Objective To develop an ELISA method for determination of heparin-induced thrombocytopenia (HIT)antibody. Methods The compound formed between human platelet factor 4 (PF4)and heparin was used as the coating antigen,incu-bating the patients plasma with the coating antigen in the well,after washing,the second antibody labeled HRP was added in the well to incubate and washing again,the chromogenic substrates was added in the well to incubate,when the stop reaction was finished,the absorbance A450/A630 was detected,and the test results were judged according to standard,this method was compared with IBL method and was optimized and evaluated the performance.Results An indirect ELISA method was de-velop with the purified human PF4,the optimal dilution of sample and second antibody were 1∶100 and 1∶1 500 which de-tected by the orthogonal test,the intra-and inter-assay average coefficients of variation were 7.66% and 7.76%(<10%) respectively that detected by repeated measurement the three positive standard plasma.Through measureing the 100 healthy human plasm with no history of using heparin,the positive and negative predictive reference values were 0.304 and 0.456. IBL and this method detected 100 hemodialysis patients samples at the same time,and the result of statistical analysis was that,the sensitivity,speciality and accuracy of this method were 90%,97.78% and 90%,respectively.The negative and posi-tive predictive value were 81.8% and 98.88% respectively,and the difference was statistically significant [K=0.84(0.81~1)and Pexac=0.012<0.05].The difference was statistically significant,consistency was optimal,95% confidence interval was 92.59%~92.59%.Conclusion Comparing with the IBL,the method reported by this article had the similar perform-ance and good consistency,and it could satisfy the clinical detection and diagnosis of HIT patients.

Objective To screen the distribution frequency of Mur blood group among voluntary blood donors in Hezhou,Guangxi,and further analyze the molecular basis of of Mur antigen positive samples.Methods The Mur pheno-type of voluntary blood donors in Hezhou was serologically screened using microplate method,and the distribution frequency of Mur antigens in different ethnic groups was analyzed.Genetic typing was performed on these positive samples with PCR-SSP method to verify the accuracy of the serological method,and the genetic background was sequenced and analyzed.Re-sults Among 3 298 samples from voluntary blood donors in Hezhou,432(13.10%,432/3 298)were screened positive for Mur antigen,and PCR-SSP genotyping validation showed that all 432 samples were electrophoretic positive.Among them,the proportion of Han blood donors with positive Mur antigen was12.79%(331/2 587),Yao ethnic group was13.25%(64/483),Zhuang ethnic group was 16.51%(36/218),and no statistically significant difference was found in the three groups(P＞0.05).Further sequencing results showed that 428 samples were GYP(B-A-B)Mur,also known as GYP.Mur type(12.98%,428/3 298),the other 4 samples were GYP(B-A-B)Bun,also known as GYP.Bun type(0.12%,4/3 298).Conclusion The Mur blood type frequency is high in the voluntary blood donors in Hezhou,Guangxi,and is predominant characterized by GYP.Mur genotype.Due to ethnic integration,no significant difference was noticed in the frequency of Mur blood type distribution between Han,Zhuang and Yao population.Therefore,conducting extensive Mur blood group antigen and antibody testing in Hezhou is of great significance for ensuring clinical blood transfusion safety.

【Objective】 To prepare and preliminarily identify the specific monoclonal antibody (McAb) against-AB blood group antigen. 【Methods】 The human AB red blood cells were used to immunize BALB/c mice for producing monoclonal antibodies against human red cell AB antigens. The cell lines that can secrete AB monoclonal antibodies were obtained by cell fusion and screening using hybridoma cell technology.The specificity and recognized epitopes of anti-AB McAb were preliminarily identified by blood group serological assay. The antibody was also used to detect the clinical samples. 【Results】 One hybridoma cell line secreting IgM monoclonal antibody against human AB antigens was obtained. This anti-AB McAb could agglutinate with human red blood cells of type A, B and AB, but not agglutinate with type O red blood cells. The red blood cell absorption and elution test confirmed that the antibody recognized the common epitope of A and B. The anti-AB McAb obtained and the commercial anti-A and anti-B reagents were used to detect 567 clinical samples in parallel, with the concordance rate at 100%. 【Conclusion】 A hybridoma cell line secreting anti-AB McAb can be successfully prepared.

