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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.
Content may be subject to copyright.
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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Clinical Chemistry and Laboratory Medicine
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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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63. Fisher RS, Chan DW, Bare M, Lesser RP. Capillary prolactin measurement for
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65. Zimmermann MB, Moretti D, Chaouki N, Torresani T. Development of a dried whole-
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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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... The concept for preservation of dried human biological samples as a spot is attributed to Ivar Christian Bang in 1913 [39,40]. Typically, these microsamples can be collected via a skin prick with a lancet or created by transferring from a phlebotomy tube with a micro-pipette [30,41]. Currently, such samples are collected on a specialized filter paper which can contain anywhere from 15-50 µL of blood, and take approximately three to four hours to completely dry at room temperature [39]. ...
... This can make DBSs amenable to collections where coldchain shipping protocols may not be feasible. However, it should be noted that belowzero temperatures (-20˚C, -80˚C) are still recommended for the long-term storage of DBSs [39] [30,41]. ...
Preprint
Full-text available
Microsamples (collections usually less than 50 µL) have been introduced in pre-clinical, clinical, and research settings to overcome obstacles in sampling via traditional venipuncture. However , venipuncture remains the sampling gold standard for metabolic phenotyping of blood. This presents several challenges in metabolic phenotyping workflows: accessibility for individuals in rural and remote underserved areas (due to the need for trained personnel), the unamenable nature to frequent sampling protocols in longitudinal research (for its invasive nature), and sample collection difficulty in the young and elderly. Furthermore, venous sample stability may be compromised when temperate conditions necessary for cold-chain transport are beyond control. Alternatively, research utilising microsamples extends phenotyping possibilities to inborn errors of metabolism, therapeutic drug monitoring, nutrition, as well as sport and anti-doping. Although the application of microsamples in metabolic phenotyping exists, it is still in its infancy, with whole blood being overwhelmingly the primary biofluid collected through the collection method of dried blood spots. Research into metabolic phenotyping of microsamples is limited; however, with advances in commercially available microsampling devices, common barriers such as volumetric inaccuracies and the 'haematocrit effect' in dried blood spot microsampling can be overcome. In this review, we provide an overview of the common uses and workflows for microsampling in metabolic phenotyping research. We discuss the advancements in technologies, highlighting key considerations and remaining knowledge gaps for employment of microsamples in metabolic phenotyping research. This review supports the translation of research from the 'bench to the community'.
... One possibility to engage a wider range of phenotypes is to use home-sampled dried blood spots (DBS). This strategy can facilitate a sample collection across hard-to-reach population groups and reduce the risk to the bias in the study design (9). DBS sample collection is in use since the 60's and has a large usage in clinical chemistry applications particularly for newborn screening. ...
... The observed increase in levels of circulating CHRDL2 may support recent findings of an active role of bone marrow as immune regulatory organ and indicate active proliferation of cells involved in adaptive immune response (38) An inherent limitation of studying DBS samples is the need for very sensitive methods for quantification. (9). In this study we chose to focus on stable proteins occurring at medium to high abundance levels in the circulation. ...
Preprint
Full-text available
Self-sampled blood provided valuable information about the COVID-19 seroprevalence in the general population. To enable an even deeper understanding of pathophysiological processes following SARS-CoV-2 infections, 276 circulating proteins were quantified by proximity extension assays in dried blood spots (DBS). Samples from undiagnosed individuals collected during the first wave of the pandemic were selected based on their serological immune response and matched on self-reported symptoms. We stratified these as seropositive (IgM+IgG+; N = 41) or seronegative (IgM-IgG-; N = 37), and to represent the acute (IgM+IgG-; N = 26) and convalescent phases (IgM-IgG+; N = 40). This revealed proteins from a variety of clinical processes including inflammation and immune response (MBL2, MMP3, IL2RA, FCGR2A, CCL5), haemostasis (GP1BA, VWF), stress response (ANG), virus entry (SDC4) or nerve regeneration (CHL1). The presented approach complements clinical surveys and enables a deep molecular and population-wide analysis of COVID-19 from blood specimens collected outside a hospital setting.
... 2 of 15 pool rich in specific metabolite concentrations in a linear range [14]. The reliable QC materials with obtained values from NSQAP participating laboratories within a similar MS/MS method and instrumentation help to maintain QA by quantitative internal QC monitoring [8]. ...
