Current And future use of “dried blood spot” analyses in clinical chemistry

Abstract

Abstract The analysis of blood spotted and dried on a matrix (i.e., "dried blood spot" or DBS) has been used since the 1960s in clinical chemistry; mostly for neonatal screening. Since then, many clinical analytes, including nucleic acids, small molecules and lipids, have been successfully measured using DBS. Although this pre-analytical approach represents an interesting alternative to classical venous blood sampling, its routine use is limited. Here, we review the application of DBS technology in clinical chemistry, and evaluate its future role supported by new analytical methods such as mass spectrometry.

Full text

For Review Only
Current and future us
e of “Dried Blood Spot” analyses in
clinical chemistry
Journal:
Clinical Chemistry and Laboratory Medicine
Manuscript ID:
CCLM.2013.0228
Manuscript Type:
Review
Date Submitted by the Author:
26-Mar-2013
Complete List of Authors:
Lehmann, Sylvain; CHU Montpellier, IRB
DELABY, Constance; CHU Montpellier, IRB
VIALARET, Jérôme; CHU Montpellier, IRB
DUCOS, Jacques; CHU Montpellier, Unité de Virologie Lapeyronie
HIRTZ, Christophe; CHU Montpellier, IRB
Section/Category:
General Clinical Chemistry and Laboratory Medicine
Classifications:
70.101 Blood sampling < 70.100 Sampling < 70 Reference Values, 70.105
Preanalytical phase < 70.100 Sampling < 70 Reference Values, 70.107
Sample handing < 70.100 Sampling < 70 Reference Values
Keywords:
Dry Blood Spot, Preanalytics, Mass spectrometry
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Current and future use of “Dried Blood Spot” analyses in clinical chemistry
Sylvain LEHMANN
1
, Constance DELABY
1,2
, Jérôme VIALARET
1
, Jacques DUCOS
3
and
Christophe HIRTZ
1
Affiliations
(1) CHU Montpellier, Institut de Recherche en Biothérapie, hôpital St Eloi, Laboratoire de
Biochimie Protéomique Clinique et CCBHM, Montpellier, F-34000 France. Université
MONTPELLIER 1, Montpellier, F-34000 France. INSERM U1040, Montpellier, F-34000
France.
(2) Université Paris 7-Denis Diderot, France.
(3) CHU Montpellier, Unité de Virologie Lapeyronie, Montpellier, F-34000 France. INSERM
U1058, Montpellier, F-34000 France.
2204 words, 1 table, 2 figures, 123 references
Running title: DBS in clinical chemistry
3-6 Keywords: Dry Blood Spot, Preanalytics, Mass spectrometry.
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Abstract
The analysis of blood spotted and dried on a matrix (i.e. "Dried Blood Spot" or DBS) has
been used since the 1960s in clinical chemistry; mostly for neonatal screening. Since then,
many clinical analytes, including nucleic acids, small molecules and lipids, have been
successfully measured using DBS. Although this pre-analytical approach represents an
interesting alternative to classical venous blood sampling, its routine use is limited. Here, we
review the application of DBS technology in clinical chemistry, and evaluate its future role
supported by new analytical methods such as mass spectrometry.
Introduction
Over a century since a new blood sampling method based on the use of a dry matrix was first
described by Ivar Bang (1), the interest in DBS 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 (2). Set up for the
first time in Scotland, this use of DBS spread to the UK in the seventies, 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
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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 (3).
Conservation
Once dry, the DBS cards are moved into a waterproof plastic bag, possibly along with a
desiccant and a humidity indicator (4). 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 one week for
proteins (5), to one year or more for nucleic acids (6). As far as serology is concerned, the
blotting papers are usually kept at -20°C upon receipt (7). For long term preservation (up to
several years) the blotting papers are stored either at -20°C or -80°C (8, 9).
Extraction
Extraction of the analytes from DBS specimens needs to be achieved using a standard
procedure. One or more 2 to 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
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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 (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 PCR to genomic chips (10).