【Objective】 To understand the frequency and significance of anti-" Mi^a" (anti-" Mi^a" mixtures of antibodies) in local population in Zhongshan, and the influence of different experimental conditions on the activity of human anti-" Mi^a" . 【Methods】 The microplate-based agglutination assay and polybrene method were used to screen anti-" Mi^a" in 3 587 blood samples from voluntary blood donors and patients using O type red blood cells with positive Mi^a antigen, then.rechecked by tube method and microcolumn gel card method. 【Results】 The frequency of anti-" Mi^a" was 1.06% (38/3 587), among which 60.5% (23/38) were IgM and 39.5% (15/38) were mixture of IgM and IgG; 0.61% (13/2 135) in local blood donors and 1.72% (25/1 452) in patients(P<0.01). 65.8% (25/38) of the population carrying anti-" Mi^a" had a history of immunity. 57.9% (22/38) were identified to be anti-" Mur" and 42.1% (16/38) anti-" Mi^a" using GP.Vw erythrocyte. The appropriate incubation time for anti-" Mi^a" test was 10 min. 【Conclusion】 The frequency of anti-" Mi^a" was relatively high among blood donors and patients in Zhongshan, and most of the anti-" Mi^a" carriers had a history of immunity. Most anti-Mi^a antibodies were active in saline, and some of them were mixture of IgM and IgG. It may be helpful to include Mi^a positive red blood cells in the irregular antibody screening cell panel to improve the safety of blood transfusion.

【Objective】 To establish a highly sensitive detection method of platelet HPA-3 and HPA-15 genotyping by droplet digital PCR (ddPCR), and to explore the feasibility of applying it to the detection of human platelet antigen (HPA) compatibility in maternal peripheral blood fetal free DNA. 【Methods】 For SNP mutation sites of HPA-3 and HPA-15, specific primers and MGB probes were designed, and amplification conditions such as annealing temperature and primer concentration of ddPCR were optimized to establish the optimal reaction system and clarify the test procedures. The methodological performance of the assay was evaluated, including specificity, sensitivity, repeatability and stability. ddPCR was used to detect 67 clinical blood samples, and the allele typing results were compared with the gene sequencing results. The fetal free DNA HPA antigen of 52 maternal peripheral blood samples was detected. 【Results】 The ddPCR method for detecting platelet HPA-3 and HPA-15 showed good specificity of primers and probes. The optimal annealing temperatures for HPA-3 and HPA-15 were 61.6℃ and 60.2℃, respectively. The optimal concentrations of primers were 900 nM and 700 nM respectively. The final concentration of the probe was 250 nM. The quantitative detection range of copy number was 2 to 20 000 copies, with lower limit of detection of 0.1 copies/μL, and the linearity is good. In low copy number samples, the intra - and inter batch coefficient of variation (CV) of actual detection values for HPA-3 and HPA-15 were both lower than 5%. The detection results of HPA-3 and HPA-15 genotypes of 67 blood samples were consistent with the gene sequencing results, and its application in fetomaternal platelet HPA-3, HPA-15 genotype detection met expectations. 【Conclusion】 The HPA-3 and HPA-15 ddPCR detection system constructed in this study has high accuracy, good repeatability, stability and sensitivity, and can be applied to the establishment of platelet HPA-3 and HPA-15 genotype donor pool, gene matching and fetomaternal platelet compatibility detection.

【Objective】 To study the incidence and specificity of platelet antibody in blood donors in Suzhou, analyze the distribution characteristics of platelet antibody in blood donors in this area, and explore the significance of platelet antibody detection in blood donors to reduce the adverse reactions toplatelet transfusion in clinical. 【Methods】 Platelet antibody detection was performed in 2178 blood donors in this area by solid-phase immunosorbent assay. The antibody specificity of the positive samples was analyzed by commercial kit, and the anti-CD36 antibody positive samples were further identified by flow cytometry and gene sequencing. 【Results】 Twelve positive samples were detected by platelet antibody screening, with a positive rate of 0.55%(12/2 178), including 5 males (0.33%, 5/2 178)and 7 females(1.06%, 7/2 178). Among the positive samples, anti-HLA-Ⅰ antibody was identified in 2 cases, anti-CD36 antibody in 1 case, and the antibody specificity was not identified in the other 9 cases. In one case, the positive rate of anti-HLA-Ⅰ antibody PRA was 31.31%(31/ 99), which was mainly specific to anti-B15, anti-B35 and anti-B40. The positive rate of anti-HLA-Ⅰ antibody PRA in the other case was 45.45%(45/ 99), which was mainly specific to anti-A2, anti-A11, anti-A24, anti-A29, anti-A33, anti-A66, anti-B15 and anti-B35. The blood donor with anti-CD36 antibody was type I CD36 deficiency, and 329_330delAC mutation occurred in exon 5. 【Conclusion】 Through antibody screening and specificity identification, the positive rate of platelet antibody in females was significantly higher than that in males(P<0.05). In addition to the common anti-HLA-I antibodies, anti-CD36 antibody was also detected in type I CD36 deficient blood donor. Therefore, the detection of platelet antibodies in blood donors is of certain clinical significance to reduce the adverse reactions to blood transfusion caused by antibodies in platelet products.

