A novel high-prevalence antigen in the Lutheran system, LUGA (LU24), and an updated, full-length 3D BCAM model.

BACKGROUND: The basal cell adhesion molecule (BCAM) carries the antigens of the Lutheran (LU, ISBT005) system. We report a novel Lutheran antigen and propose an updated, full-length 3D model of BCAM. STUDY DESIGN AND METHODS: Red blood cell testing, antibody identification, and BCAM genomic DNA sequencing were done by standard methods. Multi-template homology modeling of BCAM used structural templates selected for coverage, highest sequence identity, and protein domain family. All variants causing the loss or gain of a Lutheran antigen were analyzed for residue accessibility and intraprotein interactions. RESULTS: An antibody to a high-prevalence antigen in the plasma of a pregnant woman was determined to be directed at a novel Lutheran antigen. Sequencing of BCAM found three homozygous changes: c.212G > A (p.Arg71His) and two silent, c.711C > T and c.714C > T. The model was built from the first two immunoglobulin crystallized domains of BCAM (D1, D2), three other templates (for D3, D4 and D5 with a higher sequence identity with the target than those used for the model proposed by Burton and Brady in 2008, and for the transmembrane region) and RaptorX (for the intracellular domain). All residues associated with a Lutheran antigen were found to be exposed in wild-type or variant proteins, except p.447 associated with loss of Lu13 expression. CONCLUSION: The c.212G > A change results in the loss of LUGA (LU24) antigen. Whole genome sequencing continues to reveal polymorphisms with uncertain immunogenicity. This model and demonstration that nearly all residues associated with the expression of a Lutheran antigen are exposed will help evaluate the significance of new polymorphisms.

Monoclonal anti-CD47 interference in red cell and platelet testing.

BACKGROUND: Anti-CD47 (Hu5F9-G4) is a human monoclonal immunoglobulin G (IgG)4 antibody that is in clinical trials to treat hematologic or solid malignancies. CD47, a glycoprotein expressed on all cells, binds to signal-regulatory protein α on macrophages and regulates phagocytosis. Blocking CD47 is thought to enhance phagocytosis and promote antitumor responses. Here, we evaluate drug interference in pretransfusion testing, determine mitigation strategies, and compare interference with anti-CD38 (Daratumumab). STUDY DESIGN AND METHODS: Samples from four patients were tested by standard methods. Anti-IgG (Immucor monoclonal Gamma-clone and Ortho BioClone) were used, and dithiothreitol and enzyme-treated RBCs were tested. Allo-adsorption was performed with papain treated RBCs, pooled platelets, or with commercial human platelet concentrate. Platelet antibody testing was performed according to manufacturer's instructions. RESULTS: All plasma samples reacted 3+ to 4+ in all phases with all red blood cells (RBCs) by all methods including immediate spin. Stronger reactivity was observed with D- RBCs with titers as high as 16,384 at indirect antiglobulin testing. Reactivity at indirect antiglobulin testing using Gamma-clone anti-IgG (which does not detect IgG4) was only weakly positive and confirmed to be carryover agglutination. Plasma reacted with dithiothreitol, trypsin, papain, α-chymotrypsin, or warm autoantibody removal medium (W.A.R.M., Immucor) treated RBCs. Direct antiglobulin testing and autocontrol were negative or weak with 3+ reactive eluates. Reactivity was removed by multiple alloadsorptions with papain-treated cells or pooled platelets. Polyethylene glycol adsorption was invalid due to precipitation of antibody. CONCLUSION: Anti-CD47 (Hu5F9-G4) interferes with all phases of pretransfusion testing, including ABO reverse typing. To remove interference requires multiple RBC alloadsorptions and/or the use of monoclonal Gamma-clone anti-IgG in the indirect antiglobulin testing.