The pre-analytical processing of blood samples for detecting biomarkers on protein microarrays

• Immune-profiling by protein microarrays can be improved using purified IgG.
• IgG but not diluted serum/plasma enables extended linear quantitative measures.
• Purified IgG best preserves differentially reactive antigen profiles.
• Using IgG avoids matrix effects thus ensures high correlations between replicates.

Specimen collection method and quality insurance are pivotal in biomarker discovery. Pre-analytical variables concerning blood collection and sample handling might affect analytical results and should be standardised prior application. In this study, we examine pre-analytical characteristics of blood samples using protein microarray. The influences of 1) standby times until centrifugation (1 h, 4 h, 24 h and 48 h), 2) four blood collection methods, and 3) IgG purified from those samples on differentially reactive antigens between samples (“DIRAGs”) were investigated. Spearman correlation analyses of intra-individual arrays demonstrated remarkable differences (0.75–0.98 vs. 0.5–0.75) of antibody reactivities within and between serum and plasma samples. Class comparison showed that reactive antigen profiles were best preserved using IgG purified samples of serum tubes without separation gel as after 24 h 83% of the 1 h baseline DIRAGs were re-found. Testing dilution series with protein microarrays and Luminex xMap® Technology, we found linear correlations (R2 = 0.94–0.99) between IgG concentration and read-out when using purified IgG instead of serum. Therefore, we highly recommend standardising pre-analytics and proposing the use of purified IgG for autoantibody immune-profiling with protein microarrays to reduce potential unspecific binding of matrix proteins abundant in serum and plasma samples.SignificanceAlthough purified IgG cannot completely compensate the influence of pre-analytics, in highly parallel immune-profiling IgG enables reduction of unspecific effects, which occur when using serum or plasma for analysis on protein microarrays. Reproducibility problems due to pre-analytical processing of blood samples might explain discrepant results reported in various biomarker studies.

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