Detection of alloimmunization in Glanzmann Thrombasthenia and Bernard-Soulier Syndrome: Data from a Brazilian Center

ABSTRACT Introduction: The Glanzmann Thrombasthenia (GT) and Bernard-Soulier Syndrome (BSS) are rare hereditary disorders of platelet function. Their treatment often requires platelet transfusion, which can lead to the development of alloantibodies. Objective: In this study, we aim to develop a strategy for alloantibody detection and to describe the frequency of alloimmunization in a patient population from a single center in southeastern Brazil. Methods: Samples from patients with GT or BSS were tested using the Platelet Immunofluorescence Test (PIFT). If a positive result was obtained, a confirmatory step using the Monoclonal Antibody Immobilization of Platelet Antigens (MAIPA) and Luminex bead-based platelet assay (PAKLx) was executed. Main results: Among 11 patients with GT, we detected the presence of alloantibodies in 5 using PIFT, with confirmation through MAIPA and PAKLx in 2 (1 anti-HLA and 1 anti-HPA), resulting in a frequency of 18.1%. Among 4 patients with BSS, PIFT was positive in 3, with confirmation by MAIPA and PAKLx in 1 (anti-HLA), showing a frequency of 25%. The two patients with anti-HLA antibodies exhibited a panel reactive antibody (PRA-HLA) testing greater than 97%. Conclusion: Our study highlights the importance of identifying platelet alloimmunization in this patient population. The proposed algorithm for platelet alloantibodies detection allows resource optimization.

Research progress of platelet lysates in the field of regenerative medicine and cell therapy / 中国输血杂志

Human platelet lysates(HPL), as a new type of biomaterial, can promote tissue repair, cell proliferation and inflammation control. This paper introduced the development of HPL in the field of regenerative medicine and cell therapy and summarizes their application. The potential of HPL to promote cell proliferation was used as an entry point to show its advantages as a supplement of cell culture medium. Since there is currently no standard procedure for HPL preparation, this paper sorts out the standardization elements such as raw materials source, donor variability and preparation technology, in order to provide reference for the establishment of standards of relevant industry in the future.

Study on the extensibility of platelet donor gene database in Shaanxi / 中国输血杂志

【Objective】 To analyze the commonality and characteristics between voluntary blood donors and hematopoietic stem cell donors in this region, and explore the potential for integration and development between China Marrow Donors Program (CMDP) and voluntary blood donors, especially platelet donor databases, so as to improve recruitment success rate and inventory rate. 【Methods】 The database modeling and comparison methods were used to screen and stratify the matching and integration degree between the voluntary blood donors in recent 10 years and the marrow donors in the Shaanxi Branch of CMDP. The frequencies of HLA-A,-B alleles, HPA alleles and haplotypes were calculated with Arlequin 3. 5. 2. 2 software, and the matching probability of different platelet donor reserve pools was conducted according to the phenotypic frequencies. 【Results】 Among the voluntary donors with known HLA genotypes in this region, according to their blood donation behavior,the active blood donors excavated were divided into the first, second, third and fourth echelons of platelet donor reserve pools, with 696, 2 752, 9 092 and 12 028 donors, respectively. The first echelon had the highest proportion of 10-50 times of platelet donations and 10-20 times of whole blood donations, with 13.65% and 26.01%, respectively. The second echelon had 10-20 times of whole blood donations and 10-50 times of platelet donations, accounted for 15.04% and 1.38%, respectively, which were significantly different from other echelons' blood donation characteristics (P<0.05). With a database size of the existing platelet donor bank adding the first and second echelons (n=4 955), there was a 69.02% probability of matching at least one donor with matching HLA-A-B phenotype. When considering the matching ABO and HPA phenotypes, the probability of finding at least one donor with fully matching HLA, HPA and ABO isotype (type B as an example) was 48. 73%. 【Conclusion】 The three groups of whole blood donation, apheresis platelet donation and marrow donation in Xi'an area have a large cross-distribution. Compared with expanding the storage capacity from scratch, the active blood donors in CMDP database are the largest back-up force of platelet donors. While expanding the effective storage capacity, it can minimize the cost of building platelet donor bank and the demand for resources.

