Over a century since a new blood sampling method based on the use of a dry matrix was first described by Ivar Bang , the interest in dried blood spot technology has continuously evolved. This alternative approach, based on collecting blood spots on blotting paper and drying them, is called “dried blood spot” or DBS. In 1963, Robert Guthrie used this technique to develop systematic neonatal screening for the metabolic disease, phenylketonuria . Set up for the first time in Scotland, this use of DBS spread to the UK in the 1970s, mainly to detect any innate errors in metabolism that were treatable. Of note, the use of DBS remains almost exclusively limited to this type of neonatal screening, even though many studies demonstrate its potential application in clinical biology, as well as in research. Indeed, classical clinical chemistry methods, small molecule and lipid analysis or molecular biology approaches, are all perfectly suited to the use of DBS. However, one limitation is represented by the small blood volumes associated with DBS sampling (5–10 µL) and therefore the need for very sensitive methods. Recent technological advances, in microfluidics, multiplex immunological/genomic detection systems, and mass spectrometry, could however settle most sensitivity problems. In this overview we will summarize the pros and cons of this particular biological sampling method and evaluate its future role in clinical biology.
General DBS procedure
Collection and sampling
The collection area (finger, heel) has to be first disinfected. The skin is then punctured with a sterile lancet (Figure 1). The first blood drop is dabbed and subsequent drops are placed on blotting paper marked with circles to be filled. Once all the required circles are filled, the blotting paper is left to dry for a few hours at room temperature on a non-absorbent surface. The drying time is very important as residual humidity favors bacterial development or molds and modifies the extraction stage .
Once dry, the DBS cards are moved into a waterproof plastic bag, possibly along with a desiccant and a humidity indicator . The purpose of the desiccant is to finalize the drying process, which also minimizes any risk of infection associated with sampling. Periods of storage at room temperature vary according to the biological factor, from 1 week for proteins , to 1 year or more for nucleic acids . As far as serology is concerned, the blotting papers are usually kept at –20°C upon receipt . For long-term preservation (up to several years) the blotting papers are stored either at −20°C or –80°C [8, 9].
Extraction of the analytes from DBS specimens needs to be achieved using a standard procedure. One or more 2–8 mm diameter discs are then created with a specific punch. These small “spots” are placed in an elution buffer for variable time spans according to the procedure. The DBS extraction is then treated as a hemolyzed whole blood sample, and tested with methods often intended for plasma or serum. The elution buffer plays a major role in re-solubilizing the analytes to be tested. A wide variety of buffers are described in the literature. The most common are saline/phosphate buffers, often with added detergents (Tween, Triton…), carrier proteins and chelators [ethylene diamine tetra acetic acid (EDTA)], as well as organic buffers with methanol, acetonitrile or ethanol. For nucleic acids, standard commercial kits exist which are compatible with molecular biology tests, from polymerase chain reaction (PCR) to genomic chips .
Pros and cons of DBS
One of the main advantages of using DBS technology is that it allows access to samples in pre-analytical situations were standard blood collection is challenging (problem with sampling, storage). The typical DBS contains approximately 50 µL of whole blood on an average surface of 12 mm2 (Figure 2). It enables the testing of various analytes such as nucleic acids, proteins, lipids, or small organic and non-organic molecules (Table 1). Two types of DBS are mostly available: cotton paper filters of different qualities (Whatmann 903 Protein Saver Cards Whatmann, Springfield Mill, UK; Perkin Elmer 226 Spot Saver Card, Perkin Elmer, Waltham, USA) and glass microfiber filter papers (Agilent Bond Elut DMS, Santa Clara, CA, USA; Sartorius Glass Microfiber Filters, Goettingen, Germany). The main difference between the two supports is that the glass fiber does not soak up reagents, which diminishes non-specific analyte adsorption on the membrane.
