Expanding the Utility of High-Sensitivity Dried Blood Spot Immunoassay Testing with Single Molecule Counting

Abstract

Background: Dried blood spot (DBS) testing has been used for years in newborn screening and for other applications when obtaining blood by venipuncture is impractical or expensive. However, several technical challenges have restricted the use of DBS testing to qualitative assays or to analytes that are present in relatively high concentrations. The application of high-sensitivity detection using single molecule counting (SMC™) technology can potentially overcome the limitations of DBS as specimen source. Methods: A method was developed for reproducibly collecting, storing, and subsequently reconstituting DBS samples to be used with assays based on the SMC technology. Before extraction, DBS samples were scanned, and the blood spot area was calculated to normalize for sample volume and spot variability. DBS sample extraction was done using an efficient high-salt extraction buffer. DBS samples were tested using SMC-based cardiac troponin I (cTnI), prostate-specific antigen (PSA), and C-reactive protein (CRP) assays. Results: The SMC-DBS assays showed reproducible sensitivity, precision, and the stability required for quantifying low-abundance biomarkers. These assays were not significantly impacted by normal variations in hematocrit or sample collection technique. Correlation coefficients obtained from method comparisons between SMC-DBS and laboratorydeveloped tests or Food and Drug Administration-cleared tests using traditional sample types were 1.08, 1.04, and 0.99 for cTnI, PSA, and high-sensitivity CRP, respectively. Conclusions: Combining DBS finger-stick blood collection with next-generation immunoassay technology will aid the expansion of DBS testing to protein biomarkers that are in low abundance or to low-volume samples, and will enable the development and adoption of DBS testing to far-reaching applications. IMPACT STATEMENT We have developed a novel method that combines the convenience and stability of dried blood spot (DBS) sampling with the sensitivity of single molecule counting (SMC) technology to create SMC-DBS assays with the precision and accuracy of traditional serum-based methods. The methodology will be useful for those with chronic conditions, in whom repeated testing of biomarkers is required; in neonatal populations, for which the sample volume is scarce; and in demographic studies done in the field, where sample storage and shipment facilities are suboptimal. This methodology allows for the expansion of DBS testing to low-abundance protein biomarkers and enables a more widespread adoption and application of DBS testing.

Hematocrit, blood volume, and surface area of dried blood spots – a quantitative model

“…We suggest that future assay methods involve the scanning and calculation of DBS surface area and blood volume calculated as a quality control step when the hematocrit is known or calculated in clinical studies for the measurement of different drugs. Another study by Mukherjee et al used the ImageJ ® software to find the correlation between surface area and blood volume,25 however, in our study we found the correlation between blood volume, surface area, and hematocrit level all together in one model makes the prediction of either HCT or BV easier.Variation in hematocrit levels between patients and inaccuraciesin blood volumes applied to Guthrie cards can have a marked effect on analyte concentrations measured in DBS samples. The current…”

“…Blank blood from healthy volunteers was collected in the EDTA tubes then centrifuged, separated and accurate amounts of red blood cells and plasma were mixed to obtain blood samples of different hematocrit (HCT) levels (25,30,35,40,45, 50, and 55%), This range was selected to cover the HCT levels expected in patients. Aliquots Health, USA).…”

Instant on-paper protein digestion during blood spot sampling

“…However, recent efforts do suggest the applicability towards larger biomolecules177,178 . Some investigations has also utilized DBS and DMS for proteomic investigations as concept studies87,[179][180][181][182] . Nevertheless, DMS and DBS are not extensively reported for analysis of low abundance biomarkers.…”

Point-of-Care Testing and Home Testing: Pragmatic Considerations for Widespread Incorporation of Stool Tests, Serum Tests, and Intestinal Ultrasound

“…However, rapid serum point-of-care tests are not yet accessible in routine clinical practice. [32][33][34] Here, we review the current state, pragmatic considerations, and future implications of point-of-care testing and home testing for noninvasive IBD monitoring in the tight control model.…”