The relevance of a bank with genotyped platelets donors

ABSTRACT Objective: Describe the clinical and laboratory characteristics and the transfusion strategy of patients at Hospital Israelita Albert Einstein with platelet refractoriness and identify their etiological characteristics. Standardize the platelet immunofluorescence technique by flow cytometry as a test for platelet compatibility in immune platelet refractoriness in transfusion support. Methods: Review of medical records of refractory platelet patients followed at HIAE from January 2011 to May 2017. Clinical-demographic data, laboratory data and identification of the use of compatible genotyped platelets for patients in need of transfusion therapy were collected. The analyzed patients were classified according to the etiology of their platelet refractoriness. To standardize the FC-PIFT technique, blood group O platelets were incubated with serum from blood group AB donors and anti-IgG monoclonal antibody to determine the negative control. In order to verify the influence of the ABO system, monoclonal anti-IgG antibodies were incubated with blood group A or B platelets and with blood group O donor serum with isohemagglutinins below and above 1/64. Results: A total of 47 patients were evaluated, a 51% (24/47) preponderance of associated immune and non-immune factors (NIPR + IPR). The most common causes of NIPR + IPR were splenomegaly (54%) and the development of HLA antibodies (88%), consistent with the literature. For patients who required therapeutic transfusion, only a small portion received compatible genotyped platelets. Conclusion: Although 60% of patients could benefit from the therapeutic transfusion of genotyped platelets, only 10% were actually transfused with this type of blood component. This reaffirms the need for investments in a bank of genotyped platelet donors.

The polymorphism of HPA-1-6w, HPA-15 and HPA-32bw-35bw in platelet donors from Deyang, Sichuan / 中国输血杂志

【Objective】 To explore the polymorphism of HPA-1-6w, HPA-15 and 32bw-35bw in platelet donors in Deyang, Sichuan, and estimate whether to include the detection of 32bw-35bw in the platelet bank. 【Methods】 Polymerase chain reaction with sequenced based typing (PCR-SBT) was used to sequence the HPA-1-6w, HPA-15 and 32bw-35bw loci of 205 platelet donors in Deyang. Allele frequencies were calculated by the direct counting method. The frequencies of HPA-1-6 and 15 alleles in northern and southern Chinese, Japanese and Australian population were compared, and those HPA loci and HPA-32bw-35bw were searched in the Chinese Millionome Database (CMDB) and genomAD to obtain the polymorphism data. Then the Chi-square test was performed with the data of this study through GraphPad Prism 9 software. 【Results】 The allele frequencies of HPA-1b, 2b, 3b, 5b, 6bw and HPA-15b were 0.005(2/410), 0.037(15/410), 0.471(193/410), 0.020(8/410), 0.010(4/410) and 0.461(189/410), respectively, b allele of HPA-32bw-35bw and HPA-4 was not detected. Statistical significance was observed between the HPA-1b allele frequency of this study and northern Chinese, Australian population and genomAD global population sample (P< 0.05, 0.005 vs 0.014 vs 0.145 vs 0.122). The frequency of HPA-2b alleles in this study, Japanese population and genomAD global population samples was 0.037 vs 0.120 vs 0.100, with statistical difference(P<0.05). Comparison of HPA-5b and HPA-6bw allele frequencies with those of genomAD global population showed a statistical difference (P<0.05, 0.020 vs 0.089 and 0.010 vs 0.000 008, respectively). 【Conclusion】 The polymorphisms of HPA-1-6w and HPA-15 of donors in Deyang has characteristics of the southern Chinese. The frequencies of HPA-32bw-35bw were extremely low, which could be excluded from the platelet bank in Deyang.

The capacity evaluation and utilization strategy of the platelet donor database with known HLA/HPA genotype in Shaanxi / 中国输血杂志