... In the 1990's, tandem mass spectrometry (MS/MS)-based NST for amino acid (AA) and acylcarnitine (AC) was developed and was implemented as a routine test for the simultaneous screening of various IEM to be carried out in an efficient and cost-effective manner [6,7]. Dried blood spots (DBS) have advantages such as minimizing blood collection volume and the distress to neonate [8], therefore DBS has been used for MS/MS-based NST [9,10]. Biobanking for residual DBS specimens may be useful for additional diagnostic use in unexpected causes of IEM for the child and family, research projects, and in the development of new NBS assays [11]. ...
Article
Full-text available
Dried blood spots (DBS) have advantages such as minimizing blood collection volume and the distress to neonate. DBS have been used for tandem mass spectrometry (MS/MS)-based newborn screening tests (NST) of amino acid (AA) and acylcarnitine. The Newborn Screening Quality Assurance Program (NSQAP) have been provided quality control (QC) materials for MS/MS, as DBS cards. The NSQAP is generally provided within 14 months of the shelf life and the recommended storage condition is at −10 °C to −30 °C. Previously, several accelerated degradation studies had been performed to determine the transportation stability and short-term stability of AAs and acylcarnitines in DBS. However, the experimental condition is markedly different to the storage condition. We performed long-term monitoring for the real-time stability of seven AAs and 14 acylcarnitines from three levels of 2012 NSQAP QC materials across a time period of 788 days. Arginine suddenly yielded a catastrophic degeneration pattern, which started around D300. When comparing this with previous accelerated degradation studies, methionine, tyrosine, citrulline, and acetylcarnitine did not show a remarkable measurand drift for the real-time stability, except for arginine. Our study showed that arginine would require intensive QC monitoring in routine practice, and should be used for the assessment of the stability in long-term storage of DBS samples for biobanking.
... In contrast to venous blood sampling, only a few drops of blood (approximately 60 µl per circle) are needed. DBS sampling usually do not require cooling, extensive storage space, or even medically trained personnel, therefore increasing the feasibility of this method for studies of large populations or in remote areas with limited laboratory infrastructure (Basu et al., 2017;Chaudhuri et al., 2009;Lehmann et al., 2013;McDade et al., 2007;Nelson et al., 2016;Ostler et al., 2014;Santa-Rios et al., 2020). ...
... However, DBS sampling has so far not been used for biomonitoring of total Hg in blood in the general population in countries with an expected low exposure, e.g., Germany, in combination with direct Hg analysis. The implementation of DBS sampling for Hg biomonitoring comes with multiple challenges such as the background contamination of DBS cards as well as potential contamination or loss of Hg during sampling, transport, and storage (Basu et al., 2017;Chaudhuri et al., 2009;Funk et al., 2013Funk et al., , 2015Lehmann et al., 2013;Nyanza et al., 2019;Santa-Rios et al., 2020). ...
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Venous blood is a preferred matrix for the determination of total mercury (Hg) in human biomonitoring but has some drawbacks such as the requirement for an uninterrupted cold chain for transport and storage and the need of medical personnel for sample collection. Therefore, we tested and implemented a simpler and less expensive method for measuring Hg in human blood using dried blood spots (DBS). For method development, we investigated the influence of different storage conditions (temperature, storage vessel, time) on DBS samples. For method validation, we compared DBS and venous blood and investigated whether DBS sampling is suitable for measuring Hg in the general population in countries with low Hg exposure such as Germany. Based on our results, we found that pre-cleaned glass tubes were most suitable for storage of DBS samples, as this allowed the samples to remain stable for at least 4 weeks even at high temperatures (40 °C). When comparing venous blood and DBS, a very good correlation (r = 0.95, p < 0.01, Spearman-Rho) and high precision of DBS (mean relative standard deviation 8.2% vs. 7.2% in venous blood samples) were observed. Comparing the recoveries of both matrices in different concentration ranges, the variation of the recoveries decreases with increasing Hg concentration. The mean recoveries also decreased with increasing Hg concentration. Overall, we found comparable results for DBS and venous blood using direct Hg analysis. Furthermore, we demonstrated that DBS are suitable for Hg biomonitoring in the general population in Germany and improved the storage conditions for the DBS.
