Production of recombinant humanized monoclonal anti-human neutrophil antigen (HNA) antibodies with potential applicability as standard antibodies.

BACKGROUND: Antibodies against human neutrophil antigen (HNA) are involved in the pathogenesis of neonatal alloimmune neutropenia, autoimmune neutropenia, and transfusion-related acute lung injury. The present methods for anti-HNA antibody identification strongly depend on the presence of standard antisera with known allo/isospecificities. Here, we aimed to produce recombinant humanized antibodies to HNA from available mouse monoclonal antibodies (MoAbs). STUDY DESIGN AND METHODS: RNAs were extracted from available hybridoma cells producing mouse anti-HNA antibodies recognizing HNA-1a (TAG-1), -1b (TAG-2), -2 (TAG-4), and FcγRIIIb, and the cDNA was synthesized. Recombinant fragments consisting of the variable regions of the H and L chains of the mouse MoAb ligated to the constant region of human IgG were incorporated into an expression vector and transfected into CHO cells. Antibody specificity of the selected humanized monoclonal antibodies was confirmed, and tested by the participants of the ISBT Granulocyte Immunobiology Working Party (GIWP) workshop 2020. RESULTS: GIFT results confirmed the specific reactivity of TAGH-1 to -4, except for a cross-reactivity of TAGH-2 with HNA-1a/a neutrophils, only in flow-cytometry. MAIGA results showed clear specificity of all humanized antibodies, but the selection of the appropriate capture monoclonal antibody was essential for the test. The results of the ISBT GIWP showed high concordance among the labs. CONCLUSIONS: These are the first humanized monoclonal antibodies to HNA-1 and HNA-2 antigens produced and they will be important standard reagents for laboratories testing for neutrophil antibodies. We plan to have these humanized MoAbs available through WHO.

Risk factors for T-cell lymphopenia in frequent platelet donors: The BEST collaborative study.

BACKGROUND: Severe T-cell lymphopenia of uncertain clinical significance has been observed in frequent apheresis platelet donors. Two commonly used plateletpheresis instruments are the Trima Accel, which uses a leukoreduction system (LRS) chamber to trap leukocytes and the Fenwal Amicus, which does not use an LRS chamber. STUDY DESIGN AND METHODS: We performed an international, multicenter, observational study comparing T-cell populations in frequent platelet donors collected exclusively using the Trima instrument (n = 131) or the Amicus instrument (n = 77). Age- and sex-matched whole blood donors (n = 126) served as controls. RESULTS: CD4+ T-cell counts <200 cells/µL were found in 9.9% of frequent Trima (LRS+) platelet donors, 4.4% of frequent Amicus (LRS-) platelet donors, and 0 whole blood donors (p < .0001). CD4+ T-cell counts <200 cells/µL were only seen in platelet donors with ≥200 lifetime donations. In multivariable analysis, age, lifetime donations, and instrument (Trima vs. Amicus) were independent risk factors for lymphopenia. In 40 Trima platelet donors, a plasma rinseback procedure was routinely performed following platelet collections. No Trima platelet donors receiving plasma rinseback had a CD4+ T-cell count <200 cells/µL versus 13/91 Trima platelet donors not receiving plasma rinseback (p = .01). DISCUSSION: Recurrent bulk lymphocyte removal appears to contribute to the development of T-cell lymphopenia in frequent, long-term platelet donors. Lymphopenia is more common when an LRS chamber is used during platelet collection but can occur without an LRS chamber. Blood centers using LRS chambers can mitigate donor lymphopenia by performing plasma rinseback.

The utility of a monocyte monolayer assay in the assessment of intravenous immunoglobulin-associated hemolysis.

BACKGROUND: Hemolysis following the administration of intravenous immunoglobulin (IVIG) is an important adverse event (AE). While the monocyte monolayer assay (MMA) has been used to predict in vivo hemolysis when serologically incompatible blood may be transfused, it has also been shown to correlate with IVIG-associated hemolysis. In this study, the MMA was examined for its utility in assessing the risk of hemolysis after IVIG. STUDY DESIGN AND METHODS: Forty-two non-blood group O patients receiving high-dose IVIG (≥2 g/kg) were examined using an autologous and allogeneic MMA. Hemolysis was defined by a drop in hemoglobin of ≥1 g/L, a positive direct antiglobulin test (DAT) and eluate, and a decrease in haptoglobin or increase in lactate dehydrogenase and/or reticulocytes. RESULTS: Forty-two patients provided 50 assessable postinfusion samples, with hemolysis observed in 20 (40%) of cases. Autologous MMA using post-IVIG red blood cells significantly correlated with clinical outcomes when compared to allogeneic MMA (P = .0320 vs .5806, t test). No significant difference in receiver operating characteristics was observed when comparing autologous MMA testing against DAT for the diagnosis of IVIG-associated hemolysis. However, when using samples collected 5 to 10 days after receipt of high-dose IVIG, the autologous MMA had higher sensitivity than the DAT. CONCLUSION: MMA testing with autologous monocytes collected 5 to 10 days after receipt of high-dose IVIG can be used for the diagnosis of IVIG-associated hemolysis and may be of particular value in cases in which the Day 5 to 10 DAT is negative.