【Objective】 To evaluate the appropriate optimal capacity and matching probability of the platelet donor database with known HLA/HPA genotype in Shaanxi aera, and provide data support for subsequent construction, maintenance and application of the local platelet donor database. 【Methods】 A total of 11 755 individuals from the Shaanxi Branch of China Marrow Donor Program, 401 and 249 unrelated random platelet donors in Shaanxi aera were enrolled to the population study of HLA-A, -B polymorphisms, HPA genotyping and CD36 antigen expression, respectively. The frequencies of HLA-A, -B alleles, HPA alleles and haplotypes were calculated with Arlequin 3. 5. 2. 2 software; matching probability and capacity evaluation of platelet donor database was conducted according to the phenotypic frequencies. 【Results】 The population genetic and phenotypic polymorphisms data of HLA-A, -B and HPA1-6, 10, 15, 21 in Shaanxi aera were obtained. The frequency of CD36 type Ⅰ or Ⅱ deficiency was 0.40%(1/249). According to the subsequent calculating and deriving, with a database size of 194 donors, the patient having approximate 95% probability could achieve matching of HPA1-6, 10, 15, 21 genotype. With a database size of 1500 donors, there is a 95% probability of matching at least one donor with HLA-A-B phenotype frequency >0.002 or haplotype frequency >0.001; meanwhile, the probability of matching a cross-reactive group donor should be 44.95%-97.57%. Based on database size of 8 856 and 15 033, the probabilities of matching HLA-A, -B phenotype were about 80% and 90%, respectively. 【Conclusion】 The differences in the distribution of HLA/HPA polymorphism in different regions make the establishment mode and optimal capacity of platelet donor database different. It is necessary to apply a variety of platelet matching transfusion strategies to expand the range of donor selection, thereby effectively reducing the database construction cost and resource requirements.

Analysis and prospect of platelet compatible transfusion / 中国输血杂志

Platelet compatible transfusion can effectively solve the immune mediated platelet transfusion refractoriness (PTR), save platelet resources and improve blood safety. This paper comments and prospects the compatibility modes of HLA, HPA and CD36, HLA antibody titer, antigen immunogenicity and the development of platelet compatible transfusion. The pattern of HLA compatible platelets involves the matching in the alleles, antigens and epitopes levels, respectively, as well as avoidance donor specificity antibody (DSA) method. While setting the mean fluorescence intensity (MFI) threshold of avoidance DSA needs to be explored when using the DSA prediction method. Allele specific HLA antibodies can be found in the patients with PTR. Therefore, the patients and donors should be genotyped for HLA-A, -B loci at high-resolution level in order to avoid allele specific HLA antibodies. The immunogenicity of various antigens or epitopes at HLA-A and -B loci are different. Selecting donor platelets with low antigen expression or low immunogenicity may be a way of HLA compatible platelets. As the probability and type of HPA antibody production are different in the various populations, the approaching of compatibility HPA involves allele matching and avoidance DSA. As to CD36, the compatibility mode mainly refers to avoidance DSA, which means blood donors with CD36 antigen type Ⅰdeficiency are preferentially selected, and then those with CD36 antigen type Ⅱ deficiency. In the future, more attention should be paid to the scale up of database capacity and update of the information construction. The time waiting for compatible platelets transfusion in clinical could be significantly shortened if the requiring and matching are only conducted within the inventory and candidate platelets.

Correlation between human platelet antigen polymorphisms and platelet parameters / 中国输血杂志

【Objective】 To investigate the correlation between human platelet antigens (HPA) polymorphisms and platelet parameters. 【Methods】 The HPA-2, HPA-3, HPA-5 and HPA-15 genotypes of 139 healthy Chinese Han individuals were detected using TaqMan-MGB probe real-time PCR, while platelet parameters including platelet count (PLT), mean platelet volume (MPV), platelet distribution width (PDW) and platelet-large cell ratio (P-LCR) were measured using hematology cell analyzer. 【Results】 The PLT was significantly lower in the individuals with HPA-2aa genotype compared to those with HPA-2ab [(234.35±50.10)×103/μL vs (269.58±41.66)×103/μL, P<0.05], while the PLT was significantly higher in individuals with HPA-5aa and HPA-15aa genotypes compared to those with HPA-5ab and HPA-15ab/bb [HPA-5: (239.36±49.81)×103/μL vs (200.29±48.02)×103/μL; HPA-15: (251.00±58.41)×103/μL vs (231.29±45.20)×103/μL, P<0.05], respectively. The MPV, PDW and P-LCR were significantly lower in individuals with HPA-5aa genotype compared to those with HPA-5ab [mpv: (10.01±0.72)fL vs (10.94±1.01)fL; PDV: (11.94%±1.35%) vs (14.25%±2.78%); P-LCR: (25.32%±5.03%) vs (31.73%±6.39%), P<0.05], but did not differ among the HPA-2 and HPA-15 genotypes. Besides, no significant differences in platelet parameters of individuals with HPA-3aa and HPA-3ab/bb genotypes were notable(P>0.05). HPA-2, -5 and -15 polymorphisms were identified as independent factors for platelet count, and HPA-5 polymorphism was an independent factor for platelet volume, revealed by multiple linear regression analysis. 【Conclusion】 HPA-2, -5 and -15 polymorphisms are correlated with platelet count, and HPA-5 polymorphism is correlated with platelet volume.

