Background:
HLA Class I (HLA-CI) antigens are expressed on platelets. Immune factors are found in 10-25% of platelet refractoriness patients, with antibodies to HLA-CI the most common cause.
These patients can become highly immunised, making compatibility searches difficult because of limited number and availability of suitable donors.
HLA-CI antigen depletion from the surface of platelets using citric acid may provide an alternative product to support these highly immunised patients.
Aim:
To investigate the effect of HLA-CI antigen depletion in platelet components using flow cytometry (IFT) and ELISA assays.
Method:
One unit from a double apheresis platelet collection (n=8 pairs) was treated with citric acid solution (pH 3.0) and the other with saline (control). Components were sampled on day 5 post-collection and tested.
Platelet glycoprotein (GP) expression levels were assessed by flow cytometry. Sera containing strong and weak HLA-CI antibodies were tested by flow cytometry and ELISA. Sera containing antibodies to HPA-1a, HPA-5a and -5b was also tested.
Results:
A significant reduction in HLA-ABC was observed following citric acid treatment (Table 1). Significant reductions in GPIIb/IIIa (CD41) and GPIb/IX (CD42a) expression were also observed.
Weak and strong HLA-CI antisera reactivity showed significant reductions by IFT and ELISA. Significant ELISA reductions were not observed, nor expected, with HPA-1a, -5a and -5b antisera due to the ELISA assay ability to discriminate between HPA and HLA antibodies. IFT cannot discriminate between the two.
IFT (average MFI)
Glycoprotein expression Saline ACID p-value
CD41 (GPIIb/IIIa) 4330 ± 1527 3647 ± 1521 0.004*
CD42a (GPIb/IX) 4469 ± 2692 4174 ± 2505 0.015*
HLA-ABC 6162 ± 1259 1037 ± 447 <0.0001*
IFT (average MFI) ELISA (Ratio)
Antisera Saline ACID p-value Saline ACID p-value
HPA-1a 12.0 ± 6.8 7.0 ± 2.3 0.025* 80.3 ± 31.3 58.3 ± 12.3 0.054348448
HPA-5a 7.1± 4.3 4.8 ± 2.3 0.022* 17.9 ± 10.5 15.7 ± 9.7 0.326267511
HPA-5b 3.9 ± 1.8 3.0 ± 1.2 0.043* 5.9 ± 11.3 4.2 ± 7.4 0.259711613
HLA class I (weak) 1.9 ± 0.9 0.9 ± 0.2 0.009* 7.61 ± 5.8 2.8 ± 2.8 0.011*
HLA class I (strong) 18.4 ± 7.5 5.6 ± 1.9 0.0004* 60.2 ± 15.1 17.9 ± 9.3 0.00015*
Table 1 Platelet glycoprotein expression and antisera reactivity changes after citric acid solution treatment. Data were analysed using paired t-tests, *indicates p<0.05 was considered significant. Data represent mean ± standard deviation (n=8).
Conclusion:
Significantly reduced HLA-CI antibody reactivity was seen in both IFT and ELISA assays following citric acid treatment. Further testing of acid treated platelets against a larger set of clinical platelet refractory samples is needed to progress development of this modified blood product.