The preparing and processing of dried blood spots (DBS) for their final analysis are still poorly standardized for most diagnostic applications. To overcome this shortcoming, a comprehensive step-by-step protocol is suggested and subsequently evaluated with regard to its effectiveness for detecting markers of viral infections.
The idea of collecting blood on a paper card and subsequently using the dried blood spots (DBS) for diagnostic purposes originated a century ago. Since then, DBS testing for decades has remained predominantly focused on the diagnosis of infectious diseases especially in resource-limited settings or the systematic screening of newborns for inherited metabolic disorders and only recently have a variety of new and innovative DBS applications begun to emerge. For many years, pre-analytical variables were only inappropriately considered in the field of DBS testing and even today, with the exception of newborn screening, the entire pre-analytical phase, which comprises the preparation and processing of DBS for their final analysis has not been standardized. Given this background, a comprehensive step-by-step protocol, which covers al the essential phases, is proposed, i.e., collection of blood; preparation of blood spots; drying of blood spots; storage and transportation of DBS; elution of DBS, and finally analyses of DBS eluates. The effectiveness of this protocol was first evaluated with 1,762 coupled serum/DBS pairs for detecting markers of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus infections on an automated analytical platform. In a second step, the protocol was utilized during a pilot study, which was conducted on active drug users in the German cities of Berlin and Essen.
The idea of using blood collected on a paper card made of cellulose is ascribed to Ivar Christian Bang (1869 – 1918), the father of modern clinical microanalysis1, 2. In 1913, Bang determined glucose from eluates of dried blood spots (DBS)3 and, later, also performed nitrogen measurements using the Kjeldahl method with this filter paper technique2. Subsequently, several investigators reported on the use of DBS for serological testing to diagnose syphilis2. As early as in 1924, Chapman summarized the advantages of DBS testing when he particularly stressed four items, which are still valid today: (1) compared to conventional venipuncture, less blood volume is required and this fact was most important in pediatric diagnostics; (2) blood collection is simple, non invasive, and inexpensive; (3) the risk of bacterial contamination or hemolysis is minimal; and (4) DBS can be preserved for long periods with almost no deterioration of the analytes2, 4. Besides its use in testing for syphilis, further early applications of the DBS technique included, e.g., the detection of antibodies against measles, mumps, poliovirus, parainfluenza virus, and respiratory syncytial virus (RSV) in 19532, the identification of Shigella in feces dried onto filter paper and shipped by regular mail from Indonesia to Leiden in the Netherlands as well as the detection of antibodies to Schistosoma in DBS taken in endemic areas and analyzed more than three months later5. In 1963, fifty years after Bang’s original communication2, 6, Guthrie finally published his famous method for the diagnosis of phenylketonuria from DBS obtained by a heel prick from newborns7, 8.
Although from that time onwards, DBS were regarded as a commonly applicable method for collecting, storing, transporting, and analyzing a variety of human body fluids5, their use in diagnostics still remained predominantly focused on the diagnosis of infections especially in resource-limited settings and the systematic screening of newborns for inherited metabolic disorders for decades9, 10. Since 2005, however, a variety of new and innovative DBS applications have begun to emerge. This resulted in an almost exponential increase in the number of respective scientific publications on DBS from about 50 to almost 450 annually at present. Among the emerging applications are such diverse fields as toxico- and pharmacokinetic studies, metabolic profiling, therapeutic drug monitoring, forensic toxicology, or environmental contamination control10, 11.
DBS testing has, thus, made a triumphal march through clinical laboratory diagnostics during the past 100 years2. As in clinical chemistry12, however, during this march pre-analytical variables were not adequately considered for many years. Indeed, even today, after such eminent activities as the CDC’s filter paper evaluation project13 or the formulation of a national standard for blood collection on filter paper in the framework of newborn screening14, the pre-analytical phase is still largely undervalued in most of the other fields, in which DBS testing is applied5, 10.
