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

DBS have been used for 100 years² but, surprisingly, there is still no general consensus about their preparation and processing. To date, a sufficient standardization of this important pre-analytical phase has only been achieved in the field of newborn screening¹⁴, whereas a variety of different protocols exists for all other applications of DBS testing^5, 16, 23. To overcome this remarkable heterogeneity, a comprehensive step-by-step instruction for preparing and processing DBS to be utilized in immunoassays and by molecular techniques is presented in this communication and evaluated with regard to its effectiveness for detecting markers of HBV, HCV and HIV infections. The focus of the following discussion is primarily placed on the different steps of the suggested protocol.

In the history of DBS testing many different filter paper cards have been used² but today only two commercial sources are approved by the FDA as class II medical devices for blood collection^5, 16. These filter card systems are highly uniform and have very similar absorption characteristics so that analytical results obtained from capillary blood prepared on either of them do not differ by more than 4% – 5%²⁸. Not surprisingly, Masciotra and co-workers²⁹ therefore detected HIV-1 RNA equally well with a qualitative assay after elution from blood collection cards from different sources. Given these data, it can be virtually excluded that any of the discrepancies observed when testing coupled serum/DBS pairs for markers of HBV, HCV and HIV infections¹⁷ was caused by the choice of the filter card alone. However, when accurate quantification of an analyte is indispensable, source-to-source variation may no longer be negligible and more sophisticated techniques, e.g., perforated DBS (PDBS) as a method for microsampling³⁰ or the preparation of dried serum spots (DSS)³¹ may have to be applied instead of conventional DBS testing¹⁰.

Since only small quantities of blood are used for DBS testing (one drop of capillary blood consists of approximately 50 µl)^5, 16, variations in the sample volume are crucial and, admittedly, at least some of the discrepancies between serological results and participant’s self-reported HBV and HCV status recorded during the piloting of the “DRUCK Study” are attributable to this variable. The single most important factor for minimizing “fluctuations” of the sample volume is undoubtedly a correct technique of capillary blood collection, which can only be achieved by careful and ongoing training of the technical personnel²². Furthermore, as a measure of quality control, all laboratories working with DBS should have already initiated procedures for identifying and subsequently excluding DBS specimens that have to be considered as unsatisfactory or invalid¹⁶.

Studies conducted primarily in the context of HIV³² and newborn screening³³ have shown that high humidity may lead to a degradation of analytes, but hitherto no consensus has been reached regarding the question of how long DBS should be air-dried. An interval of at least 4 hr or preferably O/N is proposed in this communication, which therefore adopted conditions that were used in the vast majority of all relevant publications^32, 34 Data on storage of DBS to be subsequently used for HBV and HCV testing are conflicting. In 1981, Villa and co-workers³⁵, who applied contemporary analytical techniques, reported that storage at RT did not affect the results of HBV analyses during the entire observation period of 180 days if antibody titers were > 1/1,000, but that DBS became borderline-positive or even negative after 15 days when titers in serum were only 1/100. Storage at -20 °C or 4 °C did not result in a substantial improvement. In contrast, a study conducted thirty years later³⁶ tested replicates of a HBV-positive sample, and the authors found that anti-HBc as well as anti-HBs antibodies were stable for up to 183 days at RT, whereas HBsAg under the same conditions became false-negative already after 63 days. With regard to HBV DNA detection from DBS eluates, the concentration of the viral nucleic acid was stable for at least seven days at 37 °C³⁷ or proved to be “resistant” to storage at RT for up to three weeks³⁸. Testing for anti-HCV antibodies using two commercially available third generation immunoassays³⁹ provided accurate results for a period of 117 days using DBS samples stored at -20 °C, 2 – 8 °C, and 20 – 25 °C, respectively. Storage at -20 °C, however, resulted in the lowest variation of optical densities. Applying a fourth generation anti-HCV ELISA, i.e., Monolisa HCV-Ag-Ab-ULTRA, in the context of DBS testing, Larrat et al.⁴⁰ observed a sharp decrease in analytical specificity after storing the DBS specimens for more than three days at RT. On the other hand, precise testing results were obtained with the same kit utilizing samples deposited for 60 days under various conditions (-20 °C, 2 – 8 °C, and 22 – 26 °C) by Brandao and co-workers⁴¹. Observations on the decline of HCV RNA concentrations in DBS under various storage conditions range from no significant alteration at RT for up to one year⁴² to a tenfold change after four weeks at ambient temperature⁴³. 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 short-term 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 volume²³. 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 sites²³. Only a few reports, which directly compare different elution buffers in the context of DBS testing, are available. Villar et al.³⁶, e.g., recorded almost equivalent elution capacities for all buffers used, but PBS/BSA 0.5% resulted in the lowest level of non-specific reactivity. A very similar observation was made by Croom and co-workers⁴⁴ when applying the specimen diluent of the Genetics Systems rLAV EIA for the elution of anti-HCV antibodies from DBS. 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 morning²³. 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. However, a careful optimization of the elution conditions for every single analyte was not possible in the preceding evaluation because a high sample throughput had to be guaranteed in a comparatively short time during the “DRUCK Study”¹⁷. Consequently, the rather unfavorable volume of 1,000 µl of PBS-based buffer was used in order to complete the whole elution process for all parameters in two separate operations.

This approach one the one hand fails “completely in the anti-HBc/anti-HBs system for those individuals infected with HIV due to the low antibody concentrations; … and it required molecular biological procedures with optimal analytical sensitivity with regard to HBV DNA and HCV RNA tests.”¹⁷ On the other hand, the high elution volume proved to be in no way disadvantageous for the determination of HBsAg, anti-HCV, and anti-HIV by the kits used throughout the evaluation. “The detection of HBsAg positive materials from whole blood eluates succeeded with a sensitivity of 98.6% to a similarly high degree as in previous studies, which had in part used a much smaller elution volume of 100 µl, 250 µl, or 600 µl, or 500 µl”^{17, 36, 45, 46}. The sensitivity of 97.8% determined in the investigation of 179 serum/DBS pairs for anti-HCV antibodies corresponded to the results of already existing reports^{24, 44, 47, 48, 49}, which had worked with an elution volume that was lower by a factor of 5 to 10. “In addition, the protocols for anti-HCV detection had been appropriately optimised … by stipulating their own cut-off points”^{17, 24, 48, 49} “or by increasing the sample volumes from 20 µl to 100 µl.”^17, 49 Finally, the analytical specificity and sensitivity (100% each) established for anti-HIV detection were equal or superior to the performance characteristics of other immunoassays specifically adapted to DBS testing^50, 51 “or a stepwise procedure with the combined use of several anti-HIV tests”^17, 52. “They, indeed, also exceeded the performance record of an assay that had been specially developed and optimised for the detection of anti-HIV antibodies in DBS eluates (Q-Prevent HIV 1 + 2 DBS kit).”^17, 53

Taken together, the comprehensive step-by-step protocol presented in this communication proved to be a feasible and user-friendly tool for preparing and processing DBS and can, thus, be used reliably in diagnostic virology. It allows approaches using automation ⁵⁴ and due to its excellent performance characteristics has the potential to serve as a kind of foundation-stone for a future generally accepted consensus protocol in the global field of DBS testing in laboratory medicine.