PB1213 - The Development of a Mini-scale Euglobulin Clot Lysis Assay

Background: Fibrinolysis protects the vasculature from excessive thrombus formation, with alterations contributing to abnormal thrombosis or bleeding. The original euglobulin clot lysis test, first described in 1948, is a fibrinolytic test designed to assess the time to fibrin clot lysis. An automated euglobulin clot lysis assay (ECLA) (Smith et al., 2003), utilizes spectrophotometric readings of absorbance to determine lysis times and is available clinically in selected specialized coagulation laboratories as a screening/diagnostic test for hyper- and hypo-fibrinolysis. However, the ECLA has limited clinical application due to significant manual sample processing, low throughput, and high variability.

Aims: We describe a modified ECLA (the "mini-ECLA") based on 85% less plasma consumption, increased automation, and higher throughput using a 96-well microplate.

Methods: The method involves a series of acidification, centrifugation, washing, clot stimulation, and absorbance reading as shown in Figure 1. The mini-ECLA clot lysis time is calculated as the time the absorbance reaches the median OD value (50% clot lysis), minus the time at max OD reading (full clot formation), with the clot lysis ratio determined by dividing the clot lysis time of the sample by the control plasma.

Results: Clot lysis ratios were determined in a representative sample of commercial control and deficient plasmas, as follows: Factor Assay Control Plasma (FACT) [George King Biomedical, 0020-0], 1.0 ± 0.09 (n=21), with an inter-assay CV=9% and an intra-assay CV=7%, plasminogen deficient [Affinity Biologicals, PG-DP], >25 ± N/A (n=9), alpha-2-antiplasmin deficient [Affinity Biologicals, A2AP-DP], 0.58 ± 0.09, (n=10), and plasminogen activator inhibitor-1 (PAI-1) deficient [Affinity Biologicals, PAI-DP], 0.57 ± 0.07, (n=16).

Conclusion(s): The mini-ECLA shows analytic sensitivity to hypo- and hyper-fibrinolytic states while allowing for significant sample preservation, greater automation, and higher throughput. Future directions include additional calculations of assay metrics indexed to control values and validation of analytical sensitivity to additional impaired fibrinolytic states and antifibrinolytic agents.