CLB-09449; No.

of pages: 7; 4C:
Clinical Biochemistry xxx (2016) xxxxxx

Contents lists available at ScienceDirect

Clinical Biochemistry

journal homepage: www.elsevier.com/locate/clinbiochem

Comparison between dried blood spot and plasma sampling for

therapeutic drug monitoring of antiepileptic drugs in children with
epilepsy: A step towards home sampling
Camilla Linder a,b,, Katarina Wide c,d, Malin Walander c,d, Olof Beck a,b, Lars L Gustafsson a,b, Anton Pohanka a,b
a
Department of Laboratory Medicine, Division of Clinical Pharmacology, Karolinska Institutet, Stockholm, Sweden
b
Department of Clinical Pharmacology, Karolinska University Hospital, Stockholm, Sweden
c
Department of Clinical Science, Technology and Intervention (CLINTEC), Karolinska Institutet, Stockholm, Sweden
d
Astrid Lindgren Children Hospital, Karolinska University Hospital, Stockholm, Sweden

a r t i c l e i n f o a b s t r a c t

Article history: Objectives: To investigate if dried blood spots could be used for therapeutic drug monitoring of the antiepi-
Received 13 October 2016 leptic drugs, carbamazepine, lamotrigine and valproic acid in children with epilepsy.
Received in revised form 12 December 2016 Methods: Fingerprick blood samples from 46 children at a neuropediatric outpatient clinic was collected on
Accepted 22 December 2016 lterpaper at the same time as capillary plasma sampling. A validated dried blood spot liquid chromatography
Available online xxxx
tandem mass spectrometry method for carbamazepine, lamotrigine and valproic acid was compared with the
routine plasma laboratory methods. Method agreement was evaluated and plasma concentrations were estimat-
Keywords:
Red blood cell/plasma ratio
ed by different conversion approaches.
Conversion factor Results: Strong correlation was shown between dried blood spot and plasma concentrations for all three
Estimated plasma concentrations drugs, with R2 values N 0.89. Regression analysis showed a proportional bias with 35% lower dried blood spot
Hematocrit concentrations for valproic acid (n = 33) and concentrations were 18% higher for carbamazepine (n = 17). A
LC-MS/MS ratio approach was used to make a conversion from dried blood spots to estimated plasma for these two
drugs. Dried blood spot concentrations were directly comparable with plasma for lamotrigine (n = 20).
Conclusions: This study supports that dried blood spot concentrations can be used as an alternative to plas-
ma in a children population for three commonly used antiepileptic drugs with the possibility to expand by adding
other antiepileptic drugs. Clinical decisions can be made based on converted (carbamazepine, valproic acid) or
unconverted (lamotrigine) dried blood spot concentrations. Dried blood spot sampling, in the future taken at
home, will simplify an effective therapeutic drug monitoring for this group of patients who often have concom-
itant disorders and also reduce costs for society.
2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

1. Introduction children on antiepileptic drug polytherapy, drug-drug interactions, sub-

tle adverse drug reactions or co-morbidities in chronically disabled chil-
The most commonly used antiepileptic drugs (AEDs) for the treat- dren [3,4].
ment of children with epilepsy in Sweden and elsewhere are carbamaz- Presently TDM is usually performed using venous or capillary blood
epine (CBZ), lamotrigine (LTG), valproic acid (VPA) and levetiracetam sampling at out-patient clinics, measuring the trough drug concentra-
[1,2]. Therapeutic drug monitoring (TDM) can assist in individualizing tions in plasma before the morning dose [3]. Blood sampling at a clinic
dosages in pediatric care due to pharmacokinetic variations between is time-consuming and sometimes stressful, especially for children
with chronic diseases or disabilities. Self-collection at home of a capil-
lary dried blood spot (DBS) sample should be of advantage for many
Abbreviations: ADD, attention decit disorder; ADHD, Attention decit hyperactivity
syndrome; AEDs, antiepileptic drugs; b/p, blood-to-plasma ratio; CBZ, carbamazepine; of these patients and their guardians [5,6]. Although DBS has been
CI, condence interval; DBS, dried blood spots; EMA, European Medicines Agency; HCT, used in different qualitative assays since the 60's, it is a new matrix for
hematocrit; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LTG, the TDM laboratory [7,8]. In quantitative bioanalysis based on DBS, he-
lamotrigine; RBC, red blood cell; RBC/p, red blood cell-to-plasma ratio; TDM, therapeutic matocrit (HCT), spot homogeneity, blood-to-plasma ratio of the drug
drug monitoring; VPA, valproic acid.
Corresponding author at: Department of Clinical Pharmacology C1:68, Karolinska
and sampling a correct volume are factors that may affect the result
University Hospital, SE-141 86 Stockholm, Sweden. [9]. There has been a rapid development of assay technologies with
E-mail address: [email protected] (C. Linder). much improved sensitivity such as liquid chromatography-tandem

http://dx.doi.org/10.1016/j.clinbiochem.2016.12.008
0009-9120/ 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Please cite this article as: C. Linder, et al., Comparison between dried blood spot and plasma sampling for therapeutic drug monitoring of
antiepileptic drugs in children with ep..., Clin Biochem (2016), http://dx.doi.org/10.1016/j.clinbiochem.2016.12.008
2 C. Linder et al. / Clinical Biochemistry xxx (2016) xxxxxx