... Many countries carry out screening program for inborn errors of metabolism (IEM) conditions utilizing dried blood spot (DBS) [1]. DBS analysis provides advantages of significantly small volume of blood, minimal sample preparation, and relatively long term stability of analytes through drying [2]. Although individual IEM is rare, the collective incidence could be up to one in 500 to 4,000, posing severe public health problem [3]. ...
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Objectives Next generation sequencing (NGS) technology has allowed cost-effective massive parallel DNA sequencing. To evaluate the utility of NGS for newborn screening (NBS) of inborn errors of metabolism (IEM), a custom panel was designed to target 87 disease-related genes. The pilot study was primarily proposed for second-tier testing under the NBSIEM program in Hong Kong. Methods The validation of the panel was performed with two reference genomes and an external quality assurance (EQA) sample. Sequencing libraries were synthesized with amplicon-based approach. The libraries were pooled, spiked-in with 2% PhiX DNA as technical control, for 16-plex sequencing runs. Sequenced reads were analyzed using a commercially available pipeline. Results The average target region coverage was 208× and the fraction of region with target depth ≥20× was 95.7%, with a sensitivity of 91.2%. There were 85 out of 87 genes with acceptable coverage, and EQA result was satisfactory. The turnaround time from DNA extraction to completion of variant calling and quality control (QC) procedures was 2.5 days. Conclusions The NGS approach with the amplicon-based panel has been validated for analytical performance and is suitable for second-tier NBSIEM test.
... ii. Evaluation of plasma micro-sampling for dried plasma spots (DPS) in quantitative LCMS/MS bioanalysis using ritonavir as a model compound 47 . Chromatographic separation was achieved on a reverse-phase C18 column with 1 mM ammonium acetate in water / acetonitrile using a solvent gradient at a flow rate of 400 mL / min and detection was by TSQ quantum access triple quadrupole mass spectrometer equipped with a heated electrospray ionization source. ...
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Bioanalysis is a sub-discipline of analytical chemistry covering the quantitative measurement of xenobiotics (drugs and their metabolites and biological molecules in unnatural locations or concentration) and biotics (macromolecules, proteins, DNA, large molecule drugs, metabolites) in biological system. The focus of bioanalysis in the pharmaceutical industry is to provide as quantitative measure of the active drug and/or its metabolite(s) for the purpose of pharmacokinetics, toxicokinetics, bioequivalence and exposure response (pharmacokinetics / pharmacodynamics studies). Over the past several years dried matrix spot (DMS) sampling technique has emerged as a pertinent method in both qualitative and quantitative bioanalysis context. There are many types of DMS techniques such as dried blood spot (DBS), dried plasma spot (DPS), dried urine spot (DUS) and dried breast milk spot (DBMS). The most commonly used technique is DBS wherein the blood sample is directly soaked on to a paper (with or without treatment) and after drying it can be analyzed by modern analytical, immunological or genomic detection systems. Several advantages of DMS techniques such as low sample requirement, transportation and storage without special treatment, better analytes stability, enhanced clinical cooperation in clinical trials and reduced unforeseeable exposure of biohazard to analysts, make it the most appropriate sampling technique for bioanalysis. This review illustrates the available information on DBS, DPS, DUS and DBMS methods which may serve for investigators in the field of bioanalysis. Further, the proficiency and appliance of DMS method in pharmacokinetic (PK), therapeutic drug monitoring (TDM), toxicokinetic (TK), metabolomic and disease diagnosis is explored.
... Conventional newborn screening with DBS begins with the addition of capillary blood droplets, derived either from heel-or finger-stick within 24-48 hours after birth, onto a solid, cellulose based filter card which is then passively dried in ambient conditions. In this context, pre-analytical issues emerge as blood in a non-uniform manner dries in the presence of cells, and differential blood cells lyse and introduce contaminants 6 . Hematocrit variation 7 , as well as the inconsistency between successive blood drops from finger-sticks often used to increase total spotting volumes 8 , add to pre-analytical variation. ...
Article
Newborn screening using dried plasma spots offers preanalytical advantages over conventional cards for plasma-associated targets of interest. Herein we present dried plasma spot-based methods for measuring metabolites using a 250+ compound liquid chromatography tandem mass spectrometry library. Quality assurance reduced this library to 134, and from these, 30 compounds determined the normal newborn reference ranges.
