Dried Blood Spot Testing: Innovations and Applications

By Robert Lerman, M.D. | Published 2/9/2020 0

dried blood spot testing

Photo source: iStock

During the first ten days of December 2019, two very different headlines highlighted news reports that seemingly had nothing in common. But in actuality, they did. They both illustrated the potential new benefits from a well-established, but previously limited, clinical laboratory technique called Dried Blood Spot Testing or DBS.

Here are the stories:

  • On December 1, World AIDS Day was commemorated. It provided an opportunity for people around the globe to unite in the fight against HIV. It reminded us to support the 36.7 million people living with the virus. And, to remember those who died from AIDS-related illness.
  • Eight days later, the World Anti-Doping Agency (WADA) issued a four-year penalty against the Russian national athletics team [1], banning them from all international sporting competitions, including the Olympics,. The ban was because of their continued participation in the years-long doping scandal.

Here’s a closer look at the role of DBS in those stories:

In October of 2019, the WADA announced a collaboration with seven Anti-Doping Organizations [2] The collaboration centered on the development and implementation of DBS for drug testing in sports in time for the 2022 Winter Olympic and Paralympic Games in Beijing, China.


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Meanwhile, UNAIDS was approaching the final stretch of their 90-90-90 initiative [3] that included three goals to accomplish by the end of 2020.

Ninety percent of all people: 

  • living with HIV should know their HIV status,
  • diagnosed with HIV should receive sustained antiretroviral therapy (ART)
  • receiving ART should have viral suppression.

DBS testing has shown promise for both HIV screening and viral load testing [4], potentially facilitating the achievement of those goals.

Advantages of DBS testing

DBS testing is a form of blood sampling that uses capillary blood obtained using a heel stick or finger prick instead of serum or plasma drawn from a vein.[5] The blood is then blotted and dried onto filter paper that is used for testing.

DBS offers practical, clinical, and financial advantages as compared to conventional blood testing:

DBS testing was developed in 1963 and became widely used for the first time in 1969-70 for neonatal screening for the congenital metabolic disease, phenylketonuria (PKU) [7].

Challenges related to DBS testing

DBS has been the standard of care for neonatal screening for a variety of diseases, including PKU, sickle cell, hypothyroidism, and HIV infection.

However, the utilization of DBS beyond neonatal screening was limited for many years [8] due to a variety of technical issues. First and foremost, the quantity of blood collected with each sample is small (only 2-3 drops of blood). Therefore, successful analysis is dependent on highly sensitive and reliable analytic techniques.

Other contributing factors that make consistent DBS sampling a challenge include the following:

  • cumbersome drying procedures
  • differences in blood sample viscosity
  • distribution on the filter paper
  • stability of the sample during transport and storage (especially at high temperatures and humidity)

More work than conventional blood tests

The actual work of processing and analyzing the DBS samples involves more labor than with conventional serum samples, often requiring manual processes.

The utility of DBS as a diagnostic tool is also dependent upon the cross-validation of the method with a reference plasma/serum-based assay for the same target substance. Reference intervals need to be established in order to properly interpret the results of DBS testing. This is because the values for whole blood used in DBS often differ from those derived from serum or plasma.

Taken together, these technical challenges have played a role in limiting the adoption of DBS testing despite its potential advantages. Historically, few clinical laboratories have been equipped to perform DBS testing.

Related content:
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Current use case: HIV viral load monitoring

The World Health Organization recommends viral load monitoring to test the efficacy of ART in HIV patients. However, although countries in sub-Saharan Africa are scaling up their efforts to monitor ART, most African countries are not able to provide monitoring for more than 50% of all affected patients. In order to provide full access to testing services, countries will need to address the “Last Mile”, or patients in the most remote areas. This use case illustrates a challenge that can potentially benefit from the use of DBS. 

In the Journal of the International AIDS Society [9], Nichols and colleagues recently reported on the incremental costs of accessing the most remote 20% of patients in Zambia. It was done by expanding the network required to transport blood samples from ART clinics to centralized laboratories.

