Analysis of ADAMTS13 Activity in Plasma Products Using a Modified FRETS-VWF73 Assay.

Abstract

INTRODUCTION: Many cases of idiopathic thrombotic thrombocytopenic purpura (TTP) are characterized by very low levels of the metalloprotease ADAMTS13, presumably resulting in dysregulation of platelet/vWF interactions and microvascular thrombosis. Treatment for TTP begins with therapeutic plasma exchange (TPE), allowing replacement of ADAMTS13 and removal of autoantibody, when present. Fresh frozen plasma (FFP) and plasma cryoprecipitate reduced (also known as cryoprecipitate poor plasma [CPP]) have been used as replacement fluids. While current AABB standards allow thawed FFP and CPP components to be stored at 1–6°C for up to 5 days before use, the amount of ADAMTS13 activity in these products has not previously been measured nor has its stability been evaluated over the 5 days. Given the labor-intensive nature of our standard collagen-binding assay for ADAMTS13 activity, we chose to analyze ADAMTS13 activity in FFP and CPP components using a modified FRETS-VWF73 assay recently developed in our laboratory. Preliminary data indicate that the two assays correlate well.METHODS: Ten whole blood (WB) units (5 group O and 5 group A) were centrifuged within 8 hours of collection. Prior to freezing of the FFP, an aliquot of plasma was obtained from each component. The aliquots and FFP components were frozen at <-18°C. Following AABB guidelines, each FFP unit was processed into cryoprecipitated AHF and CPP components. The FFP aliquots and their related CPP components were stored frozen at <-18°C for 46 days and then thawed per AABB standards. Once thawed, aliquots and components were placed in a 1–6°C monitored refrigerator for 30 minutes. Each FFP aliquot (approximately 6 ml) was divided in half; one tube (day 0) was frozen at <-70°C, while the other was stored at 1–6°C until expiration of the product (day 5) and then frozen at <-70°C. Samples were sterilely collected from each CPP unit (mean volume 235 ml) 30 minutes after thaw (day 0), and then every 24 hours until expiration (days 1–5). Immediately after sampling, all CPP samples were stored at <-70°C until assayed. Day 0 and Day 5 FFP and CPP samples derived from the same WB units were analyzed for ADAMTS13 activity using a modified VWF73 FRET-labeled peptide assay (Kokame, 2005).RESULTS: Using the 2-sample t-test assuming equal variances, no statistical difference in ADAMTS13 activity was observed between the FFP and CPP products, nor was there a statistical difference observed between day 0 and day 5 samples (Table). Mean levels of ADAMTS13 activity were statistically similar to 20 normal donor (Precision BioLogic) samples (127 ±27 v. 125 ±24, p=NS). However, the mean activity level in group O units was statistically higher when compared to group A units (149 ±13 v. 104 ±16, p <0.00001).ADAMTS13 Activity*Plasma Type (n)Day 0Day 5pTotal FFP (10)127 ± 24122 ± 28NSFFP A (5)106 ± 12108 ± 23NSFFP O (5)148 ± 14144 ± 22NSTotal CPP (5)126 ± 29123 ± 25NSCPP A (5)102 ± 19102 ± 16NSCPP O (5)151 ± 12145 ± 11NS*Mean ± SDCONCLUSION: Our data indicate that both FFP and CPP products have similar ADAMTS13 activity, suggesting that both products should be equally effective in restoring ADAMTS13 activity in TTP patients. The data also indicate that ADAMTS13 is stable at 1–6°C, showing consistent efficacy of thawed FFP and CPP products for ADAMTS13 replacement throughout the refrigerated shelf life of the thawed product. These results reinforce previously reported data that there is a difference in ADAMTS13 activity between blood group O and A individuals.