In this issue of Blood, Zhang et al expand on a previous study that described the detection of antibodies against human platelet alloantigens by using gene-edited stem cell–derived target cells.1 

Antibodies against human platelet alloantigens (HPAs) present on membrane glycoproteins represent a major health problem because they are capable of generating an immune response against platelets that can, in turn, cause a variety of bleeding disorders.2,3  In addition to genotyping, phenotypic detection of anti-HPA alloantibodies is important for the proper diagnosis, treatment, and/or prevention of diseases such as fetal and neonatal alloimmune thrombocytopenia, posttransfusion purpura, and platelet transfusion refractoriness, which can have severe clinical outcomes. Currently, platelet alloantigen phenotyping is performed by detecting antibodies against whole platelets as a source of antigens or by monoclonal antibody-based antigen capture assays, each of which have problems with specificity and practicality.4 

Most HPAs are the result of single nucleotide polymorphisms that have been described on 6 platelet glycoproteins. To date, 6 biallelic HPA isoforms (HPA-1, -2, -3, -4, -5, and -15) and 26 single low-frequency antigens have been identified. Detection of alloantibodies against HPA-3 and HPA-9, which both reside on the C terminus of GPIIb, has proved particularly challenging. Despite being commonly associated with neonatal alloimmune thrombocytopenia, antibodies against these antigens are relatively rare. Furthermore, previous studies have reported difficulties with the specificity of alloantibody detection using standard practices because of epitope complexity.5-8  For example, the HPA-3 epitope is sensitive to 3-dimensional changes in GPIIb structure, and its recognition depends on glycosylation of nearby residues, which can be lost during platelet storage.

Detection of anti-HPA alloantibodies is further complicated by the fact that antibodies against class I HLA using whole platelet detection can interfere with alloantibody binding to HPA. More specific monoclonal antibody-based antigen capture techniques that can detect antibodies against glyco-modifications on alloantigens are also problematic because they are performed only by select laboratories and can be technically challenging to perform.

Because of these challenges, there is a significant need for alternative, practical methods that can detect evasive alloantibodies with high specificity and sensitivity. A previous article by Zhang et al9  used CRISPR/Cas9 technology in human induced pluripotent stem cells (iPSCs) to engineer megakaryocyte (MK) progenitors to express low-frequency platelet-specific alloantigens. That proof-of-concept study introduced the possibility of using gene editing of stem cells to express clinically relevant antigens for the detection of specific alloantibodies with 1-step flow cytometric analysis, which uses relatively small amounts of patient sample and is less cumbersome to perform than other types of analyses. However, that study was limited to expressing HPA-1 and did not directly address the issue of class I HLA antibody interference.

In the Zhang et al article in this issue, the authors expand on their original study by engineering iPSC-derived MKs to lack class I HLA antigen expression. These cells were also modified to be type O to avoid blood type incompatibility. HLA-negative, type O founder iPSCs were further edited to express HPA-3 or HPA-9 polymorphisms using the established CRISPR/Cas9 methodology. Importantly, the resulting designer cells were capable of detecting alloantibodies present in patient serum with high specificity and, in some cases, they demonstrated improved sensitivity compared with antigen capture methods. The clinical potential of this method was further demonstrated by showing that anti-HPA-9b alloantibodies in patient sera from unresolved neonatal alloimmune thrombocytopenia cases could be detected.

The authors envision using gene-edited iPSCs for alloantibody detection against additional disease-associated HPAs. Their approach provides clear advantages over existing methods by enabling the expression of particular alloantigens in a whole-cell context without class I HLA interference for highly specific flow cytometric antibody detection. Reports on the utility of this new technology in the clinical setting will surely be eagerly anticipated. Thus, this new study by Zhang et al represents another promising stride toward improving platelet alloantibody detection.

Conflict of interest statement: J.E.I. has financial interest in and is a founder of Platelet BioGenesis, a company that aims to produce donor-independent human platelets from human-induced pluripotent stem cells at scale. He is an inventor on this patent. The interests of J.E.I. were reviewed and are managed by the Brigham and Women's Hospital and Partners HealthCare in accordance with their conflict-of-interest policies.

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