The last mile is costly

They concluded that “providing sample transport services to the most remote clinics in low- and middle-income countries is likely to be cost-prohibitive. Other strategies are needed to reduce the cost and increase the feasibility of making viral load monitoring available to the last 10% of patients.”

They recommended evaluation of the cost of alternative testing methods, such as dried blood/plasma spot specimens, point-of-care testing or drones. Point-of-care devices show promise but still require lab facilities. Viral load monitoring using DBS still needs to be validated before being scaled up, but the first field testing of a CE marked protocol for HIV RNA testing using DBS [10] has been reported.

Technological innovations in DBS testing

The technological improvements that have made DBS testing more practical and more promising have come in both the “pre-analytical” and “analytical” phases [11] of DBS testing.

  • Pre-analytical phase

The pre-analytical phase of DBS testing includes:

        1. Blood collection from the patient 
        2. Application of the drop(s) of blood onto the filter paper 
        3. Drying
        4. Transport and storage of the DBS sample.

Each component needs to be performed correctly and consistently:

      • Sample collection – if the drop(s) of blood are too large or too small or not properly applied, the sample may not be suitable for analysis.
      • Paper thickness and density can influence the rate of adsorption and dispersion.
      • The patient’s hemoglobin can influence the distribution of biomarkers within a sample.
      • DBS samples must be allowed to dry completely before transport and storage (typically takes from 90 minutes to four hours and is influenced by room temperature, humidity, and exposure to sunlight). 

Innovative products

Innovative products, such as the HemaSpot devices [12], have simplified DBS specimen collection, transport, and processing. HemaSpot utilizes a self-contained device (12) consisting of an absorbent paper and desiccant covered with an application surface housed within a plastic cartridge.

Two to three drops of blood are absorbed by the filter paper after passing through a small opening in the application surface. The cartridge is closed immediately, preventing contamination and reducing biohazard risk.

The devices can be immediately shipped and stored at ambient temperature without additional drying. Other new devices claim accurate volume measurement of samples, enabling better quantitation of analyzed substances.

  • Analytical phase

In the analytic phase of DBS testing, the substance being measured needs to be removed from the filter paper. Originally this was done by placing the disc in a solvent or buffer [13].

In the 1990s, the ability of DBS to be used for profiling and quantification of biomolecules and synthetic drugs expanded greatly with the development and adoption of liquid chromatography [14]and mass spectrometry [15] to separate and identify molecules within a sample.

Surface sampling technology[16] has now been developed to allow for direct sampling of DBS without the need for a change to liquid form. This eliminates another source of potential inconsistency.

Several companies now manufacture robotic end-to-end hardware [17] for DBS handling: extraction, purification, concentration, and introduction into the analysis system.

Expanded indications for DBS

With current technology, DBS testing is currently used for a range of applications. These include widespread neonatal screening, drug toxicology, and sports doping screening.

DBS samples are compatible with a large number of bioanalytical methods. Zakaria and colleagues reported on 121 distinct biomarkers (biological molecules like peptides, proteins, and lipids) determined from DBS samples using mass spectrometry (11). Immunoassays can also be used to detect nucleic acids from and antibodies to viruses. These can be used to screen for such diseases as cytomegalovirus, herpes simplex virus, hepatitis A, hepatitis C, and HIV.

The 2017 WHO Guidelines [18] on hepatitis B and C testing recommended consideration of DBS testing in the following circumstances:

    • There are no facilities or expertise are available for conventional venipuncture
    • Rapid diagnostic tests are not available
    • It is being used for persons with poor venous access, such as those in drug treatment programs or prisons.

Finally, genomic testing using DBS [19] has allowed the detection of mutations responsible for fragile X syndrome, thalassemia, and some cancers.

The range of emerging DBS applications is vast

The range of emerging DBS applications [20] is vast and includes the following:

  • preclinical drug development
  • toxicokinetic (TK) and pharmacokinetic (PK) studies
  • clinical pharmacology
  • metabolic profiling
  • therapeutic drug monitoring
  • forensic toxicology
  • environmental contaminant control
  • epidemiological disease surveillance

Related Content: What You Need to Know About Hepatitis

Excitement for the potential of DBS

Further collaboration around standardization for both collection and transport as well as analysis and storage of DBS is required. But the excitement over the potential of DBS is typified by the comments of Dr. Olivier Rabin (2), WADA Senior Executive Director, Sciences and International Partnerships. When discussing the utilization of DBS testing in sports anti-doping programs, he said,

“The possible advantages of DBS are clear. It has the potential to add to the current global anti-doping program by complementing existing urine and blood testing to expand upon the program’s testing coverage and capacity to better reveal doping practices.

