Development and Performance of the GTI ADAMTS-13 Activity Assay.

Thrombotic Thrombocytopenic Purpura (TTP) is a disease characterized by the sudden onset of a classical pentad of symptoms: thrombocytopenia, fever, renal insufficiency, neurologic deficit, and microangiopathic hemolytic anemia. It was recently discovered that ADAMTS-13 is the protease responsible for cleaving von Willebrand Factor; deficiency of ADAMTS-13 activity has been demonstrated in the plasma of TTP patients. The lack of ADAMTS-13 activity results in the accumulation of multimers of vWF in the plasma and ultimately intravascular platelet aggregation resulting in the clinical symptoms associated with TTP. Currently there are no commercially available diagnostic assays for TTP. Several laboratories have developed “in house” assays that measure the activity of the ADAMTS-13 protease. However, these assays are cumbersome, complex, difficult to perform, and there is a long turn around time, as they require long incubations. The results are difficult to interpret and are not easily quantitated. In addition, there is no standardization of results from laboratory to laboratory, mainly because the assays use various technologies. Determining the extent to which the ADAMTS-13 enzyme activity is decreased is believed to be important in distinguishing a patient with TTP from those presenting with similar clinical symptoms. Until ADAMTS-13 assays are more quantitative, there is not a good method for studying patients with severe decreases in ADAMTS-13 levels versus those who have some intermediate but less than normal level of enzyme activity. Furthermore, the lack of standardized results makes the assays less useful in monitoring the effectiveness of treatment of the TTP patients. GTI has developed a rapid, quantitative assay for ADAMTS-13 activity that is based on the cleavage of a substrate which consists of a peptide fragment of von Willebrand Factor and subsequent fluorescent detection. An initial study using plasma (obtained from the Blood Center of Wisconsin) from 5 TTP patients demonstrated good correlation between the GTI assay and the collagen binding assay, thus demonstrating feasibility of the assay. A more extensive evaluation using 40 normal samples and 55 samples from suspected TTP patients was initiated, the results of which will be reported. Assay sensitivity/limit of detection (based on dilution studies) was 3% normal ADAMTS-13 activity. Assay precision using 3 samples (mean values of 96%, 55%, and 9% normal ADAMTS-13 activity) run in duplicate over 8 days was evaluated. Assay imprecision was less than 10% cv for all samples tested.