Genotyping of HPA-1-29bw in platelet donors in Nanjing, China / 中国输血杂志

【Objective】 To analyze the allele frequencies of the human platelet antigens 1-29 system (HPA-1-29bw) in Nanjing Han platelet donors, so as to provide references for compatible platelet transfusion. 【Methods】 HPA genotyping was performed by Sanger sequencing method in 900 Nanjing Han regular platelet donors who donated at Jiangsu Province Blood Center from February to September 2019. The frequencies of alleles and genotype were calculated using direct counting method. 【Results】 The HPA allele frequencies in Nanjing Han platelet donors were HPA-1a 0.9950, 1b 0.0050, 2a 0.9467, 2b 0.0533, 3a 0.5850, 3b 0.4150, 4a 0.9989, 4b 0.0011, 5a 0.9822, 5b 0.0178, 6a 0.9828, 6b 0.0172, 11a 0.9994, 11b 0.0006, 15a 0.5317, 15b 0.4683, 21a 0.9928 and 21b 0.0072, respectively. Only a allele was detected in HPA-7-10w, -12-14w, -16-20w and -22-29bw systems.The highest mismatch rate of HPA genes in 900 platelet donors was HPA-15 system, followed by HPA-3 system, with the rate of 37.40%(337/900) and 36.77%(331/900), respectively. One heterozygote was detected in HPA-11w system. 【Conclusion】 The chracteristics of HPA alleles frequencies in Nanjing Han platelet donors is that HPA-15 and HPA-3 are the most common heterozygotes, which should be paid attention to in local clinical transfusion.

Human platelet lysate efficiency, stability, and optimal heparin concentration required in culture of mammalian cells

BACKGROUND: Fetal bovine serum (FBS) has been used to support the growth and proliferation of mammalian cells for decades. Owing to several risk factors associated with FBS, several trials have been conducted to evaluate substitutes to FBS with the same efficiency and the lower risk issues.METHODS: In this study, human platelet lysate (HPL) derived from activated human platelets was evaluated as an alternative to FBS due to the associated risk factors. To evaluate the efficiency of the preparation process, platelet count was performed before and after activation. The concentrations of several growth factors and proteins were measured to investigate HPL efficiency. HPL stability was studied at regular intervals, and optimal heparin concentration required to prevent gel formation in various media was determined. The biological activity of HPL and FBS was compared by evaluating the growth performance of Vero and Hep-2 cell lines.RESULTS: Result of platelet count assay revealed the efficiency of HPL preparation process. Growth factor concentrations in HPL were significantly higher than those in FBS, while the protein content of HPL was lower than that of FBS. Stability study data showed that the prepared HPL was stable for up to 15 months at −20℃. Ideal heparin concentration to be used in different media was dependent on calcium concentration. Results of cell viability assay showed that HPL was superior to FBS in supporting the growth and proliferation of Vero and Hep-2 cells.CONCLUSION: The HPL prepared by the mechanical activation of platelets may serve as an efficient alternative to FBS in cell culture process.

Human platelet antigen-3 polymorphism as a risk factor for rheumatological manifestations in hepatitis C

Abstract INTRODUCTION: Hepatitis C virus (HCV) infection is involved in the pathogenesis of autoimmune and rheumatic disorders. Although the human platelet antigens (HPA) polymorphism are associated with HCV persistence, they have not been investigated in rheumatological manifestations (RM). This study focused on verifying associations between allele and genotype HPA and RM in patients with chronic hepatitis C. METHODS: Patients (159) with chronic hepatitis C of both genders were analyzed. RESULTS: Women showed association between HPA-3 polymorphisms and RM. CONCLUSIONS: An unprecedented strong association between rheumatological manifestations and HPA-3 polymorphism, possibly predisposing women to complications during the disease course, was observed.