In comparison to conventional blood testing, DBS offers practical, clinical and financial advantages. Firstly, DBS collection is easy to perform and relatively painless (Figure 1). It can be carried out by the patient at home, without the need for specialized structures such as medical laboratories. This sampling procedure is far less invasive than venipuncture, therefore is better suited for patients requiring numerous blood tests, such as those with damaged/altered veins, the elderly or infants. The use of DBS also minimizes the volume of blood taken from patients. It has been shown that drying the blood spot on blotting paper damages the capsid of viruses [HIV, Cytomegalovirus (CMV), hepatitis C virus (HCV), human T-lymphotropic virus (HTLV)]  reducing any possible risk of contamination for medical or paramedical staff . In addition, it enables the shipping of samples by regular mail with no particular risk of contamination. This represents a valuable asset for sampling in remote communities either located far away from a testing laboratory or with limited technical infrastructure available, therefore provides added value compared to standard blood sampling . Through its small size and stacking capacity, DBS is also a valuable solution for reducing and facilitating storage in clinical laboratories and biobanks . It is noteworthy that in case of storage, an individual bagging or a separation using a sturdy paper will be important to avoid the possibility of cross-contamination between cards . These properties of DBS have been utilized in experimental research, by facilitating pharmacological studies and pharmacokinetics on small animals with very limited volumes of biological liquids. This follows the regulations aimed at protecting small animals (decreasing sample volume and sophistication of sampling methods) during pre-clinical studies . Concerning sample stability, many studies have shown that most analytes from whole blood are stable at room temperature for at least 7 days. In some cases such as opiates, DBS even increases stability during storage , and nucleic acids are a major tool for short- and long-term preservation, as they can be isolated after several months at room temperature and several years at −20°C. . From a medico-economical point of view, it is interesting to note that the use of DBS allows a significant cost reduction due to decreased requirements in trained staff, facilitated transportation, storage, and processing.
A major drawback of DBS technology resides in the nature of the biological sample itself (Figure 2). In a standard sampling procedure, either serum or plasma is analyzed, whereas DBS samples are composed of hemolyzed whole blood. Hence, interferences due to hemoglobin and the release of intracellular content could occur. The blood cells (erythrocytes, leukocytes, platelets etc.) are altered by the drying process, thus cellular hematological testing is impossible. Drying can also denature proteins and alters the enzymatic activity of blood proteins (aspartate transaminase). Any remaining cells in the samples can also change the global protein composition and therefore modify the concentration of some analytes. In some cases, clinical thresholds set up using standard blood samples need to be adapted. Hematocrit that affects blood dispersal on the blotting paper also needs to be taken into account . The small volume of samples resulting from the DBS can be a disadvantage for low sensitivity assays  and for running multiple tests.
Use of DBS in clinical chemistry
The primary use of DBS in France is systematic neonatal screening. As blood sampling in newborns is difficult, DBS technology represents a viable alternative. DBS testing was set up in 1978 by the French Association for screening and preventing disabilities in children (http://www.afdphe.org/). Sampling of newborns enables the detection of phenylketonuria, hypothyroidism, adrenal hyperplasia, cystic fibrosis and sickle cell disease (in some areas). The extension of these tests to cover a wider number of diseases, similar as in to USA, is currently under consideration . A positive result will always be confirmed or denied by further specific tests. Beyond its use for neonatal screening, many clinical analytes can be measured using DBS. These analytes are divided into four major categories as follows (see also Table 1):
Exogenous nucleic acids
The measurement of nucleic acids is typically required in the virology field. There is a growing interest in viral screening through nucleic acid detection (RNA, DNA) using DBS, as current molecular biology technologies [quantitative polymerase chain reaction (Q-PCR), reverse transcription polymerase chain reaction (RT-PCR)] are very sensitive and require only a small sample amount (<20 µL). Nevertheless, it is important to note that the amount of material available from a DBS sample is between 1 and 2 logs lower compared to a standard serum or plasma sample. The preservation of nucleic acids on blotting paper is stable for long periods , providing it is dried and stored away from humidity in a suitable plastic bag containing a desiccant. DBS nucleic acid detection is mainly used in screening for viral diseases such as cytomegalovirus , herpes simplex virus , hepatitis A , hepatitis C  and HIV .
