Effect of Blood Volume on Analytical Bias in Dried Blood Spots Prepared for Newborn Screening External Quality Assurance

Abstract

Aim: Dried blood spots (DBS) are used for the analysis of more than 2000 biomarkers. We assessed a range of analyte concentrations and diameters of DBS. Materials & methods: DBS samples were created by the application of increasing volumes of whole blood prepared by the UK NEQAS Quality Assurance Laboratory. Samples were analyzed in four separate laboratories. Results: Volumes less than 25 μl (8 mm) and more than 75 μl (14 mm) created unsatisfactory analytical biases. Results obtained from peripheral subpunches tended to be higher than those from a central subpunch. Conclusion: DBS diameters formed from nonvolumetric application of blood to filter paper can be used to assess whether measurement bias will be within acceptable limits according to the analyte being quantified. DBS received for newborn screening in the UK with diameters less than 8 mm and those more than 14 mm should be rejected.

Use of Dried Blood Spot Specimens to Monitor Patients with Inherited Metabolic Disorders

“…It is important to recognise that patient results may vary significantly and that analysis of DBS specimens may not be the first choice of specimen in certain situations. Assessing the diameter of the DBS may be used as a tool to identify samples that may give unacceptable analytical biases for analyte concentrations [39]. Laboratories should have standardised criteria to ensure that all laboratories are accepting and rejecting samples of the same size and quality [25,39].…”

“…Assessing the diameter of the DBS may be used as a tool to identify samples that may give unacceptable analytical biases for analyte concentrations [39]. Laboratories should have standardised criteria to ensure that all laboratories are accepting and rejecting samples of the same size and quality [25,39]. Only appropriately sized samples should be accepted for DBS assays, thereby ensuring accurate monitoring results.…”

“…The plasma/serum component applied occupies a greater volume of the interior of the paper fibres than that of the erythrocytes (process affected by Hct level) and this leads to the loss of homogeneity across the DBS (chromatographic effect), with the erythrocytes being concentrated at the edge of the DBS. This loss of homogeneity results in increased concentrations of analytes associated with erythrocytes in the peripheral sub-punches relative to central sub-punches taken from the DBS [25,[38][39][40].…”

“…These factors should also be taken into account when interpreting the analytical result. Furthermore, the effect of central and peripheral sub-punches on analyte concentrations should also be assessed as analyte dependent differences in results have been reported [14,25,[38][39][40]. The practical application of these results would be to make recommendations on where sub-punches should be taken from the DBS to ensure more reliable monitoring results are obtained, e.g., to take central punches from two separate DBS to obtain the most consistent duplicate results.…”

“…Relationship between volumes of blood applied to the filter paper collection devices and the measured dried blood spot (DBS) diameter. Results shown are the mean of the diameter measurements (results from[25,39]). Sub-punches (3.2 mm) were taken from central (first row) and peripheral locations (second row).…”