Dried Blood Spot Analysis


Dried blood spot (DBS) analysis has been practiced for ~100 years[1], but has gained anticipation as a feasible alternate to traditional plasma assays in specific instances only this decade. Human / Animal blood is spotted on prepared filter cards, the cards are air dried, stored in an airtight bag, and shipped under administered humidity and temperature levels[2]. For bioanalytical analysis, tiny discs are punched out of the DBS and analyzed using a validated LC / MS / MS method. DBS testing has traditionally been used for preclinical, systematic newborn screening and sample collection in resource-limiting settings. Recently, however, a variety of novel and innovative DBS applications have started to emerge. These include epidemiological surveys (e.g. HIV monitoring), therapeutic drug monitoring (TDM), as well as toxicology[3].

Benefits / Advantages

Primary advantages of DBS include lower sample volume collection, and easier handling during sample storage and transportation. Other less obvious DBS methodology advantages have been listed below.

  • Nonclinical: Fewer animals, lower sample volume, less invasive sampling, and better data quality from serial sampling as compared to composite samples
  • Clinical: Volunteer and clinical staff preference over venous cannula sampling
  • Bioanalytical: Minimal preparation, greater dry compound stability, and “Non-hazardous” classification post treatment of DBS cards
  • Processing and shipping: Simpler collection and transportation equipment


We ran the study on an antiretroviral medicine, used in conjunction with other medicines to treat HIV. Due to unavailability of cryo-freezers, it is generally difficult to properly collect, store, and maintain high integrity blood samples in remote areas. Additionally, the high shipping cost for transporting samples from these remote collection sites to far away sample analysis facilities was burdensome. Therefore, the investigators decided to use DBS sampling technique in this instance. Each sample was collected at six individual spots for accurate / redundant analysis.

We processed DBS samples, and analyzed using a LC/MS/MS method developed in our laboratory, and documented results of the analysis. Each sample was analyzed in five replicates, and results from these samples were compared. We observed the coefficient of variation (CV) among these samples to be less than 7.2%, indicating that the bioanalytical method was rugged and reproducible, and sample collection process was accurate.


Hitherto, all compounds tested with DBS have shown promise. However, we strongly recommend conducting validation and feasibility analysis prior to reporting results. Per FDA guidance, this validation should cover storage and handling temperature, homogeneity of sample spotting, hematocrit, stability, carryover, and reproducibility including ISR[4]. For good measure, concordance between DBS and traditional plasma assays should be established as well[5]. We offer the following DBS sample services from our facilities.

  • Clinical LC / MS / MS method development, validation, and analysis
  • Nonclinical LC/MS/MS bioanalytical support
  • GLP toxicology method development, validation, and analysis

[1]Hannon WH, Therell BL. Overview of the history and applications of dried blood spot samples. In: Li W, Lee MS, editors. Dried blood spots. Applications and techniques. Vol. 3. Hoboken: Wiley; 2014. pp. 3–15.

[2] Grüner N, Stambouli O, Ross RS. Dried blood spots–preparing and processing for use in immunoassays and in molecular techniques. J Vis Exp. 2015 Mar 13;(97). doi: 10.3791/52619.

[3] Stove CP1, Ingels AS, De Kesel PM, Lambert WE. Dried blood spots in toxicology: from the cradle to the grave? Crit Rev Toxicol. 2012 Mar;42(3):230-43. doi: 10.3109/10408444.2011.650790.

[4] U.S. Food and Drug Administration, Guidance for Industry – Bioanalytical Method Validation, September 2013 Biopharmaceutics Revision 1

[5] Neogi U, Gupta S, Rodridges R, Sahoo PN, Rao SD, Rewari BB, Shastri S, Costa AD, Shet A., Dried blood spot HIV-1 RNA quantification: a useful tool for viral load monitoring among HIV-infected individuals in India, Indian J Med Res. 2012 Dec;136(6):956-62