36 antibodies using the standard monoclonal antibody (mAb) immobilization of platelet antigens (MAIPA) assay is hampered by a high frequency of false-negative results, most likely due to competitive inhibition of the mAb used as the capture antibody. We generated a panel of mouse mAbs against CD36 and seven hybridomas (GZ-3, GZ-13, GZ-70, GZ-143, GZ-413, GZ-507, and GZ-608), which were selected for MAIPA assays, as they reacted with mouse and human CD36. Fourteen anti-CD36 sera were assayed; all of which showed a positive reaction in a PakPlus (Immucor GTI Diagnostics, Inc., Waukesha, WI, USA) ELISA-based screening (optical density: 0.257–2.292). When the reference anti-CD36 mAb FA6-152 was used in the MAIPA assay, only 6/14 (42.9%) sera displayed a positive reaction. In contrast, anti-CD36 antibodies were detected in 13/14 (92.9%) sera when GZ-70 and GZ-608 mAbs were used. This significant improvement resulted in the identification of anti-CD36 antibodies by an antigen capture assay. Since patient’s platelets possibly carrying rare native antigens are used, this method will facilitate the identification of new platelet antibodies against CD36 that are involved in immune-mediated thrombocytopenia and other diseases, such as transfusion-related acute lung injury.

OBJECTIVE: To construct eukaryotic expression vectors for human platelet CD36 gene 220 C>T and 429+4insg variants and analyze their expressions in HEK293T cells. METHODS: RNA was isolated from human platelets and reversely transcribed into cDNA. Sequences of 220C>T and 429+4insg variants were derived by PCR amplification. The target sequence was ligated into a pcDNA3.1/V5-His-TOPO vector by TA cloning, which was transformed into TOP10 E. coli. Positive plasmids were screened by blue-white selection. After sequencing, plasmid DNA carrying 220C>T or 429+4insg variant was used to transfect HEK293T cells with the help of effectene. Expression of CD36 protein was then analyzed by flow cytometry and Western blotting. RESULTS: An eukaryotic expression vector pcDNA3.1/V5-His-CD36 (220C>T/429+4insg) was constructed by TA cloning. After transfected into HEK293T cells, the 220C>T and 429+4insg variants resulted in CD36 deficiency in HEK cells, which was confirmed by flow cytometry and Western blotting. CONCLUSION The 220C>T and 429+4insg variants can cause CD36 deficiency in human platelets. This system may be used for assessing the effect of 220C>T, 429+4insg, and other variants on the expression of CD36.
Blood Platelets ; CD36 Antigens ; Cloning, Molecular ; Escherichia coli ; Eukaryota ; Genetic Vectors ; HEK293 Cells ; Humans ; Plasmids ; Transfection

【Objective】 To find the HLA-matched platelets from platelet donor registry and track the transfusion effect for aplastic anemia patients in pregnancy with platelet transfusion refractory (PTR) caused by anti-HLA, so as to support the childbirth and follow-up treatment of the patients. 【Methods】 Antibodies to HPA and HLA were detected by ELISA kit and Luminex, respectively. DNA of the patient and 523 platelet donors from the donor registry were extracted for high-resolution HLA genotyping. The Risk Factors Evaluation Software of PTR was used to select the ABO-compatible and HLA-matched donors, without HLA Eplets specific to the patient. After MASPAT cross matching, the patient was transfused with 1 U of platelets, and the 24h post-transfusion effect was recorded. 【Results】 Only anti-HLAⅠantibody was found in the patient serum, and the specificity Eplet was 65QIA, including HLA-B^*27∶08, B^*27∶05, B^*07∶02, B^*55∶01, B^*67∶01 and B^*54∶01; anti-HLA Ⅱ antibody was negative. The HLA genotypes of both the patient and donor were HLA-A^*02∶07, A^*11∶01, B^*46∶01, B^*46∶01, C^*01∶02, 01∶03, DRB1^*04∶05, DRB1^*0901, DQB1^*03∶03 and DQB1^*04∶01. The results of MASPAT matching were negative. HLA-matched platelets transfusion provided a satisfactory posttransfusion platelet responses in patients(1 before vs 33 ×10⁹/L after). A baby boy was delivered by cesarean section 4 weeks later, and the same donor was recruited due to the mother′s low Plt and bleeding trend. The 24h posttransfusion Plt (×10⁹ / L) rose from 5 to 37 after the secondary transfusion of 1U platelet. The vital signs of the mother and her baby were normal during the two-day follow up. 【Conclusion】 The establishment of blood donor registry and screening of HLA-matched donors is an effective approach to treat PTR caused by HLA antibodies in pregnancy complicated with aplastic anemia.