Conocimientos actuales sobre la patogénesis, presentación clínica, diagnóstico y manejo de la trombocitopenia neonatal aloinmune / Current knowledge on neonatal alloimmune thrombocytopenia's pathogenesis, clinical presentation, diagnostic and management

Introducción: La trombocitopenia neonatal aloinmune es una enfermedad producida por anticuerpos maternos contra antígenos plaquetarios fetales heredados del padre. Puede ser causa de hemorragia intracraneal y conducir a la muerte o discapacidad en el feto/neonato. Aunque es la causa más grave de trombocitopenia en el neonato y la más común en los recién nacidos a término, en general ha sido poco investigada. Objetivos: Exponer los conocimientos actuales sobre la patogénesis, presentación clínica, diagnóstico y del manejo pre- y posnatal de la trombocitopenia neonatal aloinmune, Métodos: Se realizó una revisión de la literatura, en inglés y español, a través del sitio web PubMed y el motor de búsqueda Google académico de artículos publicados en los últimos 10 años. Se hizo un análisis y resumen de la bibliografía revisada. Resultados: Los anticuerpos IgG maternos son transportados a través de la placenta a la circulación fetal, opsonizan las plaquetas fetales que son removidas por fagocitosis. Los antígenos más implicados son el HPA-1a y HPA-4a. La fisiopatología de la enfermedad es muy similar a la enfermedad hemolítica perinatal, pero aún no se han implementado programas de pesquisa y el diagnóstico se realiza después del nacimiento del niño afectado de trombocitopenia, hemorragia intracraneal o muerte in útero de causa no explicada. Consideraciones finales: El impacto clínico de la trombocitopenia neonatal aloinmune y las oportunidades de tratamiento potencian la necesidad de implantar programas de pesquisa para la detección de fetos en riesgo de padecer esta enfermedad(AU)

Introduction: Neonatal alloimmune thrombocytopenia is a disease produced by maternal antibodies against fetal platelet antigens inherited from the father. It can be a cause of intracranial hemorrhage and lead to death or disability in the fetus / neonate. Although it is the most serious cause of thrombocytopenia in newborns and the most common in full-term infants, it has generally been poorly investigated. Objectives: To approximate to current knowledge about the pathogenesis, clinical presentation, diagnosis and pre- and post-natal management of neonatal alloimmune thrombocytopenia. Methods: A review of literature, in English and Spanish, through PubMed website and Google scholar search engine of articles published in the last 10 years was conducted. An analysis and summary of the reviewed bibliography was made. Results: Maternal IgG antibodies are transported through the placenta to the fetal circulation, opsonizing fetal platelets that are removed by phagocytosis. The most involved antigens are HPA-1a and HPA-4a. The pathophysiology of this disease is very similar to perinatal hemolytic disease, but research programs have not been implemented yet and diagnosis is made after birth of children affected by thrombocytopenia, intracranial hemorrhage or in uterus death by unexplained causes. Final considerations: Clinical impacts of neonatal alloimmune thrombocytopenia and treatment opportunities enhance the need to implement screening programs for the detection of fetuses at risk of suffering from this disease(AU)

Anticuerpos antiplaquetarios en pacientes cubanos en espera de trasplante renal / Platelet antibodies in cuban patients waiting for renal transplantation

Introducción: Los antígenos específicos de plaquetas, conocidos como antígenos de plaquetas humanas (HPA, del inglés human platelet antigens), se incluyen dentro del espectro de antígenos de histocompatibilidad no-HLA, debido a que los anticuerpos anti-HPA participan en el rechazo del trasplante, además de ser causa del fenómeno de refractariedad plaquetaria. Objetivo: Caracterizar los anticuerpos contra antígenos específicos de plaquetas en pacientes cubanos en espera de trasplante renal. Métodos: Se investigaron muestras de sangre de 901 pacientes mediante la técnica de inmovilización de antígenos plaquetarios con anticuerpos monoclonales. Resultados: En 78 pacientes se detectaron anticuerpos anti-HPA, que en el 87,17 por ciento reconocían los antígenos presentes en el complejo GP-IIb/IIIa. Estos anticuerpos fueron del tipo IgG en el 78,2 por ciento, IgA en el 11,53 por ciento e IgM en el 46,15 por ciento. Conclusiones: En pacientes cubanos en espera de trasplante renal son frecuentes los Ac anti-HPA, en su mayoría del tipo IgG dirigidos contra antígenos presentes en el complejo GP-IIb/IIIa(AU)

