Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement

Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.


Pre Analytical Effects of Serum Collection and Handling in Quantitative Immunoassays for Rheumatoid Arthritis.

Qureshi3,  Ferhan, Manning3,  William, Eastman3,  Scott, Hu3,  Wayne, Centola6,  Michael, Turner7,  Mary, Carson5,  Craig W.

Crescendo Bioscience, Inc., South San Francisco, CA
Crescendo Bioscience, Inc., Edmond, OK
Crescendo Bioscience, Inc.
OK Arthritis Center, Edmond, OK
Oklahoma Arthritis Ctr, Edmond, OK
Oklahoma Med Research Foundation, Oklahoma City, OK
Oklahoma Medical Research Foundation

Background:

Pre-analytical variables related to serum processing and handling can have significant influences on the results obtained in quantitative protein immunoassays. Understanding the impact of these variables is critical for assay development, validation, and commercial testing. The effect of 2 routine serum collection methods (on the clot with ambient temperature shipping vs. centrifuged with cold chain shipping) and 2 serum handling variables (time before assay and freezing) were examined in a novel, validated, 12-biomarker RA disease activity test (DA Test) and up to 15 additional proteins being evaluated as predictors of structural damage progression in Rheumatoid Arthritis (RA).

Method:

To evaluate serum collection variables, multiple matched sets of Becton Dickinson Serum Separator Transport Tubes (BD SST™) were drawn from 10 RA patients, with varying disease activity. One set of tubes from each individual was processed per manufacturer's instructions followed by overnight shipment in a temperature controlled (2–8°C) package ("Cold chain"). A matched tube from each individual was simultaneously shipped overnight at ambient temperature while remaining on the clot (e.g. not centrifuged; OTC). Upon arrival, the OTC tube was centrifuged and all samples were aliquoted for analysis in single-plex ELISAs and mutli-plex Meso Scale Discovery immunoassays.

To evaluate post-processing serum handling effects, matched aliquots of serum were held at multiple temperatures (-80°C, -20°C, 4°C and 25°C) for extended periods of time before assaying to generate a DA Test score which was then compared to the scores obtained in a baseline measurement. Additionally, multiple aliquots of serum (including both pools and individuals) were subjected to 3 successive freeze thaw cycles at -80°C prior to analysis by immunoassay.

Results:

Significant differences in analyte concentrations were seen in >45% (13/27) of the analytes between the 2 collection methods. Twelve of thirteen biomarkers levels increased in the OTC samples, while one decreased. Of the 13 biomarkers with significant changes, 5 had poor correlation within the 10 RA patient samples (R2 < 0.7) compared to the centrifuged, cold chain samples. The individual DA Test scores varied widely, (-16% to 126%), confirming the value of onsite centrifugation and cold chain shipping. The impact of serum storage (post-processing) at the various temperatures tested and multiple freeze thaw cycles on biomarker levels was minimal. Additional studies, to characterize combinations of multiple pre-analytical variables are currently being designed.

Conclusion:

Serum collection, processing and handling methods have a significant impact on the measurable serum protein levels. These results illustrate the importance of characterizing pre-analytical variability to ensure test accuracy and for using robust serum processing and handling procedures for development, validation, and clinical testing with biomarker assays including the DA Score.

To cite this abstract, please use the following information:
Qureshi, Ferhan, Manning, William, Eastman, Scott, Hu, Wayne, Centola, Michael, Turner, Mary, et al; Pre Analytical Effects of Serum Collection and Handling in Quantitative Immunoassays for Rheumatoid Arthritis. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :1606
DOI: 10.1002/art.29372

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