Establishment And Clinical Application Of Platelet Test Strategy In Patients Who Were Refractory To Platelet Transfusion

ObjectiveThe platelet transfusion is an important part of modern blood transfusion. With the development of clinical blood transfusion, the platelet transfusion refractory (PTR) is difficult to deal with. The most important cause of PTR is the platelet alloantibodies. How to solve PTR has become one of the hottest topics in the study of transfusion. To detect and determine the specificity, frequency, correlation factors of occurrence of platelet-reactive antibodies in patients who were refractory to platelet transfusions in Foshan area, and provide a reference for strategy of platelet transfusions. To resolve the platelet transfusion refractoriness(PTR) and establish a test strategy in Foshan, we have studied the genetic polymorphism of HLA-1(A,B) and HPA-1-5and HPA-15of donors in Foshan, and established platelet gene frequency database and HPA-typed and HLA-typed platelet donor registry,provided a basis for rapid identification of suitable donors.Methods1. Serum samples from55patients who were refractory to platelet transfusions were screened with solid-phase agglutination method for platelet-reactive antibodies. The incidence of platelet-reactive antibodies, HLA antibodies, HPA antibodies were calculated. Statistical analysis of the correlation factors of the incidence of platelet-reactive antibodies were accomplished.2. HLA class I of600and HPA-1to-15system of100regular apheresis platelet donors in Foshan were genotyped by PCR-SSP, respectively. A voluntary apheresis platelet donor registry with HLA and HPA typings were established by EXCEL.3. HLA class Ⅰ and HPA-1to-15system of30patients with PTR in Foshan were genotyped by PCR-SSP, respectively.4. The effectiveness of three different platelet transfusion strategy group, i e. HLA/HPA specific matching, PLT cross-matching, and random platelet transfusion for PTR patients were compared.5. Calculation and statistical methods5.1Calculation and statistical methods of the results of platelet alloantibodies testing(1)HLA antibody positive rate=HLA antibody positive number/the total number of specimens HPA antibody positive rate=HPA antibody positive number/the total number of specimens PLT autoantibody positive rate=PLT autoantibody positive number/the total number of specimens(2) The platelet alloantibodies positive rate of male and female patients was compared by Chi-square test, P<0.05was considered statistically significant.(3) Patients were divided into four groups by numbers of transfusion(2～3time,4～6time,7～10time,>10time),and PLT autoantibody positive rate was compared by Chi-square trend test, P<0.05was considered statistically significant. 5.2Calculation of gene frequency and genotype frequency of HLA and HPA gene frequency=gene number/total gene number genotype frequency=genotype number/total genotype number5.3Calculation of platelet transfusion effectiveness evaluation parameters CCI=[platelet count after transfusion (109/L)-platelet count before transfusion (109/L)]×body surface area (m2)/total platelet number of transfusion (1011), body surface area=0.0061×height (cm)+0.0128×weight (kg)-0.01529PPR=[platelet count after transfusion (109/L)-platelet count before transfusion (109/L) J×blood volume (L)/[total platelet number of transfusion (1011) x2/3],blood volume=69mL/kg weight(male),or65mL/kg weight(female)5.4The statistical methods to compare the strategies effects of PTR testing and transfusion(1) The identical donors percentage of the first two groups were compared by Chi-square test, P<0.05was considered statistically significant.(2)the the effective rate of platelet transfusion of three groups were compared by Chi-square test, P<0.05was considered statistically significant.(3) The average test time of the first two groups were compared by t-test, P<0.05was considered statistically significant.(4) The average cost of the first two groups were compared by t-test, P<0.05was considered statistically significant.Results1. Platelet-reactive antibodies were detected in the serum of58.2%PTR patients(32/55).The incidence of HLA antibodies was50.9%(28/55), accounting for87.5%of serum of the patients with platelete alloantibodies.The HPA alloantibodies were found in21.8%(12/55)serum, of which,66.7%(8/12) occurred together with ant-HLA. The platelet-reactive autoantibodys were found in7.3%(4/55)serum. Platelet-reactive antibodies were detected more in female(20/33)than in male (12/22)with a frequency of60.6%,54.5%, respectively, but there was no statistical significant difference (X2=0.01,P>0.05).The frequency of occurrence of platelet-reactive