A rapid and simple assay for the determination of ADAMTS-13 activity
M. Griffiths, H. Vetr, N. B. Binder (Vienna, Austria)
Time: 17:15 - 18:15
Objective: In recent years the determination of ADAMTS-13 activity in plasma has become an important diagnostics test in the differentiation of Thromobocytopenic Purpura (TTP) patients from those with other forms of Thrombotic micoangiopathies. The standard laboratory test for ADAMTS-13 Activity is an ELISA assay, which has an assay which requires 4 hours to process. This assay tends to be performed by specialized laboratories, so samples are usually batch tested. Therefore for a physician to receive a patient’s ADAMTS-13 activity result can range from 24 hours to as much as one week. This delay in reporting could have a negative impact on patient care.
Methods: We have developed a rapid and simple assay which would allow the physician to have an initial reportable result for ADAMTS-13 Activity within 1 hour from the blood draw. The assay is based on flow through technology, which allows for simple, rapid processing. Plasma samples are pre-incubated with a substrate which is cleaved by ADAMTS-13 present in the plasma. This pre-treated sample is applied to the single test unit which contains a membrane with immobilized antibodies specific for the cleaved substrate. As the sample flows through the membrane into an absorbent pad the cleaved substrate binds to capture antibody. A secondary biotinylated antibody is used to detect the membrane bound complex. Using a Streptavidin-gold conjugate a red colour is produced. The colour intensity is proportional to the level of ADAMTS-13 activity in the plasma sample and compared with a colour standard card, allowing semi-quantitative analysis of the patient sample. The assay can be performed at room temperature without specialized laboratory equipment and minimal training is needed to perform the test. The assay processing time is under 30 minutes. The test is designed for evaluation of a single patient sample, with a positive control run in parallel.
Results: The designed assay cut-off value of 0.1IU/mL ADAMTS-13 Activity is clinically relevant for differentiation of TTP patients. A 100% correlation was observed when TTP patient samples (n=20) were run in the TECHNOZYM ADATMS-13 ELISA and this assay.
Conclusion: This rapid and simple assay is versatile and suitable as a cost effective screening assay enabling quick turn around for ADAMTS-13 activity reporting.