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.

BACKGROUND: In the Rh blood group system, variant RhD and RhCE express several partial antigens. We investigated RH in samples with partial DIVa that demonstrated weak and variable reactivity with anti‐C. STUDY DESIGN AND METHODS: Standard hemagglutination techniques, polymerase chain reaction–based assays, and RH sequencing were used. RESULTS: DNA analysis showed that six red blood cell (RBC) samples with weak and inconsistent reactivity with anti‐C lacked RHCE*C, but all had RHD*DIVa, which encodes partial D and Goa. We then tested RBCs from 19 Go(a+) cryopreserved samples (confirmed to have RHD*DIVa) with four anti‐C and observed weak variable reactions. RHCE genotyping found all but one of the samples with RHD*DIVa also had RHCE nt 48G>C and 1025C>T, named RHCE*ceTI. Lookback of samples referred for workup and found to have either allele revealed 47 of 55 had both RHD*DIVa and RHCE*ceTI, four had RHD*DIVa without RHCE*ceTI, and four had RHCE*ceTI without RHD*DIVa. Alloanti‐c was found in a patient with c+ RBCs and RHCE*ceTI in trans to RHCE*Ce, and alloanti‐e was found in a patient with e+ RBC and RHCE*ceTI in trans to RHCE*cE. RHD*DIVa in trans to RHD erroneously tested as RHD hemizygous. CONCLUSIONS: RHD*DIVa and RHCE*ceTI almost always, but not invariably, travel together. This haplotype is found in people of African ancestry and the RBCs can demonstrate aberrant reactivity with anti‐C. RHCE*ceTI encodes partial c and e antigens. We confirm that RHD zygosity assays are unreliable in samples with RHD*DIVa.

It is possible to elicit the formation of anti-Fy alloantibodies by IV transfusion in mice that lack Fy antigens. The transfusion of RBCs alone was adequate to stimulate alloantibody production in B6CBA-F1 recipient mice. The survival of transfused Fy(b)-positive RBCs is diminished in sensitized mice. This model will be useful in further studies of RBC alloimmunization.

Of the nine alleles we identified, three were novel and six were previously documented in the dbSNP. Of these six, only one allele was previously associated with Lan negativity. To date, 19 ABCB6 alleles that encode Lan- or Lan+(w) /-, or Lan+(w) phenotypes have been described.

In addition to their value as typing reagents, these MoAbs can be used to phenotype RBCs with a positive DAT without pre-test chemical modification.

The absence of the high-prevalence antigen STAR detected by the proband's antibody is likely associated with lysine at Position 47 of the Sc glycoprotein. This amino acid change is located on the extracellular portion of HERMAP, 10 residues upstream from the polymorphism associated with Sc1 and Sc2 (Gly57Arg). STAR expands the Sc blood group system to five antigens and has been assigned the ISBT Number 013005 (SC5).

The patient's DO genes have a single-nucleotide change, which leads to the absence of the high-prevalence antigen DOYA. The absence of this antigen is associated with 183Asp and silencing of Do(a) and weakening of Gy(a), Hy, and Jo(a) antigens.

RHCE*ceAG (c.254G, p.85Gly) encodes a partial phenotype and the absence of the high-prevalence antigen RH59 (CEAG). The allele was present in one in 11 African Americans and is most often in cis to a RHD deletion associated with discordant RHD zygosity. To further determine clinical significance, detection of this allele should be part of routine RHCE genotyping in this population.