Given this background, a comprehensive step-by-step protocol14 - 16 for use in immunoassays and in molecular techniques, which covers all of the essential steps, is suggested for preparing and processing DBS in the following communication: (1) collection of blood; (2) preparation of blood spots; (3) drying of blood spots; (4) storage and transportation; (5) elution of DBS; and finally (6) analyses of DBS eluates. The effectiveness of the protocol was first evaluated with 1,762 coupled serum/DBS pairs for detecting hepatitis B virus (HBV) surface antigen (HBsAg), antibodies to HBV core antigen (anti-HBc), antibodies to HBV surface antigen (anti-HBs), HBV DNA, antibodies to the hepatitis C virus (HCV) (anti-HCV), HCV RNA, and human immunodeficiency virus (HIV) 1-p24-antigen/anti-HIV 1/2 using either a fully automated platform or sensitive qualitative nucleic acid tests.17. In a second step, the protocol was utilized in the pilot study “Drugs and Chronic Infectious Diseases” (“DRUCK Study”) which was conducted by the Robert Koch-Institute in close collaboration with the National Reference Centre for Hepatitis C on active drug users in the German cities of Berlin and Essen18.
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 testing5, 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 collection5, 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 publications32, 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 testing15, 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 reports24, 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 testing50, 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.
The authors have nothing to disclose.
This communication is based on the results previously published in Virology Journal17 in open access mode under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0) with BioMed Central as a licensee. The authors gratefully acknowledge the fruitful collaboration in the framework of piloting the study “Drugs and Chronic Infectious Diseases” (“DRUCK Study”) with U. Marcus, W. Cai, W. Zhang, and R. Zimmermann from the Robert Koch-Institute (Berlin, Germany) and are thankful to S. Moyrer for taking the photographs assembled in Figure 1 as well as to D. F. Whybrew, Ph.D, (Göttingen, Germany) for correcting the the manuscript. They also express their appreciation for skilful technical assistance to J. Ackermann, E. Bayrambasi, U. Büttner, S. Dziubek, I. Jakobsche, B. Krellenberg, H. Krohn, P. Kusimski, A. Metcalf, J. Piejek, S. Saar, M. Schröter and K. Seidel. This study was supported in part by a grant of the German Ministry of Health to the National Reference Centre for Hepatitis C.
Latex rubber gloves | Hartmann | #4049500041515 | |
70% isopropyl alcohol | Bode Chemie | #9768050 | |
Mulifly safety cannula | Sarstedt | #85.1638 | |
EDTA blood collection tube | Sarstedt | #02.1066.001 | |
Adhesive bandage | Hartmann | #2994163 | |
Dry gauze pad | Hartmann | #143213 | |
Singel-use safety lancet | Owen Mumford | #3802421 | |
Test Card | Whatman/sigmaaldrich | #10534612 | Filter paper card Grade 903 |
Disposable pipette tips | Starlab | #S1126-7810, #S1120-8810 | |
Zipper bag | Flexico | #20219 | |
Mini desiccant bag | Tropack | #- | |
Disposable Punch | pfmmedical | #48601 | 6.0 mm Disposable Biopsy Punch |
Disposable Forceps | Servoprax | #H7301 | |
12 Well Cell Culture Plate | Greiner | #665180 | |
PBS Buffer | Gibco | #14190-136 | |
Tween 20 | Applichem | #A1389.0500 | |
Sodium Azide | Merck | #66880250 | |
Safe-Lock Tubes, 1.5 mL | Eppendorf | #30120086 | |
ARCHITECT HBsAg (Qunatitative) | Abbott | #6C3642 | |
ARCHITECT anti-HBc II | Abbott | #8L4425 | |
ARCHITECT anti-HBs | Abbott | #7C1825 | |
ARCHITECT anti-HCV | Abbott | #6C3725 | |
ARCHITECT HIV Ag/Ab Combo | Abbott | #4J2727 | |
MagNA Pure 96 DNA and Viral NA Large Volume Kit | Roche | #6374891001 | |
artus HBV LC PCR Kit | Qiagen | #4506063 (24 tests), #4506065 (96 tests) | |
VERSANT HCV TMA Assay IVD | Siemens Healthcare | #2554311 |