... In comparison with venipuncture, DBS sampling is low-cost, reduces biohazard risk during sample collection and transport, and it affords the possibility of selfsampling, with participants collecting their own blood and returning the sample through the mail, without the need for a cold chain or special handling 14 . Due to these advantages, DBS sampling is the foundation of neonatal screening programs in the US, and it is increasingly applied as a minimally-invasive alternative to venipuncture in community-and population-based health research, including several applications in infectious disease epidemiology 15,16 . ...
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The spike protein of SARS-CoV-2 engages the human angiotensin-converting enzyme 2 (ACE2) receptor to enter host cells, and neutralizing antibodies are effective at blocking this interaction to prevent infection. Widespread application of this important marker of protective immunity is limited by logistical and technical challenges associated with live virus methods and venous blood collection. To address this gap, we validated an immunoassay-based method for quantifying neutralization of the spike-ACE2 interaction in a single drop of capillary whole blood, collected on filter paper as a dried blood spot (DBS) sample. Samples are eluted overnight and incubated in the presence of spike antigen and ACE2 in a 96-well solid phase plate. Competitive immunoassay with electrochemiluminescent label is used to quantify neutralizing activity. The following measures of assay performance were evaluated: dilution series of confirmed positive and negative samples, agreement with results from matched DBS-serum samples, analysis of results from DBS samples with known COVID-19 status, and precision (intra-assay percent coefficient of variation; %CV) and reliability (inter-assay; %CV). Dilution series produced the expected pattern of dose–response. Agreement between results from serum and DBS samples was high, with concordance correlation = 0.991. Analysis of three control samples across the measurement range indicated acceptable levels of precision and reliability. Median % surrogate neutralization was 46.9 for PCR confirmed convalescent COVID-19 samples and 0.1 for negative samples. Large-scale testing is important for quantifying neutralizing antibodies that can provide protection against COVID-19 in order to estimate the level of immunity in the general population. DBS provides a minimally-invasive, low cost alternative to venous blood collection, and this scalable immunoassay-based method for quantifying inhibition of the spike-ACE2 interaction can be used as a surrogate for virus-based assays to expand testing across a wide range of settings and populations.
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Newborn screening (NBS) programs operate in many countries, processing millions of dried bloodspot (DBS) samples annually. In addition to early identification of various adverse health outcomes, these samples have considerable potential as a resource for population-based research that could address key questions related to child health. The feasibility of archival DBS samples for emerging targeted and untargeted multi-omics analysis has not been previously explored in the literature. This review aims to critically evaluate the latest advances to identify opportunities and challenges of applying omics analyses to NBS cards in a research setting. Medline, Embase and PubMed databases were searched to identify studies utilizing DBS for genomic, proteomic and metabolomic assays. A total of 800 records were identified after removing duplicates, of which 23 records were included in this review. These papers consisted of one combined genomic/metabolomic, four genomic, three epigenomic, four proteomic and 11 metabolomic studies. Together they demonstrate that the increasing sensitivity of multi-omic analytical techniques makes the broad use of NBS samples achievable for large cohort studies. Maintaining the pre-analytical integrity of the DBS sample through storage at temperatures below −20 °C will enable this important resource to be fully realized in a research capacity.
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Technologies for preservation of specimens in the absence of cold chains are essential for optimum utilization of existing laboratory services in the developing world. We present a prototype called specimen transportation tube (SPECTRA-tube) for the collection, exposure-free drying, ambient transportation, and liquid state recovery of large-volume (>1 mL) specimens. Specimens introduced into the SPECTRA-tube are dried in glass fiber membranes, which are critical for efficient liquid-state sample recovery by rehydration and centrifugation. SPECTRA-tube is demonstrated for the dry storage of sputum for tuberculosis detection. Mycobacterium smegmatis (Msm)-spiked mock sputum dried in a native Standard 17 glass fiber was stable for molecular testing after 10 day storage at 45 °C and for culture testing after 10- and 5-day storage at 37 °C and 45 °C, respectively. Compatibility with human sputum storage was demonstrated by dry storing 1.2 mL Mycobacterium bovis-spiked human sputum in a SPECTRA-tube for 5 days at room temperature. We have thus demonstrated the first workflow for dry storage of sputum followed by molecular and culture testing. Compared to existing specimen dry storage technologies, SPECTRA-tube significantly increases the volume of liquid specimens that can be transported in the dry state and enables the recovery of the entire sample in the liquid state, rendering it compatible with conventional downstream analysis methods.
