separations

Review
Dried Blood Spot in Toxicology: Current Knowledge
Agnieszka Niemiec

Department of Forensic Medicine and Forensic Toxicology, Medical University of Silesia, Poniatowskiego 15,
40-055 Katowice, Poland; [email protected] or [email protected]; Tel.: +48-53-167-27-50

Abstract: Dried Blood Spot (DBS) is becoming very popular in various medical fields, especially
in toxicology. Nowadays it is commonly used in newborn screening for inherited or congenital
diseases. This paperwork is based on a review of available literature. DBS is simple and rapid, it
does not require trained medical staff to collect the samples. Specimens can be easily and safely
transported to the laboratory. DBS provides an opportunity for roadside testing and rather quick
results. Venous blood spot, collected from a finger or a heel, is put on the special paper card, which
can result in a different distribution of blood and concentration of detecting substances. Marking
multiple substances from one spot is extremely challenging, but due to further advancements in this
area, it is only a matter of time until it becomes possible and all the disadvantages vanish. DBS is
certain to develop and become even more worldwide used.

Keywords: dried blood spot; dried blood spots; dried blood filter; dry blood spot; toxicology;
forensic; filter paper; DBS





 1. Introduction
Citation: Niemiec, A. Dried Blood Dried Blood Spot (DBS) is a technique that involves collecting small samples of venous
Spot in Toxicology: Current blood (plasma or serum are applicable as well), usually from a finger, toe, or heel, onto an
Knowledge. Separations 2021, 8, 145. absorbent filter paper. Whatman® 903 paper is frequently used since it is made from pure
https://doi.org/10.3390/ cotton fibers [1,2]. Exceptionally, at the crime scene, other materials than paper are used.
separations8090145 After the drop of blood is air-dried, it can be transported to a laboratory and analyzed.
The paper card must be stored in a gas-impermeable zipper bag along with desiccant
Academic Editor: packets. The bag itself is put into the freezer. In the laboratory, all the spots are punched
Renata Wietecha-Posluszny
out and eluted. After the formation of hemolytic supernatants and centrifugation, the
spots are ready to be analyzed using immunoassays or molecular techniques (GC-MS,
Received: 15 July 2021
LC-MS/MS) [3]. DBS is widely used for the detection of HIV, hepatitis B, or C; however,
Accepted: 31 August 2021
it is also vital in newborn screening for inherited or congenital metabolic disorders [4].
Published: 6 September 2021
Recently, this method is gaining popularity in the field of toxicology, where it can contribute
to the assessment of substances of abuse, toxins, and trace elements. This paper focuses on
Publisher’s Note: MDPI stays neutral
the aspect of using DBS in toxicology.
with regard to jurisdictional claims in
published maps and institutional affil-
2. Materials and Methods
iations.
This paper was prepared through a review of available literature found using key-
words such as DBS, toxicology, filter paper, screening, and blood collecting. There were
several websites used for this search: PubMed, Science Direct, Wiley Online Library, and
Frontiers. All articles used were published in prominent scientific journals. Most of them
Copyright: © 2021 by the author.
are listed below in the References section, whereas some did not reveal anything new or
Licensee MDPI, Basel, Switzerland.
important enough to be acknowledged as a reference. I have read over thirty articles in
This article is an open access article
total to acquire knowledge of this topic.
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

Separations 2021, 8, 145. https://doi.org/10.3390/separations8090145 https://www.mdpi.com/journal/separations

Separations 2021, 8, 145 2 of 6

3. Results
3.1. History
DBS was discovered in 1913 by Ivar Bang, who used it for glucose concentration mon-
itoring in rabbits. Half a century later, in 1963, Guthrie and Susi managed to prove blood
sampling useful in screening for phenylketonuria. In the 1970s, DBS was implemented in
serological surveillance and used to diagnose syphilis and detect antibodies for mumps and
measles. Due to the increase in HIV morbidity, DBS has attracted attention at the beginning
of the year 2000 and successfully helped to monitor HIV infection. Nowadays it is used
commonly in various fields such as medicine, pharmacy, and new technologies [5,6].