Introduction: Platelet-specific antigens, known as human platelet antigens (HPA), are included within the spectrum of non-HLA histocompatibility antigens, because HPA antibodies participate in the rejection of transplantation, besides being a cause of the phenomenon of platelet refractoriness. Objective: To characterize antibodies against platelet-specific antigens in Cuban patients awaiting kidney transplantation. Methods: The technique monoclonal antibodies immobilized platelets antigens was applied to blood samples from 901 patients. Results: HPA antibodies were detected in 78 patients, which in 87.17 percent recognized the antigens present in the GP-IIb / IIIa complex. These antibodies were in 78.2 percent of the IgG class, in 11.53 percent IgA and IgM in 46.15 percent. Conclusions: HPA antibodies, mostly of the IgG class and directed to antigens present in the GP-IIb/IIIa complex, are common in Cuban patients awaiting kidney transplantation(AU)

Allele and haplotype frequencies of human platelet and leukocyte antigens in platelet donors / Frequência alélica e haplotípica dos antígenos plaquetários e leucocitários humanos em doadores de plaquetas

ABSTRACT Objective To described the allele and haplotype frequencies of human leukocyte antigen genes at the -A, -B loci and human platelet antigen genes for human platelet antigen systems 1 to 9, 11 and 15 in blood. Methods We included 867 healthy unrelated volunteer donors who donated platelets between January 2011 and December 2014. Microarray genotyping was performed using a BeadChip microarray. Medium resolution typing of the human leukocyte antigen at loci A and B was carried out using sequence-specific oligonucleotide probe hybridization. We used multivariate analysis and our human leukocyte antigen population was compared to data from the United States national bone marrow donor program. Human platelet antigen results were compared to a literature review and data from around the world. Results Our human leukocyte antigen haplotype results were more similar to those of hispanics, followed by caucasians. Likewise, our human platelet antigen sample is more similar to those of Argentina, Rio Grande do Sul and Italy. Conclusion This was the first article that discusses human platelet antigen and human leukocyte antigen data together. Rare genotypes or antibody associations can make patient management difficult. A blood bank with genotyped donors allows for optimal transfusion and can contribute to better results. Our information can serve as basis for a database of platelet antigen polymorphisms.

RESUMO Objetivo Descrever as frequências alélicas e haplotípicas de genes dos antígenos leucocitários humanos nos loci -A,- B e dos antígenos plaquetários humanos para os sistemas HPA-1 a 9, 11 e 15. Métodos Foram incluídos 867 doadores voluntários, saudáveis, não relacionados, que doaram plaquetas por aférese entre janeiro de 2011 e dezembro de 2014. A genotipagem foi realizada usando microarray BeadChip. A tipificação de resolução intermediária dos antígenos leucocitários humanos loci A e B foi realizada por meio de hibridização com sonda para oligonucleotídeos por sequência específica. Utilizamos análises multivariadas e o antígeno leucocitário humano de nossa população foi comparado com a do programa nacional de doadores de medula óssea norte-americano. Já os resultados dos antígenos plaquetários humanos foram comparados à revisão da literatura e a dados de populações de outros países. Resultados Os resultados do haplótipo de antígenos leucocitários humanos são mais parecidos com os dos hispânicos, seguidos dos caucasianos. Igualmente, a amostra de antígenos plaquetários humanos foi mais semelhante às da Argentina, do Rio Grande do Sul e da Itália. Conclusão Este foi o primeiro artigo a discutir antígenos plaquetários e leucocitários humanos simultaneamente. Genótipos raros ou associações de anticorpos podem dificultar o manejo clínico do paciente. Um banco de sangue com doadores genotipados permite um melhor resultado e transfusão possíveis. Estas informações podem servir de base para um banco de dados sobre polimorfismos de antígenos plaquetários.