antibodies with the increase in the number of blood transfusion increased rapidly.The biggest increase appeared when the the number of blood transfusion was four to six times. The frequency of occurrence of platelet-reactive antibodies increased along with the number of blood transfusion(X2tramd=13.14, P<0.05).2. The results of genetic typing of HLA-1(A,B) in volunteer platelet donors were as follows.14alleles of HLA-A were checked out in600specimens of volunteer platelet donors. The common genes was A*2,A*11,A*24, their gene frequency was0.3000,0.1792,0.1708.27alleles of HLA-B were checked out and the common genes was B*40,B*15,B*13,B*46,B*51,B*58, their gene frequency was0.1592,0.1275,0.1000,0.0858,0.0692,0.0692.3. Establishment of HLA-1(A,B)-typed platelet donor registry.4. The results of genetic typing of HPA-1-5,15in volunteer platelet donors were as follows. The gene typings of100volunteer platelet donors in foshan were as follows.HPA-1aa0.9600,HPA-1ab0.0400,HPA-1bb0.0000,HPA-2aa0.8900,HPA-2ab0.1100,HPA-2bb0.0000,HPA-3aa0.3100,HPA-3ab0.5200,HPA-3bb0.1700,HP A-4aa0.9900,HPA-4ab0.0100,HPA-4bb0.0000,HPA-5aa0.9500,HPA-5ab0.0500, HPA-5bb0.0000,HPA-15aa0.2100,HPA-15ab0.5400,HPA-15bb0.2500. The common gene typing was HPA-laa,HPA-2aa,HPA-3ab,HPA-4aa,HPA-5aa, HPA-15ab and their gene typing frequency was0.9600,0.8900,0.5200,0.9900,0.9500,0.5400. Researches shows that,HPA-1,2,3,5,15in volunteer platelet donors in foshan is polymorphic, among which HPA-3and HPA-15has higher heterozygosity,genotypes of heterozygote was0.5200and0.5400. HPA-1,2,4,5is mainly a/a homozygous genotype,0.9600,0.8900,0.9900and0.9500respectively and no b/b homozygous genotype is detected. HPA-3,15found b/b homozygous genotype is detected, i.e.0.1700and0.2500.5. Establishment of HPA-1～5,15-typed platelet donor registry.6. The results of genetic typing of HLA-1(A,B) and HPA-1-5,15in PTR patients were as follows.6alleles of HLA-A were checked out. The common genes was A*2and A*11, their gene frequency was0.3167and0.2667.12alleles of HLA-B were checked out and the common genes was B*40and B*46, their gene frequency was0.2500and0.1667. The common gene typing was HPA-1aa,HPA-2aa,HPA-3ab,HPA-4aa,HPA-5aa,HPA-15ab and their gene typing frequency was0.9667,0.8667,0.5333,1.0000,0.9333,0.5667.7. Establishment standardized detecting techniques and technical methods in platelet compatibility. Standardized detecting techniques in platelet compatibility basing on HLA-1(A,B),HPA-1-5,15genetic typing and platelet autoantibodis testing to solve PTR in Foshan.8. The rate of agreement of the donors in the transfusion strategy group of HLA/HPA specific matching and PLT cross-matching was100%and80%respectively. The effective rate of transfusion in the group of HLA/HPA specific matching, PLT cross-matching, and random platelet transfusion was100%,62.5%,10%respectively. The effective rate of transfusion in the groupland group2had statistical significance(P<0.05). It also had statistical significance (P<0.05) in the groupland group2compared with the group3respectively. The average experimental time of the group of HLA/HPA specific matching and PLT cross-matching was7.5±1.6h and13.3±6.1h(P<0.05). The average cost was365±47yuan and696±343yuan (P<0.05). Conclusions1. The platelet-specific antibody in PTR patients was not rare although alloantibodies are predominantly anti-HLA. Autoantibodies were detected in a few PTR patients.The frequency of occurrence of platelet-reactive antibodies increased along with the number of blood transfusion and had not relative with the patient gender. The clinical blood transfusion need to be scientific and reasonable. To decrease the occurrence rate of PTR, the number of blood transfusion should be reduced.2. HLA class I system of regular apheresis platelet donors in Foshan were genotyped by PCR-SSP, respectively. A voluntary apheresis platelet donor registry with HLA typings were established.3. HPA-1-5,15system of regular apheresis platelet donors in Foshan were genotyped by PCR-SSP, respectively. A voluntary apheresis platelet donor registry with HPA typings were established.4. Establishment standardized detecting techniques in platelet compatibility basing on HLA-1(A,B),HPA-1-5,15genetic typing and platelet autoantibodis testing to solve PTR in Foshan.5. Compared with PLT cross-matching,it was easier to find out the suitable donors by HLA/HPA specific matching and the average experimental time was shorter, the average cost was lower,the effective rate of transfusion was higher. The transfusion effect was good. HLA/HPA specific matching was a good strategy in Foshan, which could resolve the PTR and was worth of appling in clinic transfusion.