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 50µl of whole blood on an
average surface of 12 mm
2
(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, Perkin Elmer 226 Spot Saver Card..) and glass microfiber filter papers
(Agilent Bond Elut DMS, Sartorius Glass Microfiber Filters…). 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 (Fig. 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 (VIH, CMV, VHC, HTLV) (11) reducing any possible risk of
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contamination for medical or paramedical staff (4). 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 (12). Through its small size and stacking capacity, DBS is also a
valuable solution for reducing and facilitating storage in clinical laboratories and biobanks
(13). 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 (14).
Concerning sample stability, many studies have shown that most analytes from whole blood
are stable at room temperature for at least seven days. In some cases such as opiates, DBS
even increases stability during storage (15), 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. (16). 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
(Fig. 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
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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 (17). The
small volume of samples resulting from the DBS can be a disadvantage for low sensitivity
assays (4) 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 to US, is currently
under consideration (18). 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 (Q-PCR, 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 (3), 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 (19), herpes simplex virus (20), hepatitis A (21), hepatitis C
(22) and HIV (23).
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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 (24). DBS is
also well adapted for the Enzyme-linked immunosorbent assay (ELISA) detection of specific
antibodies against Epstein-Barr virus (25), Rubella virus (26), dengue virus (27) or hepatitis C
(7, 12) and HIV virus (22).
An interesting evolution of mass spectrometry (LC- MS/MS) is represented by quantitative
techniques for measuring peptides and proteins (28). This approach was adapted on DBS to
measure ceruloplasmin for the neonatal screening of Wilson’s disease (18) and for peptide C
quantification (29). When used in multiplex mode (multiple reaction monitoring) this mass
spectrometry method has the potential to measure many analytes within only a few microliters
hirtz (28). For instance, Chambers et al. (30) 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 (2). Small organic molecules are
significantly less sensitive than proteins to the drying process which characterizes DBS
samples. In addition, the major progress of mass spectrometry in this field has allowed the
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quantification of many small molecules such as vitamin D (31) or lipids (32). 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 (33).
Xenobiotics
In 1993, Henderson et al. (34) 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 mass spectrometry (LC/MS) in isolated populations (35). Another
example is represented by the quantification of 9 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 (36). The development
of these new measurement techniques, based on LC/MS for xenobiotics, will greatly increase
the interest of using DBS in clinical chemistry.
Genomics
The clinical potential of DBS for genomics has been demonstrated as early as 1987 (37).
DNA micro-extraction from dried blood has allowed the detection of mutations responsible
for diseases such as cystic fibrosis (38), X fragile syndrome (39), Spinal Muscular Atrophy
(40), cancers (41) and thalassemia (42). DBS, which is a fairly inexpensive sampling and
storage method, is also a good choice for genetic material biobanks (43). For instance, the
Danish national biobank for neonatal screening (DNSB) includes over 2 million DBS which
virtually corresponds to all Danish people born since 1982.
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Conclusions
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 such as mass spectrometry and the
development of fully automated DBS solutions. 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 North-America 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.
Acknowledgments
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.
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Table 1. Overview of DBS card usage in Clinical Chemistry other than its use for neonatal
screening.
