Dried Blood Spot Analysis

Background

Dried blood spot (DBS) analysis has been practiced for ~100 years[1], but has started gaining traction as an alternate to traditional assays only this decade. Human / Animal blood is spotted on prepared filter cards, the cards are air dried, stored in airtight bags, and shipped under administered humidity and temperature levels[2]. For bioanalytical analysis, we punch tiny discs out of the DBS and analyze them using a validated LC / MS / MS method. Traditionally, scientists have used DBS for preclinical studies, newborn screening, as well as sample collection in resource-limiting settings. Recently, however, a variety of innovative DBS applications have started to emerge. These novel applications 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, as well as easier handling during sample storage and transportation. Other seemingly less obvious advantages of DBS are as listed below.

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

Study

We ran the study on an antiretroviral medicine, which is used in conjunction with other treatments for 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. They collected each sample at six individual spots for accurate / redundant analysis.

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

Conclusion

Generally, the compounds tested with DBS have shown promise. We, however, strongly recommend conducting validation and feasibility analysis prior to reporting results. As per FDA guidance, this validation should cover storage and handling temperature, homogeneity of sample spotting, hematocrit, stability, carryover, and reproducibility including ISR[4]. Even more, concordance between DBS and traditional plasma assays should be established if possible[5].

NorthEast BioLab offers the following DBS sample services.

  • 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