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Methodological and preanalytical evaluation of an HMGB1 immunoassay
Julia Lehner1, Christin Wittwer, Debora Fersching
1Institute of Clinical Chemistry, University Hospital Munich-Grosshadern, Munich, Germany.
Background:
Soluble high-mobility group box 1 (sHMGB1) is a promising biomarker for the prognosis and the monitoring of cancer and of acute diseases such as trauma and sepsis.
Materials And Methods:
We investigated the methodological characteristics of an ELISA for sHMGB1 (Shino-Test, Tokyo, Japan and IBL, Hamburg, Germany) including intra- and inter-assay imprecision, dilution linearity and differences in serum and plasma materials. Furthermore, the influence of various preanalytical factors such as time and storage temperature before and after centrifugation prior to definite deep freezing, as well as multiple freeze-thaw cycles were tested. By the use of sera from 28 healthy individuals, a reference range and the dependency on patient characteristics were established.
Results:
Intra-assay imprecision (coefficients of variation (CV)=1.2-4.8%) and inter-assay imprecision (10.3-14.0%) were in an acceptable range of manual assays. HMGB1 levels were found to be considerably lower in EDTA plasma as compared to serum samples. Linearity testing yielded satisfying results with dilution recoveries of 100-121% (mean=112.3%). sHMGB1 results were the same when samples were kept at 4°C and 25°C after centrifugation, for up to 7 days (recoveries 87-128%). Delay before centrifugation led to a considerable increase in some samples. The median values for healthy individuals was 1.3 ng/ml, and the 95th percentile was 4.1 ng/ml. HMGB1 levels correlated inversely with age (R=0.33).
Conclusion:
The sHMGB1 ELISA is a robust and safe assay producing reliable quantitative results in sera.
