Rh discrepancies caused by variable reactivity of partial and weak D types with different serologic techniques

RhD discrepancies between current and historical results are problematic to resolve. The investigation of 10 discrepancies is reported here. STUDY DESIGN: Samples identified were those that reacted by automated gel technology and were negative with an FDA-approved reagent. Reactivity with a commercially available panel of monoclonal anti-D was performed. Genomic DNA was evaluated for RHD alleles with multiplex RHD exon polymerase chain reaction (PCR), weak D PCR-restriction fragment length polymorphism, and RHD exon 5 and 7 sequence analyses. RESULTS: The monoclonal anti-D panel identified two samples as DVa, yet possessed the DAR allele. Two weak D Type 1 samples had a similar monoclonal anti-D profile, but only one reacted directly with one of two FDA-approved anti-D. Only two of four weak D Type 2 samples reacted directly with one FDA-approved anti-D, and their D epitope profile differed. CONCLUSIONS: The monoclonal anti-D reagents did not distinguish between partial and weak D Types 1 and 2. Weak D Types 1 and 2 do not show consistent reactivity with FDA-approved reagents and technology. To limit anti-D alloimmunization, it is recommended that samples yielding an immediate-spin tube test cutoff score of not more than 5 (i.e., ≤1+ agglutination) or a score of not more than 8 (i.e., ≤2+ hemagglutination) by gel technology be considered D– for transfusion and Rh immune globulin prophylaxis. That tube test anti-D reagents react poorly with some Weak D Types 1 and 2 red cells is problematic, inasmuch as they should be considered D+ for transfusion and prenatal care. Molecular tests that distinguish common partial and Weak D types provide the solution to resolving D antigen discrepancies.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75591/1/j.1537-2995.2007.01551.x.pd

Synergistic complement lysis by monoclonal antibodies to the human leukocyte common antigen requires both the classical and alternative pathways.

In a previous study we isolated a series of rat monoclonal antibodies to the human leukocyte common (LC) antigen and demonstrated that synergistic complement lysis was possible between IgG2b antibodies which recognised different epitopes. In this report we have examined the mechanisms that were involved in synergistic lysis. We found that the number of C1q binding sites increased from 30,000 to 40,000/cell using a single antibody to 90,000/cell when two IgG2b antibodies to different epitopes were used together. The affinity of C1q binding also increased approx. 3-fold for the synergistic pair, and there was a similar increase in the rate of C1 activation. Combinations of an IgG2b with IgG1 or IgG2a gave much smaller increases in the amount of C1q bound and in the rate of C1 activation. Despite the large number of C1q molecules bound with the optimal synergistic pair and the increased rate of C1 activation, lysis was inefficient in serum depleted of Factor D, suggesting a requirement for the alternative pathway. This is the first demonstration of the need for the alternative pathway in complement lysis by monoclonal antibodies

Synergistic complement lysis by monoclonal antibodies to the human leukocyte common antigen requires both the classical and alternative pathways.

In a previous study we isolated a series of rat monoclonal antibodies to the human leukocyte common (LC) antigen and demonstrated that synergistic complement lysis was possible between IgG2b antibodies which recognised different epitopes. In this report we have examined the mechanisms that were involved in synergistic lysis. We found that the number of C1q binding sites increased from 30,000 to 40,000/cell using a single antibody to 90,000/cell when two IgG2b antibodies to different epitopes were used together. The affinity of C1q binding also increased approx. 3-fold for the synergistic pair, and there was a similar increase in the rate of C1 activation. Combinations of an IgG2b with IgG1 or IgG2a gave much smaller increases in the amount of C1q bound and in the rate of C1 activation. Despite the large number of C1q molecules bound with the optimal synergistic pair and the increased rate of C1 activation, lysis was inefficient in serum depleted of Factor D, suggesting a requirement for the alternative pathway. This is the first demonstration of the need for the alternative pathway in complement lysis by monoclonal antibodies