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Dried blood spot (DBS) sampling, coupled with multiple reaction monitoring mass spectrometry (MRM-MS), is a well-established approach for quantifying a wide range of small molecule biomarkers and drugs. This sampling procedure is simpler and less-invasive than those required for traditional plasma or serum samples enabling collection by minimally trained personal. Many analytes are stable in the DBS format without refrigeration, which reduces the cost and logistical challenges of sample collection in remote locations. These advantages make DBS sample collection desirable for advancing personalized medicine through population-wide biomarker screening. Here we expand this technology by demonstrating the first multiplexed method for the quantitation of endogenous proteins in DBS samples. A panel of 60 abundant proteins in human blood was targeted by monitoring proteotypic tryptic peptides and their stable isotope-labeled analogues by MRM. Linear calibration curves were obtained for 40 of the 65 peptide targets demonstrating multiple proteins can be quantitatively extracted from DBS collection cards. The method was also highly reproducible with a coefficient of variation of <15% for all 40 peptides. Overall, this assay quantified 37 proteins spanning a range of more than 4 orders of magnitude in concentration within a single 25 min LC/MRM-MS analysis. The protein abundances of the 33 proteins quantified in matching DBS and whole blood samples showed an excellent correlation, with a slope of 0.96 and an R2 value of 0.97. Furthermore, the measured concentrations for 80% of the proteins were stable for at least 10 days when stored at -20 °C, 4 °C and 37 °C. This work represents an important first step in evaluating the integration of DBS sampling with highly-multiplexed MRM for quantitation of endogenous proteins.
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We describe a simple method for evaluating thyroxine-binding globulin capacity and concentration from a single 1-cm blood spot on filter-paper used in a screening program for neonatal hypothyroidism. This method permits prompt diagnosis of about 90% of the infants with thyroxine-binding globulin deficiency in our abnormal low-thyroxine, low-thyrotropin population. There was excellent equivalence between results obtained by our method and by the method of Chopra et al. (J. Clin. Endocrinol. Metab. 35:565, 1972), and minimal overlap between the population with low thyroxine-binding globulin and the low-thyroxine, normal thyrotropin population. We recommend this method to all programs in which a primary thyroxine measurement is used in screening for congenital hypothyroidism.
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Background: Tandem mass spectrometry (MS/MS) is rapidly being adopted by newborn screening programs to screen dried blood spots for >20 markers of disease in a single assay. Limited information is available for setting the marker cutoffs and for the resulting positive predictive values. Methods: We screened >160 000 newborns by MS/MS. The markers were extracted from blood spots into a methanol solution with deuterium-labeled internal standards and then were derivatized before analysis by MS/MS. Multiple reaction monitoring of each sample for the markers of interest was accomplished in ∼1.9 min. Cutoffs for each marker were set at 6–13 SD above the population mean. Results: We identified 22 babies with amino acid disorders (7 phenylketonuria, 11 hyperphenylalaninemia, 1 maple syrup urine disease, 1 hypermethioninemia, 1 arginosuccinate lyase deficiency, and 1 argininemia) and 20 infants with fatty and organic acid disorders (10 medium-chain acyl-CoA dehydrogenase deficiencies, 5 presumptive short-chain acyl-CoA dehydrogenase deficiencies, 2 propionic acidemias, 1 carnitine palmitoyltransferase II deficiency, 1 methylcrotonyl-CoA carboxylase deficiency, and 1 presumptive very-long chain acyl-CoA dehydrogenase deficiency). Approximately 0.3% of all newborns screened were flagged for either amino acid or acylcarnitine markers; approximately one-half of all the flagged infants were from the 5% of newborns who required neonatal intensive care or had birth weights <1500 g. Conclusions: In screening for 23 metabolic disorders by MS/MS, an mean positive predictive value of 8% can be achieved when using cutoffs for individual markers determined empirically on newborns.
