Analytical comparison of in vitro-spiked human serum and plasma for PCR-based detection of Aspergillus fumigatus DNA: a study by the European Aspergillus PCR Initiative

The use of serum or plasma for Aspergillus PCR testing facilitates automated and standardised technology. Recommendations for serum testing are available, and while serum and plasma are regularly considered inter-changeable for fungal diagnostics, differences in GM-ELISA performance have been reported and attributed to clot formation. Therefore it is important to assess plasma PCR testing to determine if previous recommendations for serum are applicable, and also compare analytical performance with serum PCR. Molecular methods testing serum and plasma were compared through multi-centre distribution of quality control panels, with additional studies to investigate the effect of clot formation and blood fractionation on DNA availability. Analytical sensitivity and time to positivity (TTP) were compared and regression analysis performed to identify variables that enhanced plasma PCR performance. When testing plasma, sample volume, pre/post-extraction volume ratio, PCR reaction volume, duplicate testing and the use of an internal control PCR were positively associated with performance. When whole blood samples were spiked then fractionated, the analytical sensitivity and TTP were superior when testing plasma. Centrifugation had no effect on DNA availability, whereas the presence of clot material significantly lowered the concentration (p = 0.028). Summary: Technically there are no major differences for molecular processing of serum and plasma, but the formation of clot material potentially reduces available DNA in serum. During disease Aspergillus DNA burdens in blood are often at the limits of PCR performance. Using plasma could improve performance while maintaining the methodological simplicity of serum testing