Exogeneous nucleic acid
Methods Parameter Clinical interest References
Real Time PCR
Q PCR
Human herpesvirus type 6
Differentiation active human
herpesvirus type 6 infection
from inherited HHV-6
(
20
,
44
)
RT-PCR Human hepatitis C Monitoring hepatitis C virus
(HCV) infection among injecting
drug users
(7, 22)
Real Time PCR
Human hepatitis B Hepatitis B virus (HBV) DNA
quantification
(45)
Real Time PCR,
Q-PCR
Cytomegalovirus Diagnosis of human
congenital cytomegalovirus
infection
(19, 46)
Nested PCR, RNA assays,
RT-PCR
HIV Virus Detection of human
immunodeficiency virus
(8, 22, 47)
Peptid
es/Proteines
ELISA HIV Virus Human immunodeficiency virus
serotyping
(48)
ELISA C-reactive protein Cardiovascular risk (49)
DELFIA free-β human chorionic gonadotrophin (free-β
hCG) and PAPP-A
Fœtal aneuploidy risk (50)
Immuno
-
fluorometric assays
Luteinizing hormone and follicle
-
stimulating
hormone
circulating gonadotropin
concentrations
(
51
)
Chemiluminescent
immunoassay
Prostate Specific Antigen (PSA) Prostate cancer screening (52)
RIA Somatedin-C (IGF-1) Screening test for growth
hormone deficiency
(53)
ELISA Apoliproteins B Hypercholesterolemia (54)
Immune nephelometry Alpha1-antitrypsin Alpha1-antitrypsin deficiency
(5)
ELISA Alpha-Fetoprotein Open neural tube
defect and Down syndrome
(55)
Enzyme assays Biotinidase Biotinidase deficiency (56)
EIA Calcitonin gene-related peptide Children with autism or mental
retardation
(57)
LC-MS/MS Ceruloplasmin Wilson disease (18)
Spectrophotometry Hemoglobin Folate analysis (58)
Turbidimetric immunoassay Glycated hemoglobin A1c Diagnosis and treatment of
diabetes
(24)
LC-MS/MS HbA2 Diagnosis of thalassemia (59)
Non
-
radiochemical
HPLC
Hypoxanthine
-
guanine phosphoribosyltransferase
adenine phosphoribosyltransferase adenosine
deaminase
Purine m
etabolism disorders
(
60
)
LC-MS/MS Iduronate 2-sulfatase Diagnosis of hunter disease (61)
ELISA, RIA insulin-like growth factor Evaluation of growth
hormone status
(62)
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ELISA Prolactin Diagnosis of epilepsy (63)
ELISA
Transferrin receptor
Iron deficiency
(
64
)
DELFIA Thyroglobulin Thyroid status (65)
ELISA CD4 CD4+ lymphocyte counts in HIV
patients
(66)
ELISA Measles and rubella IgM and IgG Detection of measles and rubella
IgM and IgG
(67)
DELFIA
Toxoplasma
gondii
-
specific
IgM
and
IgA
Screening of congenital
toxoplasmosis
(
68
)
RIA Insulin Diagnosis of hyperglycemia /
hyper-insulinemia
(69)
Enzyme assays Acid alpha-glucosidase Glycogen storage disease II (70)
Enzyme assays
8 lysosomal enzymes
Clinical
differentiation among
mucopolysaccharidosis,
oligosaccharidosis, and
mucolipidosis II and III
(
71
)
Enzyme assays α-iduronidase activity Diagnosis of alpha-L-iduronidase
deficiency
(72)
Biochemistry phytanic acid and pristanic acid Diagnosis of peroxisomal
disorders
(73)
Electro-immunodiffusion Béta-Lipoprotein Familial type II and combined
hyperlipidemia.
(74)
ELISA
Fumarylacetoacetase
Hereditary tyrosinemia type I
(
75
)
Luminex TGF-β1, (MCP-1, (MIP-1α, MIP-1β, NT-4, BDNF,
RANTES, CRP, MMP-9...
Inflammatory status (76)
Enzyme immunoassay
IgE
Allergic diseas
e and repeated
macro-parasitic infections
(
77
)
ELISA IgG and IgA Nasopharyngeal carcinoma
screening.
(25)
Enzyme assays Lysosomal b-d-galactosidase (bG; EC 3.2.1.23) Mucopolisaccharidosis type I (78)
Fluorometric immunoassay
Thyroid
-
Stimulating Hormone
Immunoreactive Trypsin, Creatine
Kinase
MM Isoenzyme
Congenital hypothyroidism,
congenital adrenal hyperplasia,
andMuscular dystrophy
(
79
)
Column chromatography Thyroxine-Binding Globulin Neonatal hypothyroidism (80)
Immunoassay
Trypsine immunoreactive (IRT)
Cystic fibrosis
(
81
)
ELISA Antibodies against hepatitis A
Hepatitis A (21, 82)
ELISA
Antibodies against hepatitis B
Hepatitis B
(
83
)
CORECELL
Maternal antibody to
hepatitis B
Infection with HBV (84).
