Comparison and stability of ADAMTS13 activity in therapeutic plasma products.

BACKGROUND: The von Willebrand factor (VWF)-cleaving protease, ADAMTS13, is often deficient in cases of thrombotic thrombocytopenic purpura (TTP). The primary treatment of TTP is therapeutic plasma exchange (TPE) utilizing a variety of plasma products that help restore ADAMTS13 activity. However, multiple replacement products are available to choose from. Thawed plasma products have a variable refrigerated shelf life depending on the product type; stability of ADAMTS13 in thawed products stored at 1 to 6 degrees C has not been determined. STUDY DESIGN AND METHODS: ADAMTS13 activity was measured in three types of plasma products and cryoprecipitate. Fresh-frozen plasma (FFP) aliquots and cryoprecipitate-poor plasma (CPP) products were produced from 10 whole-blood (WB) donations. Twenty-four-hour plasma products were manufactured from 10 additional WB donations. ADAMTS13 activity in these products at time of thaw and after 5 days of storage at 1 to 6 degrees C was measured with a modified version of the FRETS-VWF73 fluorogenic assay. ADAMTS13 activity at time of thaw was measured in 10 units of cryoprecipitate and five related CPP products. RESULTS: ADAMTS13 is present in similar amounts in FFP, CPP, and 24-hour plasma products. Storage at 1 to 6 degrees C for up to 5 days did not significantly diminish ADAMTS13 activity. The concentration of ADAMTS13 in cryoprecipitate was significantly higher than that observed in plasma products. CONCLUSION: FFP, CPP, and 24-hour plasma products should be equally effective for ADAMTS13 restoration through TPE and should remain so for the duration of the shelf life of the thawed products.

Multiplex genotyping of human minor histocompatibility antigens.

Minor histocompatibility antigens (mHAg) induce major histocompatibility complex-restricted, T cell-mediated immune responses that may contribute to increased risk of graft-versus-host disease and graft-versus-leukemia effects. Unlike human leukocyte antigen genes, mHAg are encoded by genetically and functionally unrelated genes located throughout the chromosome. The role of mHAg in stem cell transplantation and the population frequencies of mHAg alleles remain unknown due in part to the lack of suitable high throughput methods for genotyping these diverse genes. Here we describe the development and utility of a multiplexed Luminex assay for genotyping human mHAg, including HA-1, HA-2, HA-3, HA-8, HB-1, CD31(125), and CD31(563). The assay uses a multiplexed, allele-independent, gated amplification of mHAg genes followed by differential detection of allele-specific primer extension products using the MultiCode PLx system (EraGen Biosciences, Madison, WI). The alleles are interrogated using a multiplex allele-specific primer extension reaction using primers tagged with EraCodes. The products are hybridized to Luminex beads and the hybridization duplexes are detected using streptavidin-phycoerythrin. The assay resolved the mHAg genotypes of 259 Caucasian donors and provided population estimates of mHAg gene and phenotypic frequencies. All mHAg alleles evaluated in this study exhibited Hardy-Weinberg equilibrium, although some mHAg phenotypes were present in large majority of individuals tested (HA-2, HB-1). This assay will provide a valuable tool for determining mHAg frequencies in other ethnic populations, as well as for establishing the clinical importance of mHAg disparities in stem cell transplantation.