Evorpacept-Induced Interference and Application of a Novel Mitigation Agent, Evo-NR, in Pretransfusion Testing.

Introduction: Evorpacept is a CD47-blocking agent currently being developed for the treatment of various cancers. Interference by evorpacept in pretransfusion compatibility testing has been reported at limited plasma concentrations. Although various mitigation strategies have been proposed, none are practical. This in vitro study assessed evorpacept-induced interference at extended concentrations and investigated the capability of a novel mitigation agent, Evo-NR. Methods: Antibody screening tests were performed on evorpacept-spiked plasma with (anti-E and anti-Jka) or without alloantibodies at evorpacept concentrations up to 2,000 µg/mL using manual gel cards and automated analyzers. Evorpacept-coated red blood cells (RBCs) (rr [ce/ce], Fy[a+b-], S-s+) were tested by direct antiglobulin testing (DAT) and antigen typing using anti-Fyb and anti-S reagents at indirect antiglobulin testing (IAT) phase. Evo-NR was used to resolve the interference in plasma and RBC samples. Flow cytometry was used to assess the mitigation effects. Results: Evorpacept-spiked plasma showed panreactive interference in antibody screening tests using manual gel cards (2+ to 3+) and automated analyzers (4+). A carryover effect was also observed in the automated analyzers. The use of a 3- to 6-fold molar excess of Evo-NR effectively resolved the interference in the plasma and enabled accurate alloantibody identification. Although the reduction in evorpacept binding to RBCs was identified via flow cytometry, Evo-NR was incapable of resolving the serologic interference observed in DAT and antigen typing at IAT phase. Discussion: Evorpacept showed constant panreactivity and a carryover effect at high concentrations. Evo-NR successfully resolved the interference in the plasma samples and could be considered a practical and efficient mitigation solution. Implementation of Evo-NR has the potential to support RBC transfusion for patients undergoing evorpacept treatment.

Prevalence and molecular basis of null blood group phenotypes in the Korean population: Analysis using a public database.

BACKGROUND: Null phenotypes are characterized by complete absence of all antigens within a blood group system and caused by null variants (e.g., nonsense, frameshift, initiation codon, and canonical splice site variants) in the genes encoding the antigens. Knowing the prevalence and molecular basis of null phenotypes is essential to establish a rare donor program, and the aim of this study was to reveal the prevalence and molecular basis of null phenotypes using the Korean Reference Genome Database (KRGDB) containing whole-genome sequences of 1722 Korean individuals. STUDY DESIGN AND METHODS: Population allele frequencies of null alleles in 39 blood group systems except ABO, MNS, Rh, Lewis, and FORS were obtained from the KRGDB. The prevalence of null phenotypes was calculated using Hardy-Weinberg equation. RESULTS: The prevalence of null phenotypes were estimated to be less than 0.001% in all blood group systems except JR and SID. The prevalence of the Jr(a-) and Sd(a-) phenotypes were estimated to be 0.0453% and 0.2323%, respectively. The most frequent null allele of the JR system was ABCG2*01N.01, accounting for approximately 85% of null alleles. DISCUSSION: Our approach using a public database allowed us to investigate the prevalence and molecular basis of null phenotypes in the Korean population, which will serve as a guide for establishing a rare donor program in Korea. Considering the clinical significance, Jr(a-) is an important null phenotype that should be typed in the Korean population, and molecular assays targeting the most frequent allele ABCG2*01N.01 may be useful in detecting this phenotype.

Fatal Prototheca zopfii Algaemia in a Patient with Acute Lymphoblastic Leukemia: a Case Report.

BACKGROUND: Prototheca algaemia is a rare but life-threatening disease that occurs primarily in immunocompromised patients. We report a fatal case of Prototheca zopfii bloodstream infection in a 54-year-old woman receiving chemotherapy for relapsed acute lymphoblastic leukemia. METHODS: The isolate was identified using an automated biochemical identification system (VITEK 2; bioMerieux) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (VITEK MS; bioMerieux). Partial 18S and 28S rDNA sequencing was performed for definitive identification and genotyping. RESULTS: The patient had persistent neutropenic fever, and isolates from blood culture were identified as P. zopfii. Sequencing was performed and the isolate was confirmed to be P. zopfii genotype 2, which was newly named as P. bovis. The patient was treated with liposomal amphotericin B but died of septic shock. CONCLUSIONS: Prototheca spp. should be considered an emerging pathogen, especially in immunocompromised patients, due to its ubiquitous nature.

Assessing and mitigating the interference of ALX148, a novel CD47 blocking agent, in pretransfusion compatibility testing.

ALX148, a novel CD47 blocking agent, is in clinical development for the treatment of advanced solid tumors and lymphoma. Because CD47 is highly expressed on red blood cells (RBCs), its therapeutic blockade can potentially interfere with pretransfusion compatibility testing. This study describes the interference of ALX148 in pretransfusion compatibility testing and evaluates the methods used for mitigating such interference. STUDY DESIGN AND METHODS: Routine serologic tests were performed on six samples from four patients treated with ALX148. Antibody screening tests were performed on ALX148-spiked plasma, and RBC testing including antigen typing was performed on ALX148-coated RBCs. Soluble CD47 or high-affinity signal regulatory protein α (SIRPα) monomers were used to remove the false-positive reactivity of ALX148-spiked plasma with or without anti-E. RESULTS: ALX148 caused false-positive reactivity in antibody screening using indirect antiglobulin testing (IAT) and two-stage papain testing. However, false-positive reactivity was not observed at the immediate spin (IS), room temperature (RT), and 37°C phases. Direct antiglobulin testing, autologous controls, and eluates showed positive results. ALX148 did not affect blood group antigen typing performed at the IS or RT phases. The use of 50- to 100-fold molar excess of soluble CD47 or 300-fold molar excess of high-affinity SIRPα monomers removed false-positive reactivity in IAT without affecting anti-E detection. CONCLUSION: ALX148 generates false-positive reactivity in IAT, interfering with pretransfusion compatibility testing. The use of soluble CD47 or high-affinity SIRPα monomers can resolve the interference without possibly missing clinically significant alloantibodies.

Elevated extracellular trap formation and contact system activation in acute leukemia.

Leukemic cells release their nuclear contents into the extracellular space upon activation. The released nuclear contents, called extracellular traps, can activate the contact system of coagulation. This study accessed the extent of contact system activation, the levels of extracellular traps, and coagulation activation in hematologic malignancies including acute leukemia. In 154 patients with hematologic malignancies (acute leukemia, n = 29; myelodysplastic syndrome, n = 20; myeloproliferative neoplasms, n = 69; plasma cell myeloma, n = 36) and 48 normal controls, the levels of coagulation factors (fibrinogen and factor VII, VIII, IX, and XII), D-dimer, thrombin generation, extracellular trap markers (histone-DNA complex, cell-free dsDNA, leukocyte elastase), and contact system markers (activated factor XII [XIIa], high-molecular-weight kininogen, prekallikrein, bradykinin) were measured. Patients with acute leukemia showed the highest levels of peak thrombin, extracellular trap markers, and factor XIIa. Factor XIIa level was significantly associated with the presence of acute leukemia. The histone-DNA complex and cell-free dsDNA were revealed as significant associated factors with the factor XIIa level. Three markers of extracellular traps and two markers of thrombin generation significantly contributed to the hemostatic abnormalities in hematologic malignancies. Contact system was activated in acute leukemia and its activation was significantly associated with the extent of extracellular trap formation. This finding suggests that extracellular traps might be a major source of contact system activation and therapeutic strategies targeting extracellular trap formation or contact system activation may be beneficial in acute leukemia.