Identification of the factors affecting the consistency of DBS formation via experimental design and image processing methodology

In the present study we aimed at reaching a procedure for control of factors that have a potential to contribute to bioanalytical method bias and consistent dried blood spot (DBS) formation. The most influencing factors were identified via experimental design and image processing methodology. The effects of five qualitative factors temperature of blood samples, type of pipettes, pipetting technique, age of blood samples and analysts were investigated by multilevel categorical D-optimal design. Due to well-known influence of hematocrit (Hct) level on rheological characteristic of blood samples, five responses were observed through the study (RSD22%Hct, RSD30%Hct, RSD39%Hct, RSD51%Hct, RSD62%Hct) for their ability to provide insights into influence of factor settings onto DBS areas consistency. The area of blood spots was determined by image processing after scanning DBS cards with four spot replicates, corresponding to particular combination of investigated factors, as defined by experimental plan. The qualitative linear mathematical models with added appropriate two-factor interactions were derived by the principle of backward elimination. It was concluded that %RSD value of DBS, for all investigated Hct levels, is completely independent of type of pipettes and age of blood samples, but can significantly be affected by blood sample temperature, pipetting technique and training of analyst. Therefore, the procedure for precise and accurate formation of DBS of uniform area, regardless the Hct value, implies samples at body/room temperature, reversed pipetting technique for rigorous delivery of a sample volume onto the card and a properly trained analyst for handling samples. Finally, a verification experiment was performed and the adequacy of the suggested procedure was confirmed. Identification of the factors affecting the consistency of DBS formation provided the evidence that this contribution to total assay bias can be successfully controlled and reduced.