【Objective】 To investigate the frequency of CD36 deletion and gene mutation in voluntary blood donors of Zhongshan city, and to explore the possibility of establishing local CD36 negative platelet donor bank. 【Methods】 Platelet CD36 antigen was detected by ELISA in 1 654 voluntary blood donors.Some of the negative samples were confirmed by flow cytometry, and genotyping was also performed. 【Results】 Platelet CD36 antigen was negative in 27 cases, accounting for 1.6% (27/1654), among which 1.6% (18/1149) were males and 1.8% (9/505) were females.No significant difference was noticed between males and females in CD36 antigen deletion cases (P>0.05). Fifteen CD36 negative samples were randomly selected, genotyped and sequenced, with type I deletion in 1 case[ 6.7% (1/15)], type Ⅱ deletion in 14 cases[ 93.3% (14/15)], and gene mutation in exon 3-14 detected in 8 cases. 【Conclusion】 The frequency of platelet CD36 antigen deletion in Zhongshan is comparable to that in other southern regions of China.The establishment of CD36 negative platelet donor bank is conductive to improve the effectiveness of platelet transfusion.

【Objective】 To investigate the expression of CD36 antigen in Suzhou area, analyze the type of antigen deficiency and gene mutation, so as to provide references for the establishment of CD36 negative donor registry in Suzhou. 【Methods】 Anticoagulant whole blood samples (805 cases) were randomly collected from healthy blood donors in Suzhou Blood Center. The expression of CD36 antigen on platelet and monocyte was analyzed by flow cytometry to determine the type of CD36 deficiency. The gene mutation type of platelet CD36 antigen-deficient was performed by genomic DNA sequencing. 【Results】 The CD36 deficiency frequency on platelet was 2.48% (20/805), among which TypeⅠ(lacking CD36 expression both on platelet and monocyte) and TypeⅡ(lacking CD36 expression on platelet only) CD36 deficiency accounted for 10% (2/20) and 90% (18/20), respectively. CD36 gene mutations were found in 10 samples, including 3 cases of 329_330 d^elAC, 1 case of 1228_1239 d^elATTGTGCCTATT and 2 cases of 1163 A>T; 1 case of 329_330 d^elAC+ 1172_1183 d^elTATTGGTCAAGC and 287 G>C+ 329_330 d^elAC heterozygous mutation. In addition, 1 case of 745 A>G and 1 case of 806 C>T mutations were novel, and not yet reported. 【Conclusion】 Results showed that the frequency of CD36 antigen deficiency in Suzhou were similar to that reported in southern China, but the mutation sites were slightly different. The establishment of CD36 negative platelet registry could provide negative platelets for patients with transfusion reactions caused by anti-CD36 antibody and improve the effect of clinical platelet transfusion.

【Objective】 To investigate the frequency of platelet antibodies in voluntary blood donors and patients in Zhongshan, Guangdong Province, and to study the specificity and cross-matching of platelet antibodies. 【Methods】 Platelet antibodies of blood donors and patients were screened by solid-phase immunoadsorption (SPIA), rechecked by flow cytometry (FCM), and antibody specificity was identified by PakPlus enzyme immunoassay, and platelet cross-matching was simulated by SPIA. 【Results】 A total of 1 049 blood donor samples and 598 patient samples were tested, with 6 (0.57%) and 49 (8.19%) samples positive for SPIA,respectively(P<0.05); In SPIA positive samples, the positive concordance rate of FCM in blood donors and patients was 100% vs 95%, and that of enzyme immunoassay was 100% vs 88%. Among the initial screening positive samples of blood donors, 5 were anti-HLA Ⅰ antibodies, accounting for 83%, and 1 was anti CD36 antibody, accounting for 17%, with an incidence rate of 0.10%. Among the 14 samples of enzyme immunoassay positive patients, 2 were anti-GP Ⅱb/Ⅲa, 1 was anti-GP Ⅱa/Ⅱa, 8 were anti HLA Ⅰ, and 3 were mixed antibodies (HLA Ⅰ, GP Ⅱb/Ⅲa, GP Ⅰa/Ⅱa). According to the types of antibodies, HLA Ⅰ antibodies were the most common, accounting for 65% (11/17), followed by HPA related anti GP, accounting for 35% (6/17). The majority of patients had a platelet antibody positive typing rate below 30%, accounting for 71.4% (10/14). 【Conclusions】 The positive rate of platelet antibody of patients in Zhongshan area is significantly higher than that of voluntary blood donors, and most of them are anti-HLA Ⅰ and anti-GP, and the incidence of anti-CD36 is extremely low. Therefore, it is necessary to establish a known platelet antigen donor bank, and at the same time, carry out platelet antibody testing and matching of patients, which is helpful to solve the issue of platelet transfusion refractoriness.