Testing methods for platelet refractoriness due to HLA alloimmunization. (A) Solid-phase assays are increasingly used to test for anti-HLA, anti-HPA, and anticomplement antibodies. In testing for HLA antibodies, beads are coated with 1 or more HLA antigens. Patient serum is added to the beads, followed by fluorescently labeled anti–human globulin to detect bound anti-HLA antibodies. Each bead is impregnated with a different ratio of dye, which produces a unique fluorescent signature that can be identified by Luminex technology. The mean fluorescence intensity (MFI) of the antibody-bound beads is used to determine positivity. Antibody screening platforms use beads coated with more than 1 antigen, while antibody specificity testing involves the use of single antigen beads. Other techniques for HLA antibody testing include flow cytometry and enzyme-linked immunosorbent assay–based methods. Caveats to solid-phase testing include the lack of a standardized MFI cutoff for determining antibody positivity. Low-level “positive” alloantibodies may not be clinically associated with transfusion refractoriness. Despite efforts to couple solid-phase assays with functional testing, newer methods that detect C1q-binding anti-HLA antibodies have not consistently demonstrated utility in platelet refractoriness. Additionally, assays may vary in their ability to detect anti-HPA alloantibodies. (B) HLA antigens are encoded on the short arm of chromosome 6. Among the HLA class I antigens, HLA-A and HLA-B are primarily implicated in alloimmune platelet transfusion refractoriness. Low-resolution HLA typing is generally performed for platelet transfusions, although high-resolution typing is required for epitope matching. (C) Platelet crossmatching identifies donor platelets that are not reactive to patient plasma. Crossmatching may be performed using various methods, including the solid-phase red cell adherence assay (which is shown), wherein wells are coated with donor platelets available in inventory. The platelet-coated wells are then incubated with patient plasma or serum, washed, reacted with anti-IgG coated indicator red cells, centrifuged, and read visually. Compatible donor products are selected for transfusion. (D) HLA class I antigens comprise a light chain (β subunit) and a heavy chain (α subunit), which have a standard crystalline structure with regions of polymorphic amino acid sequences, termed epitopes, that are immunogenic and serve as the basis of alloimmunization. Short groups of these polymorphic amino acids on the molecular surface form eplets, which may be linear sequences or discontinuous residues that lie in close proximity in the native, 3-dimensional conformation of the antigen. Eplets may be shared across many HLA subtypes. Epitope matching uses the nonself-self paradigm, in which alloantibodies are hypothesized to be directed against unfamiliar “nonself” eplets rather than “self” eplets. Therefore, in cases where there are mismatched patient-donor HLA antigens, the degree of eplet mismatch (ie, the number of “nonself” eplets present in the donor HLA antigens) may be calculated using computer algorithms to predict the likelihood of compatibility. PCR, polymerase chain reaction.