WADA is committed to making available new ways of protecting clean sport that reduce the inconvenience or discomfort for athletes and is easier, more effective and cheaper to carry out. In that way, it could be that DBS will be a major breakthrough in global anti-doping testing capacity.”


From screening to monitoring, DBS technology has the potential to bring convenient, cost-effective blood testing to a wide variety of clinical applications for patients in all corners of the world. Innovations in sample collection, storage, and analysis provide the opportunity to greatly broaden the adoption of DBS technology beyond the few large centers that perform DBS analysis today.

Continued standardization validation of DBS reference values for a wider variety of substances and standardization of clinical processes for collection and analysis of DBS samples (including further automation) will reduce barriers to widespread adoption.


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Additional articles on diagnostic innovations:
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–The Promise of Menstrual Blood Diagnostics



[1] Panja, Tariq. (2019, December 10th). Russia Banned From Olympics and Global Sports for 4 Years Over Doping. https://www.nytimes.com/2019/12/09/sports/russia-doping-ban.html

[2] WADA (2019, October 3rd). WADA leads exciting collaboration on dried-blood-spot testing. https://www.wada-ama.org/en/media/news/2019-10/wada-leads-exciting-collaboration-on-dried-blood-spot-testing

[3] UNAIDS (2014, October). 90-90-90 An ambitious treatment target to help end the AIDS epidemic. https://www.unaids.org/sites/default/files/media_asset/90-90-90_en.pdf

[4] Neogi, Ujjwal. Gupta, Soham. Rodridges, Rashmi. Nalini Sahoo, Pravat. D. Rao, Shwetha. B. Rewari, Bharat. Shastra, Suresh. De Costa, Ayesha. Shet, Anita (2012). Dried blood spot HIV-1 RNA quantification: A useful tool for viral load monitoring among HIV-infected individuals in India (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3612324/).

[5] MM De Kesel, Pieter. Sadones, Nele. Capiau, Sara. E Lambert, Willy. P. Stove, Christophe (2013). Hemato-critical issues in quantitative analysis of dried blood spots: challenges and solutions (https://www.future-science.com/doi/10.4155/bio.13.156).

[6] Lehmann, Sylvain. Delaby, Constance. Vialaret, Jerome. Ducos, Jacques. Hirtz, Christohe (2013) Current and future use of “dried blood spot” analyses in clinical chemistry (https://www.degruyter.com/view/j/cclm.2013.51.issue-10/cclm-2013-0228/cclm-2013-0228.xml?f=&print).

[7] Guthrie, R. Susi, A (1963). A simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants (https://www.ncbi.nlm.nih.gov/pubmed/14063511)

[8] Lehmann, Sylvain. Delaby, Constance. Vialaret, Jerome. Ducos, Jacques. Hirtz, Christohe (2013) Current and future use of “dried blood spot” analyses in clinical chemistry (https://www.degruyter.com/view/j/cclm.2013.51.issue-10/cclm-2013-0228/cclm-2013-0228.xml?f=&print).

[9] E Nichols, Brooke. J Girdwood, Sarah. Crompton, Thomas. Steward-Isherwood, Lynsey. Berrie, Leigh. Chimhamiwa, Dorman. Moyo, Crispin. Kuehnle, John. Stevens, Wendy. Rosen, Sydney (2019). Monitoring viral load for the last mile: what will it cost? (https://onlinelibrary.wiley.com/doi/full/10.1002/jia2.25337).