Peptides – proteins
Concerning proteins and peptides one caveat is represented by the relative difficulty of their extraction from DBS samples, as well as the low sensitivity of certain protein dosage. The main proteins measured from DBS can be classified into two groups: standard serum proteins and antibodies. The most widely used analytical techniques are immunological assays which measure clinical analytes with good specificities and sensitivities. An example is represented by the immunoturbidimetric assay for glycated hemoglobin (to monitor glycemic balance in diabetic patients). Glycated hemoglobin measured from DBS samples correlate well with standard tests. In addition, this analyte remains stable for over 15 days on DBS . DBS is also well adapted for the enzyme-linked immunosorbent assay (ELISA) detection of specific antibodies against Epstein-Barr virus , Rubella virus , dengue virus  or hepatitis C [7, 59] and HIV virus .
An interesting evolution of liquid chromatography/mass spectrometry (LC/MS) is represented by quantitative techniques for measuring peptides and proteins . This approach was adapted on DBS to measure ceruloplasmin for the neonatal screening of Wilson’s disease  and for peptide C quantification . When used in multiplex mode (multiple reaction monitoring) this mass spectrometry method has the potential to measure many analytes within only a few microliters . For instance, Chambers et al.  have succeeded in quantifying a panel of 40 serum proteins from DBS, using this approach.
Lipids, sugars and small molecules
Phenylalanine, an amino acid measured in phenylketonuria screening of newborns, exemplifies the dosage of small molecules using DBS . Small organic molecules are significantly less sensitive than proteins to the drying process which characterizes DBS samples. In addition, the major progress of liquid chromatography/mass spectrometry (LC/MS) in this field has allowed the quantification of many small molecules such as vitamin D  or lipids . For instance, high levels of triglycerides, representing an important risk for cardiovascular and coronary diseases, can be quantified using DBS. These analytes remain stable on DBS for 30 days at room temperature and up to 90 days at 4°C. The profiling of glycans on DBS was also recently achieved using mass spectrometry .
In 1993, Henderson et al.  demonstrated the use of DBS for detecting narcotics, such as cocaine, through modification of a radioimmunoassay (RIA) commercial kit. Xenobiotic testing using DBS has since played an important role, mainly by the screening of antimalarial and antiretroviral drugs by LC/MS in isolated populations . Another example is represented by the quantification of nine antiretroviral molecules in HIV using DBS. This detection method has been validated by the Food and Drug Administration (FDA) with sample stability ranging from 12 to over 90 days at room temperature . In the field of toxicology, which is a major application of DBS , Saussereau et al.  have, for example, developed a new drug screening method based on LC/MS using on-line extraction for the quantification of opiates, cocainics or amphetamines. The development of these new measurement techniques, based on LC/MS for xenobiotics, will greatly increase the interest of using DBS in clinical chemistry.
The clinical potential of DBS for genomics has been demonstrated as early as 1987 . DNA micro-extraction from dried blood has allowed the detection of mutations responsible for diseases such as cystic fibrosis , X fragile syndrome , spinal muscular atrophy , cancers  and thalassemia . DBS, which is a fairly inexpensive sampling and storage method, is also a good choice for genetic material biobanks . For instance, the Danish national biobank for neonatal screening includes over 2 million DBS which virtually corresponds to all Danish people born since 1982.
The use of DBS has many advantages in terms of sampling, transportation, storage and biosafety when compared to classical collection methods. One interesting aspect of DBS is the possibility of simplified “self/home blood sampling”. The patient will be able to independently and safely collect a blood sample. The DBS will then be sent to the laboratory by mail. As described in this review, many clinical analytes are already available on DBS, and more are to follow, thanks to innovative approaches. Indeed, development of microfluidics, multiplex immunological/genomic detection systems, mass spectrometry and automated DBS processing open new interesting clinical prospects. The detection and follow-up of metabolic, infectious and chronic diseases could therefore rely on the use of DBS. Both the patient and society could benefit from this technology. Already, several public and commercial laboratories in both Europe and USA are offering DBS kits for a broad range of analytes often grouped into panels for hormonal, metabolic or cardiovascular diseases. This evolution could dramatically change how clinical chemistry pre-analytics are handled in the future.
