Commercially Available Blood-Group Antibodies May Be Used to Visualize Blood Group A Antigen in Paraffin-Embedded Tissue Sections (2014)

Owing to the independent inheritance of the human leukocyte antigen (HLA) and blood group antigen (ABO) systems, it is possible that a highly HLA-matched solid organ may be ABO incompatible. ABO-incompatible transplant of some solid organs (eg, liver and kidney) is far more common than others (eg, heart).1-3 This discrepancy is due to presumed differences in organ-specific rates of hyperacute rejection, which are controversial.4 We believe that under certain circumstances, including accidental ABO-incompatible transplant or cases of investigational, high-risk, ABO-incompatible transplant (such as adult cardiac transplant), assessment of blood group antigen expression on allografts may be informative.Here, we report the adaptation of 4 preparations of commercially available anti-A to a widely available commercial staining platform. The anti-A preparations were compared to hematoxylin-eosin staining (Figure, A) and negative-control staining (Figure, B). The LA2 clone (Figure, C; Quotient Biodiagnostics, Newtown, Pennsylvania), the A003 clone (Figure, D; Bio-Rad Laboratories, Hercules, California), a blend of the MH04 and A3D3 clones (Figure, E; Ortho Clinical Diagnostics, Rochester, New York), and the Birma-1 clone (Figure, F; Immucor, Norcross, Georgia) all reliably stained A antigen on epithelium, endothelium, and erythrocytes of excised, formalin-fixed, paraffin embedded tonsil on an automated slide stainer (see Figure). To aid in the interpretations of these stains, we found that glycophorin A (Ventana Medical Systems, Inc, Tucson, Arizona) was an adequate control for erythrocyte staining; CD31 or factor VIII-related antigen (Ventana) were adequate controls for endothelial staining; and E-cadherin (Ventana) was an adequate control for epithelial staining. The intensity of A antigen staining can be adjusted to suit pathologist preference by adjusting the dilution of the primary antibody.Briefly, the A antigen staining was performed with a BenchMark Ultra automated slide stainer (Ventana). After deparaffinization, cells were pretreated with cell conditioning 1 (Ventana) for 64 minutes, and endogenous peroxidase activity was blocked. Specimens were incubated with A antibody (1:500 dilution) or a negative control for 32 minutes at 37°C. Blood group A antigen expression was detected by OptiView DAB Detection Kit (Ventana). Following the chromogenic detection, all slides were counterstained with Hematoxylin II and Bluing reagent (Ventana) for 4 minutes each before coverslips were applied.We hope that these findings are informative to investigators attempting to demonstrate A antigen expression on formalin-fixed, paraffin-embedded tissue sections. Anti-B staining should also be explored, although with fewer known subtypes of blood group B (compared to blood group A), there may be very little interindividual variation in B antigen expression on organ endothelium.5 In conclusion, as interest in ABO-incompatible organ transplant continues to increase, it may be helpful for pathology laboratories to be able to offer an assessment of blood group antigen expression on paraffin-embedded tissue sections.