[10] Taleb, F. Tran Hong, T. Ho, HT. Nguyen, Thanh B. Pham Phuon, T. Viet Ta, D. Le Thi Hong, N. Ba Pham, H. Nguyen, LTH. Nguyen, HT. Tuaillon, E. Delaporte, E. Le Thi, H. Tran Thi Bich, H. Nguyen, TA. Madec, Y (2018). First field evaluation of the optimized CE marked Abbott protocol for HIV RNA testing on dried blood spot in a routine clinical setting in Vietnam (https://www.ncbi.nlm.nih.gov/pubmed/29425216).

[11]Zakaria, R. Allen, KJ. Koplin, JJ. Roche, P. Greaves, RF (2016) Advantages and Challenges of Dried Blood Spot Analysis by Mass Spectrometry Across the Total Testing Process (https://www.ncbi.nlm.nih.gov/pubmed/28149263).

[12] Spot On Sciences. Product Catalog – HemaSpot Devices (https://www.spotonsciences.com/products/).

[13] Gruner, Nico. Stambouli, Oumaima. Stefan Ross, R (2015). Dried Blood Spots – Preparing and Processing for Use in Immunoassays and in Molecular Techniques (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4397000/).

[14] Waters – The Science of What’s Possible (2020). Beginners Guide to Liquid Chromatography (https://www.waters.com/waters/en_US/HPLC—High-Performance-Liquid-Chromatography-Explained/nav.htm?locale=en_US&cid=10048919)

[15] Khan Academy (2016). Isotopes and mass spectrometry (https://www.khanacademy.org/science/chemistry/atomic-structure-and-properties/mass-spectrometry/a/isotopes-and-mass-spectrometry).

[16] R Venter, Andre. A Douglass, Kevin. T Shelley, Jacob. Hasman Jr, Gregg. Honarvar, Elahe (2013). Mechanisms of Real-Time, Proximal Sample Processing during Ambient Ionization Mass Spectrometry (https://pubs.acs.org/doi/10.1021/ac4038569#).

[17] Gaugler, Stefan. K Al-Mazrou, Maha. Y Issa, Sahar. Rykl, Jana. Grill, Matthias. Qanair, Asem. L Cebolla, Vicente (2018). Fully Automated Forensic Routine Dried Blood Spot Screening for Workplace Testing (https://academic.oup.com/jat/article-abstract/43/3/212/5112958?redirectedFrom=PDF).

[18] World Health Organization (2018). Guidelines for the care and treatment of persons diagnosed with chronic hepatitis c virus infection (https://apps.who.int/iris/bitstream/handle/10665/273174/9789241550345-eng.pdf).

[19] Lehmann, S. Delaby, C. Vialaret, J. Ducos, J. Hirtz, C (2013). Current and future use of “dried blood spot” analyses in clinical chemistry (https://www.ncbi.nlm.nih.gov/pubmed/23740687).

[20] A Demirv, Plamen (2013). Dried Blood Spots: Analysis and Applications (https://pubs.acs.org/doi/10.1021/ac303205m)

Financial disclosure: Dr. Lerman is a consultant for Spot Bioscience.

Robert Lerman, M.D.

Website: https://www.tribecacompanies.com

Dr. Rob Lerman, M.D., is a Medical Affairs consultant for Tribeca Companies, focused on leveraging technology and innovation to improve healthcare quality and cost-effectiveness. 

Rob is the Chief Medical Officer and Vice-President of Clinical Operations for LindaCare, a global digital health company focused on remote patient monitoring solutions for patients with implantable cardiac devices. Previously, he served as a corporate Vice-President in the supply chain and population health departments for Dignity Health, a 39-hospital integrated delivery network.

Before joining Dignity Health in 2012, Rob practiced clinical cardiac electrophysiology for 18 years in Southern California where he was medical director of Cardiac Electrophysiology at Dignity Health St. Bernardine Medical Center and served as an Assistant Clinical Professor of Medicine for the UCLA School of Medicine. He was active throughout his clinical career in research on implantable cardioverter defibrillator therapy.

A native of Los Angeles, Rob completed his undergraduate degree at Stanford University and earned his medical degree at Albany Medical College. He completed a residency in internal medicine at Cedars-Sinai Medical Center, followed by fellowships in Cardiovascular Disease and Clinical Cardiac Electrophysiology at Harbor-UCLA Medical Center and Good Samaritan Hospital in Los Angeles.

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