The authors thank Rachel Almeras, Bader Al Taweel, Domitille Héron and Thibault Fortane for their initial help in the writing of this review and Brigitte Lehmann for editing the manuscript.
Conflict of interest statement
Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
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About the article
Prof. Sylvain Lehmann trained as an MD/PhD (1992, Strasbourg, France). He was the recipient of a Howard Hughes fellowship for physicians and spent 4 years in Washington University, St Louis, MO, USA, as a postdoctoral fellow and a research Assistant Professor. From 1997, he was a researcher of the French National Research Institut (INSERM) and in 2003 he obtained a position as Professor of Biochemistry at the Medical School of Montpellier (France). His research focuses on neurodegenerative disorders (Alzheimer, prion, etc.) and on clinical proteomics. He is a vice-president of the French National Society of Clinical Biology (SFBC) in charge of its Scientific Committee and he chairs the Working Group “Clinical Quantitative Mass Spectrometry Proteomics” of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC).
Constance Dalaby holds a PhD in Biology and graduated from University Paris 7 (France) in 2006. In 2005, she was recruited as a research assistant in Biochemistry by the Clinical Unit of Biochemistry at Pr Jean-Charles Deybach (Hôpital Louis Mourier, Colombes, France), as a member of the French Reference Center of Porphyrias. In 2008–2009, she collaborated with the Biochemistry Unit of the Hospital Clinic of Barcelona (Dr Jordi To-Figueras, Spain) and was recruited as an Assistant Professor in Biochemistry and Molecular Biology by the University Paris 7 (France) in 2009. Since 2011, she has been part of the Clinical Chemistry Laboratory (Proteomics Platform) of Pr Sylvain Lehmann (IRB, Hôpital Saint Eloi, CHRU Montpellier).
Jérôme Vialaret was born in 1984 and studied Organic Chemistry at Montpellier II University (obtaining his Master’s degree in 2007). He specialized in proteomic while working for Pierre Fabre Laboratories, EPFL (Lausanne, Switzerland), INRA (Montpellier, France) and at Montpellier Hospital. Having amassed a wealth of experience in large scale proteomic (proteome and phosphoproteome) with dedicated quantitative methods (silac and label-free), he focused on the development of protein quantification using targeted mass spectrometry in a clinical environment. He is in charge of these method developments in the Clinical Proteomic Platform of the Laboratory of Biochemistry and Clinical Proteomic directed by Pr. Sylvain Lehmann in Montpellier.
Dr. Jacques Ducos was trained as an MD (1983, Montpellier, France) and PhD (1993, Montpellier, France). He was a resident in the Montpellier Hospital (1979–1983). He specializes in Virology (HBV/ HCV/HIV) and his research is focusing on biological markers of viral infections. He is currently responsible for the functional unit of viral hepatitis at the Montpellier Hospital (Lapeyronie, France). He is the president of the Viral Hepatitis Network of the Languedoc Roussillon Camargue (RHEVIR).
Christophe Hirtz was born in 1972 and studied Biochemistry at Paul Sabatier University in Toulouse. Having got his Doctorate and an accreditation to supervise research in the proteomic field, he was recruited in 2008 as an Associate Professor at the University Montpellier I and is specialized in Biochemistry and Clinical Proteomic. His scientific interest includes the development of a new method of protein quantification using targeted mass spectrometry in a clinical environment. He is in charge of the Clinical Proteomic Platform of the Laboratory of Biochemistry and Clinical Proteomic directed by Pr. Sylvain Lehmann in Montpellier.
Published Online: 2013-06-01
Published in Print: 2013-10-01