【Objective】 To search compatible and suitable platelets for platelet transfusion refractoriness (PTR) patient caused by compound antibodies against HLA and CD36. 【Methods】 ELISA method was used to detect the antibody against platelet antigens and the specificity of HLA-I antibody in PTR patients. The specificity of HLA-I antibody and corresponding epitopes of patients were analyzed using MATCH IT! and HLA Matchmaker software. The HLA genotype of both donor and patient was obtained by HLA-SSO method. Compatible or suitable donor platelets for PTR patients were searched through cross-reactive group (CREG) of HLA-I and HLA epitope-matched approach (Eplet). The matching degree was identified using monoclonal antibody-specific immobilization of platelet antigens (MAIPA) and the platelet suspension immunofluores-cence test (PIFT). Finally, the transfusion effect was evaluated according to the corrected count increment (CCI). 【Results】 Compound antibodies against both CD36 and HLA-I antigens were detected in two PTR patients, and their phenotype of CD36 was conformed to be type I deficient. Through LSA testing, high-frequency of HLA -I antibodies was found in these two patients, and the panel reactive antibody in patients 1 and 2 was 56% (54/96) and 53% (51/96), respectively. According to HLA-CREG and Eplet matching strategies, one donor of grade C-matching with patient 1 and one donor of grade D-matching with patient 2 were screened from the CD36 deficiency donor bank, respectively. And the selected donors avoided the antigen of HLA-I antibody epitope. These results of MAIPA and PIFT also confirmed that no immune response was detected between the patient and the donor. And a CCI of >4.5 within 24 hour of transfusion of compatible platelets was obtained in patient 2. 【Conclusion】 For PTR patients caused by HLA and CD36 compound antibodies, a combination strategy including serological cross-matching, HLA-CREG and Eplet approach should be used to select the CD36 deficient donor platelets which evaded the antigen corresponding to HLA-I antibodies and had the compatible HLA epitopes.

【Objective】 To analyze the structure of CD36, search the possible B cell epitopes and prepare multi-antigen peptides(MAP) with B cell epitopes, so as to provide a preliminary experimental basis for the preparation of CD36 antibodies using MAP with B cell epitopes. 【Methods】 The potential B cell epitopes of CD36 were analyzed by bioinformatics methods, including physical and chemical properties, secondary structure, potential phosphorylation and glycosylation sites. Eight-branch MAP with CD36 B cell epitopes were synthesized by FMOC using polylysine as the core matrix. The purity of MAPs was analyzed by reverse high-performance liquid chromatography chromatography(RP-HPLC), and the molecular weight of MAPs was determined by mass spectrometry. 【Results】 CD36 is a stable and hydrophilic alkaline protein, with multiple phosphorylation and glycosylation sites and strong antigenicity, and its secondary structure is mainly characterized by irregular curls. Four potential B cell epitopes were obtained and 4 MAPs containing potential B cell epitopes were prepared. RP-HPLC analysis showed that the purity of the MAPs were above 85%, and the molecular weight of 3 MAPs was consistent with the expected theoretical molecular weight. 【Conclusion】 CD36 on platelet has strong antigenicity. MAPs containing CD36 B cell epitopes can provide the experimental basis for the preparation and related research of CD36 antibodies.