Development and validation of assay protocols for use with dried blood spot samples

Abstract

Dried blood spots (DBS)--drops of capillary whole blood collected from finger stick--represent a minimally invasive alternative to venipuncture that facilitates the collection of blood samples from research participants in naturalistic, field-based research settings. But the number of validated assays for quantifying biomarkers in DBS samples is relatively low in comparison with serum or plasma. The objective of this review is to discuss the advantages and disadvantages of DBS sampling, and to outline the steps involved in developing and validating an immunoassay for application to DBS samples. These steps include deciding on reagents, preparing calibration and quality control material, evaluating elution protocols, optimizing sample quantity, and assessing multiple aspects of assay performance, including intra- and interassay variation, lower limit of detection, accuracy, stability, and agreement between results from matched DBS and plasma samples. The broader goal of this "how-to" approach is to encourage investigators to validate, implement, and disseminate assay protocols for DBS samples in order to advance field-based research on human biology.

Laboratory and field methods for stable isotope analysis in human biology

“…Blood may be stored frozen, or dried for SIA. If "bulk" blood is to be used as the substrate for analysis, it may be frozen or dried on glass fiber filter papers immediately upon collection (see McDade, 2014 for more information on collecting dried blood spots). It is crucial to use glass fiber filter papers, as carbon from typical filter papers will contaminate the d 13 C signal of the sample.…”

Dried Blood Spots - Preparing and Processing for Use in Immunoassays and in Molecular Techniques

“…Since the risk of sample degradation is exceedingly low in the first hours after preparation of DBS (see above), it was decided to incubate the spots O/N at ambient temperature and to support the elution process by gentle end-over-end mixing. This approach has the advantage that specimens punched out the previous day can be directly transferred to routine diagnostics early the next morning 23 . The volume of the elution buffer should be adapted to the minimal respective requirements of the assays used for subsequent analyses in order to keep the dilution factor as low as possible.…”

“…Taking this rather conflicting data on the stability of HBV and HCV antigens, nucleic acids and antibodies into account, it seemed reasonable to retract in the proposed protocol to a consensus, which was defined earlier for the storage of DBS specimens to be used for HIV testing 15,30 : For shortterm deposition (up to two weeks) antigens, viral nucleic acid, and antibodies are regarded as stable at RT, whereas optimal storage for longer periods is at frozen conditions. As a rule, three parameters should be considered when designing an elution protocol: (1) the elution buffer; (2) the duration and temperature of elution; and (3) the elution volume 23 . In the vast majority of all relevant publications phosphate-buffered saline (PBS) was used for eluting DBS, and most authors added a protein, e.g., bovine serum albumin (BSA) or Tween 20, a surfactant, in order to improve the assay signal by stabilizing proteins, as they go into solution, and simultaneously blocking non-specific binding sites 23 .…”

“…As a rule, three parameters should be considered when designing an elution protocol: (1) the elution buffer; (2) the duration and temperature of elution; and (3) the elution volume 23 . In the vast majority of all relevant publications phosphate-buffered saline (PBS) was used for eluting DBS, and most authors added a protein, e.g., bovine serum albumin (BSA) or Tween 20, a surfactant, in order to improve the assay signal by stabilizing proteins, as they go into solution, and simultaneously blocking non-specific binding sites 23 . Only a few reports, which directly compare different elution buffers in the context of DBS testing, are available.…”

Therapeutic drug monitoring of carbamazepine and its metabolite in children from dried blood spots using liquid chromatography and tandem mass spectrometry

“…It offers a number of advantages including less invasiveness, low blood sample volume requirement, absence of post-collection processing, low biohazard risks [14,15]. It also facilitates cost reduction, ease of sample storage and transport before analysis.…”