Study On The Effectiveness Of Citric Acid Stripping HLA-I Antigens Off The Platelets

Objective: The reason of platelet transfusion refractoriness(PTR)is partly due to that the patient's serum contains antibodies against human leukocyte antigen(HLA)on the surface of platelet(PLT).Our study was to investigate whether citric acid can effectively elute HLA off the PLT surface,and verify the influence of the treatment on PLT morphology,routine indices,aggregation function,and confirme the effectiveness and feasibility of acid-treatment so as to provide reference for further formulation of emergency PLT transfusion strategy for PTR patients with HLA antibodies.Methods: Firstly,optimize the laboratory conditions: The PLTs were reacted with citric acid at pH 3,4 and 5 respectively.The expression rate of HLA-I antigens on the treated-PLTs was examined.The optimum pH value for stripping the HLA-? antigens off the PLTs was selected according to the minimum expression rate of HLA-I antigens;according to the principle of factorial design,the experimental condition(reaction time and volume ratio)was optimized.Then the PLTs were divided into different groups for experiments: The acid-treated group was incubated with citric acid;PBS-treated group was incubated with PBS;in the untreated group,platelet poor plasma(PPP)was used to regulate PLT concentration,which was consistent with that in the acid-treated group and untreated group.Finally,the three groups of PLTs were analyzed as follows: The expression of HLA-I and CD62 P antigens on PLT surface was analyzed by flow cytometry,PLT morphology was observed under microscope,PLT routine indices were examined by blood cell analyzer,PLT biochemical indices were evaluated by biochemical analyzer,and the hypotonic shock response(HSR)of the PLTs was assessed by spectrophotometer,PLT aggregation function was detected by TEG analyzer.Results: 1.The expression rates of HLA-I antigen on PLTs treated with citric acid at pH 3,4 and 5 were 51.21±2.02%,79.04±2.66% and 80.91±2.31% respectively.2.The most suitable condition for acid elution was pH 3,while the volume ratio of acid and PLTs was 5:1 and reaction time was 5 min.3.Citric acid can effectively remove up to 80% of HLA-I antigen,the expression of PLT activation marker CD62 P increased from 4.17±3.54% to 8.89±5.70%(P<0.05).4.MPV and PDW in acid-treated PLTs increased from 9.05± 0.14 fL,8.53±0.28 fL to 11.54±0.81 fL,11.08±0.68 fL respectively(P<0.05).5.There was no obvious aggregation under microscope.6.HSR at acid-treated,PBS-treated and untreated groups was 51.65±6.56%?54.95±6.32%?62.05±8.14% respectively.7.The metabolism of acid-treated PLT was slightly enhanced.8.TEG showed that PLT aggregation was slightly decreased both in acid-treated and PBS-treated groups.Conclusion:Citric acid at pH 3 can effectively strip HLA-I antigens off the surface of PLTs,which caused a little damage to PLT structure and function.The advantages and disadvantages of this method should be fully weighed before amplified clinically,and it could be considered as one of the emergency transfusion strategies for PTR patients with anti-HLA antibodies,in the case of emergency transfusion.