3.2. Drugs Analysis

DBS may be significant for detecting drugs and help pharmaceutical concerns in con-
ducting research. Multiple substances can be measured in DBS: benzodiazepines, Z-drugs
(zolpidem, zopiclone), opiates (6-monoacetylmorphine, morphine, codeine, hydromor-
phone, hydrocodone, oxycodone, noroxycodone), tramadol, methadone, buprenorphine,
fentanyl, ketamine, and their respective metabolites [7] (and references therein). Moreover,
there are reports that it is possible to mark the level of ethyl glucuronide that serves as an
alcohol abuse marker [7] (and references therein). Analyzing drugs with DBS serves two
main purposes. Firstly, it can detect drug abuse in adults or measure the level of medication
(TDM—Therapeutic Drug Monitoring) and improve a follow-up. Secondly, it can evaluate
the exposure to the drugs before birth in newborns. Both therapeutic and illegal ones.
The aforementioned TDM used with DBS has proven to be highly effective in monitoring
busulfan in children, who had to undergo a hematopoietic stem cell transplantation [8]. In
addition, a study by Hahn et al. showed that topiramate TDM is also possible and highly
beneficial [9]. Finally, a study by Duthaler et al. contains good results of therapeutic drug
monitoring of antiretroviral drugs in resource-poor regions using the DBS technique [10].

3.2.1. Substances of Abuse Marker

Detecting substances of abuse via DBS is still under a lot of research. However, there
have been numerous reports that it is possible to detect cannabinoids, cocaine, and its
metabolites as well as opiates, such as morphine, non-opiate opioids, amphetamine, and its
analogs and gamma-hydroxybutyric acid [7]. Morphine may cause some issues since it is a
heroin (illicit drug) or codeine (licit drug) metabolite. Marking 6-Monoacetylmorphine is a
solution because it is one of the active heroin derivatives [11]. The vast majority of these
drugs have a short half-life, but DBS has a stabilization effect on them so they can endure
for a longer period [7]. It is also worth mentioning that a recent study by Moretti et al.
describes a successful analysis of a few psychoactive substances and their metabolites in
postmortem blood [12]. However, research conducted by Patteet et al. also confirmed that
DBS is vital in therapeutic drug monitoring of antipsychotics, but it excluded olanzapine
and N-desmethylolanzapine [13].
Driving under influence of drugs (DUID) has been discussed in the DRUID project.
The role of DBS in this matter has been evaluated, but only to some extent [7]. Oral
fluid, which is widely used for DUID testing is not a perfect method. There are some
controversies, whether the results are comparable to the ones obtained via usual blood
testing. What is more, they can be falsified by different conditions such as the use of mouth
wash [14]. DBS seems to be more reliable and its sampling is quick and does not require any
extra knowledge or experience so it can be collected by an officer during a routine control.
Additionally, there was a study by Simões et al. performed to check the use of DBS
along with UPLC-MS/MS technology in the field of forensic toxicology. As a result, DBS
was proven to be useful in the detection of illicit drugs [15].

3.2.2. Trace Elements Detection

There is a wide range of elements confirmed to be detected in DBS: Pb, As, Ba,
Be, Bi, Ca, Cd, Co, Cr, Cs, Cu, Fe, Hg, K, Li, Mg, Mn, Mo, Na, Ni, P, Rb, S, Sb, Se,
Separations 2021, 8, 145 3 of 6

Tl, V, and Zn [7] (and references therein). There is a possibility of creating a “metallic
profile” of an individual to assess the exposure to contamination and take appropriate
measures to clean the environment [16]. It may be challenging due to the differences in
the distribution of different elements on paper and the possible contamination during
collection or transportation.

3.3. Newborn Screening

Although DBS in newborn screening is widely used for detecting congenital or in-
herited diseases, it can also help in assessing the prevalence of tobacco and cocaine in
pregnant women [7]. It is performed by detecting benzoylecgonine (cocaine metabolite)
and cotinine (nicotine metabolite). Unfortunately, it will not assess the whole pregnancy
period, but only the time near the delivery due to the elimination of these substances from
the organism. However, the immature liver of newborns functions slower than in adults,
so it extends the time for detection of these substances. The first such report was published
by Henderson et al., who used redesigned urinary benzoylecgonine radioimmunoassay
screening test to assess using cocaine by the mother during pregnancy [1].
Not only measuring elements in adults corresponds with the contamination of the en-
vironment. The benzene oxide, perchlorate, organochlorine dichlorodiphenyldichloroethy-
lene (DDE), the PFCs perfluorooctane sulfonate (PFOS), and perfluorooctanoate (PFOA)
have been evaluated in newborns with the use of DBS [7].

3.4. DBS Testing in SARS-CoV-2 Serology

Nowadays the possibility of detecting SARS-CoV-2 antibodies is being thoroughly
studied. Numerous studies have proven DBS effective in antibodies detection with accuracy
comparable to serum or plasma samples [17,18]. This method is less expensive and easier,
so it can be widely used in the pandemic. It can be used not only to confirm an infection,
but also to test vaccine-stimulated antibody response [19].