Clinical significance of human platelet antibody in the treatment of children with immune thrombocytopenia / 中华实用儿科临床杂志

Objective To investigate the expression of human platelet antibodies(HPA)in children with immune thrombocytopenia(ITP)at different ages and their clinical significance in the treatment of ITP.Methods Two hundred eighty-eight cases of children who were newly diagnosed as primary ITP and detected with HPA from January 2013 to December 2015 in the First Affiliated Hospital of Zhengzhou University were selected.According to the HPA values,they were divided into <1:10 group,1:10 group and >1:10 group.According to their age,they were di-vided into <3-year-old group and ≥3-year-old group.Data were organized and the relationship among platelet antibody,age,gender and short-term efficiency of treatment was analyzed by using SPSS 21.0 statistical analysis soft-ware.Results In this study,there were 288 cases of children,with male to female ratio of about 1.3:1.0.As to the<3-year-old group and ≥ 3-year-old group,this difference of male to female ratio was statistically significant (respectively 1.93:1.00 and 1.02:1.00,χ2=6.629,P<0.05),and the difference of the HPA positive rate was statistically significant(respectively 72.5% and 59.5%,χ2=5.716,P<0.05);the HPA positive rate of boys and girls respectively was 65.9% and 63.7%,so their diffe-rence of the HPA positive rate was not statistically significant (χ2=0.143,P>0.05).Regarding the short-term efficiency,HPA in <1: 10 group,1: 10 group and >1:10 group was respectively 89.1%,89.1%,100.0%.Statistical analysis suggests:the short-term efficiency of <1:10 group and 1:10 group was basically the same(χ2=0.000,P>0.05);In comparison of <1:10 group with >1:10 group(χ2=4.268,P<0.05),and in comparison of 1:10 group with >1:10 group(χ2=4.411,P<0.05),their differences were statistically significant.Conclusions This study suggests that boys are more susceptible to ITP,espe-cially in the <3 age group.The total positive rate of HPA is higher in <3 years old group.The HPA has a certain guiding significance for the diagnosis of ITP.Compared with the other 2 groups,HPA>1:10 group may have higher short-term efficiency in clinical practice.

Comparative cultivation of single cell derived placental mesenchymal stromal cells in vitro / 上海交通大学学报（医学版）

Objective • To explore optimal placental derived mesenchymal stem cells (PMSCs) culture medium using different combination of human platelet lysate (HPL), basic fibroblast growth factor (bFGF) and traditional fetal bovine serum (FBS) for further basic and clinical study. Methods • Single cell derived PMSCs was harvested and incubated with 4 kinds of culture, i.e, FBS, FBS+bFGF, HPL and HPL+bFGF. The morphology, growth state, cell phenotype and multi-energy differentiation were observed. Cells of P1, P2, P3 and P4 generation were counted respectively. The number of units of colony formation was analyzed by inoculating P4 cells. Results • It was identified that PMSCs had the biological properties of MSCs. Quantificationally, cell density reached (1.12×107) cells/cm2 in FBS+bFGF group and (1.24×107) cells /cm2 in HPL group (P>0.05) in P4, while those in FBS group and HPL+bFGF group were (5.58×106) cells /cm2 and (8.56×106) cells /cm2, respectively. For cell morphology, the P4 PMSCs of FBS+bFGF and HPL groups kept adherent growth, but the HPL cells could not be adherent in P5 generation. The number of colony was 51/well in FBS+bFGF group, and it was 52/well in HPL group (P>0.05) in P4. The FBS+bFGF group and the HPL group were able to maintain the characteristics of MSCs and the ability of pluripotent differentiation in the P4 generation. Conclusion • PMSCs in P4 cultured in HPL medium can keep the biological characteristics and meet the clinical transplantation requirements in quality and quantity, which are preferable for their low immunogenicity to clinical applications. In long term, PMSCs cultured in FBS+bFGF medium are preferable for the longer lasting characters of MSCs and larger quantity in basic studies. HPL+bFGF medium has no advantage on quality and quantity.

Establishment of a direct detection method for human platelet antigen1-17,Cab allele whole blood / 国际检验医学杂志

Objective To establish an acurate and convenient method to distinguish human platelet antigen(HPA) SNPs based on Target Enriched Multiplex-PCR(TEM-PCR),fluorescent probe melting curve analysis and blood direct PCR.Methods Design TEM-PCR primers and probes of HPA1-17,Cab alleles,amplify target sequences of all 18 alleles by blood direct PCR and distinguish different SNPs by melting curve of probes.Results The TEM-PCR could amplify all target sequences of 18 alleles and the melting curve analysis could distinguish those SNPs,the accuracy was equal to PCR-SSP method and the process was more convenient without blood genomic DNA extraction and subsequent gel electrophoresis thus decrease the cross-contamination risk.Conclusion Successfully established a HPA1-17,Cab alleles distinguishing method based on TEM-PCR,blood direct PCR and fluorescent probe melting curve analysis technique.

