Refined Method for Collection of Plasma Samples to Evaluate the Role of Plasma Kallikrein in Various Disease States
Monday, March 7, 2016
South Exhibit Hall H (Convention Center)
Jonathan A. Bernstein, MD FAAAAI, H. James Wedner, MD FAAAAI, Paula J. Busse, MD FAAAAI, Aleena Banerji, MD, Marco Cicardi, C Sufritti, Edward G. Brooks, MD, Adam Cheifitz, Lawrence B. Schwartz, MD PhD FAAAAI, Cem Akin, MD PhD FAAAAI, Daniel Sexton, Chris Stevens, Leslie E. Stolz, Malini Viswanathan, Ryan Faucette, Joseph C. Biedenkapp, Yung H. Chyung, Burt Adelman
Rationale: Examination of plasma biomarkers of contact system activation is challenging due to inadvertent activation during blood collection and processing. We examined the utility of specialized blood collection tubes in assessing levels of cleaved high-molecular-weight kininogen (cHMWK) in plasma from healthy subjects and those with Types I/II hereditary angioedema (HAE), idiopathic angioedema or HAE with normal C1-INH (HAEnC1).

Methods: To avoid artificial activation of the contact pathway during blood sampling, the study used standardized blood collection techniques and custom tubes containing protease inhibitors. Blood samples were collected from healthy subjects and disease subjects (during periods of disease quiescence and flare) to assess the percentage of cHMWK using a Western blot assay. 

Results: In healthy subjects, levels of cHMWK were stable (<5%) at RT for at least 24 hours following blood collection into custom tubes but were elevated (12%) in plasma samples obtained from a commercial vendor, demonstrating the importance of optimizing blood collection techniques when examining contact pathway activation. cHMWK percentages were clearly elevated at baseline in subjects with HAE (n=21) compared to healthy controls (n=26) but not in plasma from subjects with idiopathic angioedema (n=4) or HAEnC1 (n=5), suggesting limited plasma kallikrein activity, although not excluding a role of contact pathway activation during acute attacks in those disorders.

Conclusions: A refined method for collection of plasma samples for evaluation of contact system activation has been developed which prevents cleavage of HMWK ex vivo.