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Our purpose was to evaluate second-trimester prenatal screening for open neural tube defects and Down syndrome by use of dried blood specimen collection and transport. A prospective study of 7497 dried blood specimens from patients <35 years old was performed. Specimens were assayed for maternal blood alpha-fetoprotein and free beta-human chorionic gonadotropin. Patient-specific risks for both disorders were calculated and used to determine whether further evaluation was indicated. The study included an evaluation of the median and SD of analyte multiple of the median levels. The initial positive rate for open neural tube defect was 4.4% adjusted to 2.7% after ultrasonographic revision and collection of a second sample. The initial positive rate for Down syndrome was 3.6% adjusted to 2.8% after ultrasonographic revision. All seven cases of open neural tube defect were detected within the increased risk group. Six of 8 (75%) cases of Down syndrome were detected. The median alpha-fetoprotein multiple of the median was 3.5 in open neural tube defect cases and 0.6 in Down syndrome cases. The median free beta-human chorionic gonadotropin multiple of the median was 2.4 in Down syndrome cases. The SD (log e) of alpha- fetoprotein and free beta-human chorionic gonadotropin in 5868 unaffected white patients was 0.4022 and 0.5635, respectively. Second-trimester dried blood screening for open neural tube defects and Down syndrome can achieve screening efficiency comparable to serum-based protocols with distinct advantages over the conventional method of blood collection.
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Simple method for a1-antitrypsin deficiency screening by use of dried blood spot speci- mens. X. Costa, R. Jardi, F. Rodriguez, M. Miravitlles, M. Cotrina, C. Gonzalez, C. Pascual, R.. Vidal. #ERS Journals Ltd 2000. ABSTRACT: The use of dried blood spot (DBS) specimens in quantitative a1-an- titrypsin (a1-AT) detection or genetic analysis is limited because protein levels in the samples are low and they contain components that can interfere with polymerase chain reaction amplification. A methodological adaptation was developed to overcome these drawbacks which is discussed here. The study population consisted of 200 healthy volunteers and 300 patients with chronic obstructive pulmonary disease (COPD). DBS specimens were tested for a1-AT concentration using a modified nephelometric assay and phenotyped with an iso- electric focusing method. Genetic diagnosis was established by deoxyribonucleic acid sequencing using a simple purification procedure to remove contaminants. The nephelometric method showed a detection limit of 0.284 mg.dL-1, correspond- ing to a serum concentration of 13 mg.dL-1. The correlation coefficient between a1-AT concentrations in DBS versus serum samples was R2=0.8674 (p1.9 mg.dL -1 , corresponding to 114 mg.dL-1 in serum samples. One hundred and twenty-five COPD patients (42%) showed a1-AT values
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Background: Pompe disease, or acid maltase deficiency, is a genetic muscle disorder caused by mutations in the gene encoding the acid alpha-glucosidase (GAA) enzyme, which is essential for the degradation of glycogen to glucose in lysosomes. The wide clinical variability is resulted from genetic heterogeneity, and many different mutations of the GAA gene have been reported. Some of these mutations are associated with specific phenotypes, such as the c. -32T>G (IVS1-13T>G) mutation seen in late-onset Pompe disease. Methods: We used a real-time PCR, after genomic DNA extraction isolated from DBS (dried blood spots) and PCR amplification. Results: Our results successfully detected in controls and patients have been 100% concordant with sequencing results. Conclusions: This assay combines simple sample processing and rapid analysis and it allows to detect the patients with a milder form and slower progression of this disease with a high reliability.
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A potential role of chemokines in the pathophysiology of Autism Spectrum Disorders (ASDs) has been previously suggested. In a recent study we examined levels of three inflammatory chemokines (MCP-1, MIP-1α and RANTES) in samples of amniotic fluid of children diagnosed later in life with ASD and controls frequency-matched to cases on gender and year of birth. In this follow-up study, levels of the same chemokines were analyzed postnatally in dried blood spot samples from the same subjects utilizing the Danish Newborn Screening Biobank. Crude estimates showed decreased levels of RANTES. In the adjusted estimates, no differences were found in levels of the three examined chemokines in ASD cases compared to controls. Our findings may cautiously suggest an altered cell-mediated immunity during the early neonatal period in ASD. Further research is needed to examine the relationship between maternal/fetal and neonatal chemokine levels and their role in ASD.