ELISA
Anti
-
HCV antibodies
Detection of antibodies to
hepatitis C virus
(
12
,
85
)
ELISA
Anti
-
malarial antibodies
Diagnosis of malaria
(
86
)
ELISA
Pseudomonas aeruginosa antibodies
Pseudomonas aeruginosa in
patients with cystic fibrosis
(87)
ELISA Thyroid antibody Thyroid-antibody screening (88)
ELISA
Antibodies against tetanus Screening of tetanus and
diphtheria toxins
(89)
ELISA
Antibodies against Brucella Diagnosis of human brucellosis (90)
ELISA
Antibodies against cysticercus Detection of anti-cysticercus
antibodies
(91)
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ELISA
Antibody against HTLV-1 and HTLV-2 Detection of the Human T-
lymphotropic virus
(92)
Immuno-fluorescence
Antibodies against to Coxiella burnetii, Bartonella
quintana, and Rickettsia conorii
Diagnosis of Rickettsial Diseases (93)
ELISA Antibody against syphilis Diagnosis of syphilis (94)
Indirect hemagglutination
test
Antibody against treponema Diagnosis of syphilis (95)
ELISA
Antibody against Trypanosoma cruzi
Diagnosis Trypanosoma
cruzi infections
(
96
)
ELISA
Antibody against Trichomonas vaginalis Seroepidemiology of
Trichomonas vaginalis
(97)
Fluorescent Galactose-1-phosphate uridyltransferase (GALT) Galactosemia (98)
ELISA Epstein Barr Virus Epstein-barr virus
immunoglobulin G (IgG) serology
(25)
EIA
Rubella Virus
Detection of congenital Rubella
virus
(
26
)
EIA
Dengue Virus
Dengue virus diagnosis
(
27
)
ELISA antibodies against hepatitis A
Hepatitis A (21, 82)
ELISA Antibodies against hepatitis B Hepatitis B (83)
CORECELL
Maternal antibody to
hepatitis B
Infection with HBV (84)
ELISA
Anti-HCV antibodies Detection of antibodies to
hepatitis C virus
(12, 85)
Multiplex ligation-
dependent probe
amplification on DNA
(MLPA)
Detecting 22q11.2 deletions
Manifestations associated with
DiGeorge Syndrome
(99)
PCR GSTM1 et GSTT1 gene variant Researching paediatric cancer
susceptibility genes.
(41)
ELISA multiplex
Human papillomaviruses (HPV), Helicobacter
pylori (H. pylori ), hepatitis C virus (HCV), and JC
polyomavirus (JCV).
Infections of human
papillomaviruses, Helicobacter
pylori, Hepatitis C Virus, and JC
Virus.
(100)
Lipids and Small molecules
Densitometry Phenylalanine Phenylketonuria
(2)
Enzymatic method Triglycerides Evaluation of the
cardiometabolic risk
(32)
LC-MS/MS Amino, organic, and fatty acid
Metabolic disorders (101)
Fluorimetric HPLC method
Homocysteine
Homocysteinuria
(
102
)
Enzymic methods Determination of glucose Monitoring of diabetic patients (103)
LC-MS/MS 17-OHP, androstenedione Congenital adrenal hyperplasia
(104)
HPLC Retinol Retinol analysis (105)
LC-MS/MS Thyroxin (T4) and TSH Congenital hypothyroidism (106)
Chemiluminescence Free thyroxine (FT4) Assessment of thyroid status (107)
LC-MS/MS Free carnitine Inborn errors of metabolism (108)
GC
-
MS
Methylcitrate
Newborn screening
for
propionic
acidaemia
(
109
)
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GC-MS Octanoate, decanoate, cis -4-decenoic acid
(C10:1) et cis -5-tetradecenoic acid
Free fatty acids (110)
LC-MS/MS Succinylacetone Hepatorenal tyrosinemia
(111)
FIA-ESI-MS/MS Guanidinoacetate and creatine
Primary creatine disorders (112)
Xenobiotics
LC-MS HIV antiretroviral drugs
(NVP, SQV, ATV, APV, DRV, RTV, LPV, EFV, ETV)
HIV Therapeutic follow up (36, 113)
RIA
Cocaine metabolite (benzoylecgonine)
Information on newborns and
maternal exposures to various
substances, including drugs of
abuse
(
114
)
LC/MS
quinine, mefloquine, sulfadoxine, pyrime
thamine,
lumefantrine, chloroquine
Blood levels of drugs
administered for Malaria and
pneumonia treatment
(
35
,
115
)
Capillary gas
