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.

Objective To investigate the biomechanical properties of our self-designed 4 cannulated screws in 4 configurations for fixation of extremely unstable femoral neck fractures.Methods Twelve adult cadaveric femoral specimens were randomly divided into 4 equal groups (n =3) and made into models of extremely unstable femoral neck fracture combined with comminution (Pauwels type Ⅲ).Group A was subjected to fixation in configuration of “double axial compressions plus double stabilizations”,group B to configuration of “positive triangle parallel compression plus small angle screwing”,group C to configuration of “inverted triangle parallel compression plus small angle screwing”,and group D to configuration of “diamond pattern screwing”.Static compression tests,cyclic loading tests and limit load tests were carried out for the 4 groups on a biomechanical testing machine.Results For groups A,B C and D,the axial compression stiffness was respectively 995.29 ±34.16 N/mm,509.89 ± 138.90 N/mm,559.28 ± 111.25 N/mm and 610.18 ±232.35 N/mm,and the limit load was respectively 3,225.33 ±461.31 N,2,008.67 ±237.27 N,2,705.67 ±496.39 N and 2,395.33 ±403.71 N,showing significant differences between the 4 groups (P ＜ 0.05).For groups A,B C and D,the displacement was respectively 0.46 ± 0.10 mm,1.47 ± 0.72 mm,1.14 ±0.24 mm and 1.22 ±0.22 mm,and the limit stiffness was respectively 1,139.28 ±342.09 N/mm,843.56 ±408.91 N/mm,585.98 ± 81.60 N/mm and 729.96 ±251.37 N/mm,showing no significant differences between the 4 groups (P ＞ 0.05).Conclusions In the fixation of extremely unstable femoral neck fracture with our self-designed 4 cannulated screws,the configuration of “double axial compressions plus double stabilizations” may lead to the greatest biomechanical advantage while the configuration of “positive triangle parallel compression plus small angle screwing” may result in the poorest biomechanical properties.

Objective:To investigate the clinical efficacy of proximal femoral nail anti-rotation (PFNA) assisted by the "3-2-1" surface positioning method in the treatment of femoral subtrochanteric fractures.Methods:A total of 97 patients with subtrochanteric fractures admitted to the Second Hospital of Fuzhou from January 2015 to December 2020 were retrospectively analyzed. They were divided into two groups according to whether the "3-2-1" surface positioning method (3 longitudinal axes, 2 preset incisions, and 1 auxiliary incision) was used. There were 44 patients in the surface positioning group, including 25 males and 19 females, aged 61.59±18.43 years (range, 22-90 years). According to the Seinsheimer classification, there were 13 cases of type II, 11 cases of type III, 6 cases of type IV, and 14 cases of type V. The mechanism of injury was low energy injury in 26 cases and high energy injury in 18 cases. There were 53 patients in the traditional positioning group, including 30 males and 20 females, aged 56.38±17.24 years (range, 24-90 years). According to the Seinsheimer classification, there were 9 cases of type II, 22 cases of type III, 9 cases of type IV, and 13 cases of type V. According to the mechanism of injury, there were 30 cases of low energy injury and 23 cases of high energy injury. The length of incision, operation time, and blood loss were recorded. At 1, 3, 6, and 12 months after operation, the anteroposterior and lateral X-ray films of the hip were taken to evaluate the imaging indicators (neck-shaft angle, anteroposterior and lateral displacement, and angulation), fracture healing, and complications (infection, malunion, loosening and breakage of the internal fixation, and periprosthetic fracture). The Harris hip score and EuroQol five dimensions questionnaire (EQ-5D) were evaluated.Results:All patients successfully completed the operation and were followed up for 15.12±1.54 months (range, 12-18 months). The operation time, incision length, dominant blood loss and hidden blood loss in the surface positioning group were 1.78(1.50, 2.00) h, 8(8, 9) cm, 300(200, 400) ml and 843(629, 1 130) ml, respectively, which were less than 2.10(1.69, 2.38) h, 10(9, 12) cm, 400(300, 500) ml and 1 030(954, 1 266) ml in the traditional positioning group, and the difference was statistically significant ( P<0.05). The neck-shaft angle in the surface positioning group was 135.54°±2.83°, which was larger than 132.33°±3.37° in the traditional positioning group, and the difference was statistically significant ( t=5.02, P<0.001). The anterolateral and lateral displacement and lateral image angle in the surface positioning group were 4.70±1.60 cm, 4.52±1.71 cm and 9.36°±2.94°, respectively, which were lower than 6.14±2.57 cm, 5.98±2.70 cm and 11.46°±4.68° in the traditional positioning group, and the difference was statistically significant ( P<0.05). One year after operation, the Harris hip score and EQ-5D score of the surface positioning group were 92(84, 99) points and 0.90(0.73, 1.00) points, respectively, which were higher than 88(74, 96) points and 0.81(0.72, 0.94) points of the traditional positioning group ( P<0.05). Conclusion:The "3-2-1" surface positioning method assisted PFNA internal fixation in the treatment of femoral subtrochanteric fracture can improve the quality of reduction, reduce intraoperative blood loss, and improve hip function and quality of life.

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 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 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 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.