3.5. Advantages of DBS

DBS has several advantages. Firstly, it can be easily collected. It is simple and there
is no need for trained medical staff to perform sampling. The extraction procedure is
simplified, economical, and cost-effective. There is a possibility for automatization, which
would increase the speed of this already rapid process. Secondly, there is a low biohazard
risk during transportation. There is no leakage because paper cards with blood are dry
so it is safe for the personnel. Safety is also maintained due to the loss of infectivity of
some viruses during the drying process. That applies to the HIV-1 and -2 viruses, the
human T-cell leukemia/lymphoma virus-I and -II, and the hepatitis C virus [20]. Thirdly,
DBS has a stabilizing effect on drugs and it inhibits degradation of the substance [21].
The other advantage of DBS that is worth mentioning is the ability to assess the acute
state of the patient. This technique is quick and can provide crucial information about
a patient’s health, to treat him as fast as possible [20]. Another important advantage is
gender neutrality and lack of adulteration issues [22]. The other asset to be enumerated is
the ability to keep DBS paper cards as evidence, even when the case is closed and other
evidence is discarded. Their storage is uncomplicated and they do not require a lot of
space. The downside of this idea is that paper cards cannot be stored forever due to their
limited preservation time [7] (and references therein). Therapeutic drug monitoring is
a perfect place for DBS. Due to easy sampling and speed, DBS can be performed before
the doctor’s arrival and show the concentration of certain substances (such as antibiotics,
antidepressants, immunosuppressants, and antiretrovirals) in the patient’s blood [23].
Liquid extraction or solid-phase extraction requires more blood (around 100–2000 µL) than
DBS (10–100 µL), which makes DBS more convenient [21]. Finally, there is an opportunity
to perform roadside testing for impaired driving. Collecting blood on the crime scene is
reliable and practical [21].
Separations 2021, 8, 145 4 of 6

3.6. Disadvantages of DBS

DBS may seem flawless, but it has some downsides as well. The drying time is
quite long and lasts around 2 h, depending on the conditions such as the type of card
and blood volume. Also, the blood can coagulate or lyze, which makes the distribution
on the paper card differ [24]. The viscosity of the blood has a similar impact on blood
distribution [2]. Then, the results may be disturbed and not reliable [21]. Moreover,
detecting some substances may be difficult. Few substances have been measured with the
DBS technique. Further research is needed. Due to the small volume of blood collected,
it may be impossible to detect multiple drug groups at once. It would require a few DBS
samples, so the whole process would be elongated and more complicated [1].

3.7. Future Perspectives

DBS has enormous potential for massive development. It is constantly being automa-
tized and new robots are being designed to make DBS less dependent on human beings.
Direct analysis technologies are evolving and filter paper is being brought to perfection.
Probably, it will be good enough to provide the same blood distribution within the spot
and preserve the substances for a longer period. Standardization of the whole process
may contribute to the increase of results reliability. Hopefully, in the future, there will be
extensive research of postmortem samples, that are hemolyzed and putrefied [25]. Further-
more, there are derivatives of the DBS technique such as perforated DBS (PDBS), bilayer
DPS card, and Hemaspot technology [26]. They may replace usual DBS and create new
possibilities for substances detection. Especially, the pharmaceutical sector is bound to use
DBS even more and it may improve their clinical studies [27]. Also, worldwide preparing
DBS kits divided into panels for metabolic, hormonal, and cardiovascular disorders can
speed along the diagnostics.

4. Discussion
DBS is a developing technology that provides wide possibilities for various medical
fields. Its simplicity makes it available for nearly everyone. It is safe and cost-effective.
Stabilization of the substances in blood on paper cards elongates the time before detection
and prevents de novo formation of substances, which can change the results. This factor is
also vital in keeping the evidence after the case is closed since paper cards do not require
much place. The storage and transportation of the biological material are safe and rather
fast. Results come quickly so they can help treat the patient in the right way. Therapeutic
drug monitoring is easier and more accessible due to the DBS. Roadside testing (especially
for alcohol) with DBS can dodge results’ disturbances such as a mouth wash. It is already
commonly used in newborn screening and it is being introduced to pharmaceutical compa-
nies, which benefit a lot from this technology. Creating a “metallic profile” of an individual
makes DBS contribute to the environment as well. The aforementioned disturbances do
not seem to be impossible to overcome. Further research is needed to implement DBS on a
wider scale, but it looks very promising for all fields, especially toxicology.

Funding: This research received no external funding.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Acknowledgments: Koło Naukowe Medycyny Sadowej
˛ SUM.
Conflicts of Interest: The authors declare no conflict of interest.

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