Efficacy of Human Platelet Rich Fibrin Exudate vs Fetal Bovine Serum on Proliferation and Differentiation of Dental Pulp Stem Cells

BACKGROUND AND OBJECTIVES: The imperative role of dental pulp stem cells (DPSCs) in regenerative therapy demands an in-vitro expansion which must deal with the safety and ethical problems associated with fetal bovine serum (FBS). The primary aim of this study was to compare the effects of human platelet rich fibrin (hPRF) exudate Vs FBS on proliferation and osteodifferentiation of human dental pulp stem cells (hDPSCs). The secondary one was to determine the optimum concentration of hPRF exudate inducing hDPSCs proliferation and osteodifferentiation. METHODS: The direct method was used to prepare hPRF exudate. hDPSCs were isolated from impacted mandibular third molars of twelve donors by the outgrowth method. For cell viability and proliferation rate testing, 96 well plates were used and the assay was done in duplicate and the trial repeated four times under the same conditions. Six wells were used to contain 10% FBS, serum free media, 1%, 5%, 10% and 20% concentrations of hPRF exudates, respectively. The proliferation assay was carried out by MTS tetrazolium cell proliferation assay kit and Elisa reader. The study design for osteodifferentiation protocol was exactly as the proliferation one and instead the assay was carried out by alizarin red with Elisa reader. RESULTS: Compared to 10% FBS, 10% hPRF exudate was the optimum concentration for hDPSCs proliferation, while 1% hPRF exudate was the optimum concentration for osteodifferentiation of hDPSCs. CONCLUSIONS: Avoiding the risk of zoonosis which may be occurred with FBS, it is recommended to use 10% hPRF exudate for proliferation and 1% for osteodifferentiation.

Optimization of Recombinant Human Platelet-Derived Growth Factor-BB Encapsulated in Poly (Lactic-co-Glycolic Acid) Microspheres for Applications in Wound Healing

Growth factors play multiple and critical roles in wound repair processes. Platelet-derived growth factor (PDGF) is a potent growth factor that is particularly important in the early inflammatory phase of wound healing. In order to extend the half-life of PDGF, polymeric encapsulation is used. In the current study, Poly (lactic-co-glycolic acid) (PLGA) microspheres containing recombinant human (rh) PDGF-BB were prepared to prolong the effectiveness of this growth factor. PLGA microspheres were optimized using a modified w/o/w-double-emulsion/solvent evaporation method by changing the processing conditions of stirring speed and emulsifier (polyvinyl alcohol) concentration. Microspheres prepared using the optimized method released rhPDGF-BB for up to three weeks. An in vitro migration assay showed a significant decrease in the wound area in cells treated with rhPDGF-BB microspheres compared to control cells. These findings demonstrate the potential of rhPDGF-BB encapsulated in microspheres to enhance wound healing.

Research on the clinical value between gene polymorphism of HPA 1-6,9,15 and platelet transfusion refractoriness / 中国生化药物杂志

Objective To research the clinical value between gene polymorphism of human platelet alloantigens (HPA) 1-6, 9, 15 and platelet transfusion refractoriness ( PTR) .Methods Totally 40 patients with platelet transfusion refractoriness( PTR) were randomly selected, and patients and donors’ peripheral blood specimens were collected and tested these samples with platelet GP specific antibodies, and judged the results of platelet transfusion, and with the help of the combination of PCR and direct sequencing for classification of HPA 1-6,9,15 antigens and observe the percent platelet recovery ( PPR ) after the same type of platelet transfusion, and explore the relationship between HPA gene polymorphism and PTR. Results There was no HPA-b gene was found neither on patients and donors’ HPA 1,4,9, showed the distribution of aa homozygous form; HPA 5,6 were mainly aa homozygous form, little bb homozygous form was discorvered.And HPA 2,3,15 were distributed of polymorphism, the frequency of HPA 2,3,5,6,15 were found with polymorphism.Conclusion For these patients who were happened with PRT many times, in addition to taking HLA into account, HPA gene polymorphism are also need to be considered.Most people only need to test patients and donors’ HPA2,3,15 gene to decrease the occurrence of PTR significantly when making HPA matching.