chromatography
Dichlorodiphenyldichloroethylene
Newborns' body burden of
environmental pollutants
(
116
)
Fluorescence polarization
immunoassay
Theophylline Therapeutic drug monitoring (117)
Genomics
PCR Mutations of Factor V G1691A (FVL), prothrombin
(PT) G20210A, 5'10'methylenetetrahydrofolate
reductase (MTHFR) C677T, and methionine
synthase (MS) A2756G
Susceptibility to venous
thromboenbolism
(118)
Real-Time PCR Mutation C.-32T>G (IVS1-13>G) Acid Maltase deficiency (119)
DNA based assay Mutation (IVS4+919G->A) Fabry disease (120)
DHPLC Substitution (c.840C>T) Spinal muscular distrophy (121)
Specific restriction digest
method
Mutation (c.985A>G) Medium Chain acyl-coA
dehydrogenase deficiency
(MCADD)
(122)
PCR Mutation of Cystic Fibrosis Transmembrane
Conductance Regulator (CFTR)
Cystic Fibrosis (123)
PCR DNA mutation Beta thalassemia (42)
PCR
Real-time PCR
SMN1 exon 7 deletions
Copy number variations of SMN1 and SMN2
Spinal muscular atrophy (40)
PCR FMR1 methylation Fragile X syndrome (39)
Multiplex ligation
-
dependent probe
amplification on DNA
(MLPA)
Detecting 22q11.2 deletions
Manifestations associated with
DiGeorge Syndrome
(
99
)
PCR GSTM1 et GSTT1 gene variant Researching paediatric cancer
susceptibility genes.
(41)
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Legend
Table 1: Overview of DBS card usage in Clinical Chemistry other than its use for neonatal
screening.
Figure 1: DBS collection process: Peripheral blood is collecting by the patient at home. He
disinfects the area (finger) and pierces the skin using a sterile lancet before blotting the blood
onto high quality filter paper. The DBS is drying 1 to 3 hours at room temperature and
mailing using classical envelope. At the laboratory, the DBS is stored at room temperature.
The sample is punched (2-6 mm) and the analytes are extracted using an appropriate buffer
before analysis.
Figure 2: Comparison of the use of classical blood sampling vs DBS sampling resulting in a
100 fold reduction in blood volume and an ease of storage.
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59. Daniel YA, Turner C, Haynes RM, Hunt BJ, Dalton RN. Quantification of
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chemistry 2002;324:135-9.
61. Wang D, Wood T, Sadilek M, Scott CR, Turecek F, Gelb MH. Tandem mass
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62. Diamandi A, Khosravi MJ, Mistry J, Martinez V, Guevara-Aguirre J. Filter paper
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63. Fisher RS, Chan DW, Bare M, Lesser RP. Capillary prolactin measurement for
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64. McDade TW, Shell-Duncan B. Whole blood collected on filter paper provides a
minimally invasive method for assessing human transferrin receptor level. J Nutr
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65. Zimmermann MB, Moretti D, Chaouki N, Torresani T. Development of a dried whole-
blood spot thyroglobulin assay and its evaluation as an indicator of thyroid status in
goitrous children receiving iodized salt. Am J Clin Nutr 2003;77:1453-8.
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Patient
Disinfection of
the sampling
area
Prick with a
lancet
Deposit on
filter paper
Drying 1 to 3
hours at RT
Transport/
Mailing
Punch (2-6 mm
diameter)
Extraction with
Appropriate
buffer
Analyses
Figure 1
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1500 rpm
5 x 50µl
5-10 ml
VS. Dry
Storage 4°C
Storage Ambient T°
Whole blood with
hemolysis
Serum
or
Plasma
Whole blood with
cell preservation
Fig.1
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