mass spectrometry (LC-MS/MS), allowing the measurements of multi- capillary blood was collected in a Li-heparin capillary tube for routine
ple of exogenous small compounds in a drop of blood [10]. Break- plasma analysis. An additional 300500 L was collected in a capillary
throughs with prelaboratory preparation of microliter whole blood tube for HCT measurement (K2 EDTA Microvette, both tubes from
samples to dried plasma and the design of micro-devices for collection Sarstedts, Nmbrecht, Germany).
of a dened sample volume, makes it likely that DBS will become an at- HCT was analyzed on a SYSMEX XE-5000 (Sysmex, Kobe, Japan), De-
tractive sampling technique for quantitative analysis [1114]. partment of Clinical Chemistry, Karolinska University Hospital, Hud-
Drugs vary in their distribution between the cell fraction and plasma dinge. Whatman cards were left drying at room temperature for at
in whole blood, described as the specic blood-to-plasma ratio (b/p- least 3 h and then stored in zip-lock bags (Joka 11-68, VWR, Radnor,
ratio) or red blood cell/plasma ratio (RBC/p-ratio) of the drug [6,9,15 PA) kept at 4 C with desiccant packages (Millipore, Darmstadt, Germa-
17]. Apart from the RBC/p-ratio, which can be concentration dependent, ny) until analysis. On arrival in the laboratory DBS samples were visual-
the patients individual HCT should in theory also affect the relation be- ly inspected and blood spots with a diameter of b7 mm, corresponding
tween DBS and plasma concentrations. In order to compare DBS con- to an approximate volume of b15 L were excluded since they were not
centrations with traditional plasma concentrations, a conversion within validation ranges [29].
algorithm may be required to calculate an estimated plasma value
based on the DBS concentration. Different approaches, with experimen- 2.3. Analytical methods
tal data of paired plasma and DBS concentrations, corrected for sample
HCT [18] or not corrected for HCT [19,20] have been used to calculate CBZ, LTG and VPA concentrations in DBS samples were measured
a ratio which is then used as a conversion factor. Theoretical approaches using a recently developed LC-MS/MS method [29]. The method could
constructing algorithms considering RBC/p ratios and individual patient be applied in the hematocrit range 0.300.60 in CBZ and LTG, VPA
HCT levels have also been presented [21]. 0.350.60 and for volumes between 15 and 50 L.
VPA is mainly distributed in plasma and yields lower concentrations In the analysis of plasma concentrations, LTG and CBZ were analyzed
in whole blood, e.g. using DBS [2022]. In CBZ and LTG, the distribution by immunochemical methods, QMS LTG and CEDIA CBZ II on an Indiko
between whole blood and plasma is more equal since the RBC/p ratio is Plus analyzer (all from Thermo Scientic, Waltham, MA). The methods
close to one and direct comparisons have been proposed for these drugs were performed according to the manufacturer's protocols and kit-in-
[21,2326]. Clinical validations are needed to introduce DBS as an alter- serts. An accredited in-house LC/MS method was used for routine VPA
native matrix in routine TDM [27]. We present an approach on how analysis [22].
TDM decisions can be made with DBS concentrations.
In this study on children with epilepsy and concomitant neurological 2.4. Statistics
diagnoses, we evaluated capillary DBS concentrations of three common-
ly used AEDs with concentrations of simultaneously collected capillary Capillary DBS concentrations and plasma concentrations were com-
plasma. DBS concentrations were analyzed using a recently developed pared using Passing and Bablok regression analysis. No constant bias be-
liquid chromatography tandem mass spectrometry (LC-MS/MS) meth- tween the methods was dened as when the 95% condence interval
od while plasma concentrations from capillary blood were analyzed (CI) of the intercept of the regression line included zero. In analogy,
with the routine laboratory methods. We also investigated if conversion no proportional bias was dened as when the 95% condence interval
factors were needed to calculate estimated plasma concentrations from for the slope of the regression line included one. Bland-Altman plots
DBS concentrations for the use in clinical practice TDM. were used to identify outliers or tendencies. Criteria for cross validation
from European Medicines Agency (EMA) guidelines on bioanalytical
2. Material & method method validation (67% of the samples should have a difference within
20% of the mean) [30], were applied on unconverted DBS concentra-
2.1. Patients tions. Drugs that needed conversion were evaluated with these criteria
a second time, after being converted to estimated plasma
This study was approved by the local research ethics committee concentrations.
(Regionala etikprvningsnmnden EPN at Karolinska Institutet, Stock- All calculations, analyses and gures were made using Microsoft
holm, 2012/2146-3) and the work conducted in accordance with the Excel 2013 and Addinsoft XLSTAT, 2016.
Declaration of Helsinki. Patients and/or their guardians approved partic-
ipation in the study by informed consent prior to blood sampling. Inclu- 2.5. Conversion of DBS concentrations to estimated plasma concentrations
sion criteria for the study population were children and adolescents
aged 2 to 18 years and treated for epilepsy with CBZ, LTG or VPA as a sin- Two different approaches were used to calculate estimated plasma
gle or combined drug therapy at Department of Neuropediatrics, concentrations based on DBS concentrations. The rst approach was to
Karolinska University Hospital, Huddinge. use the average ratio between measured plasma concentrations and
DBS concentrations and multiply the DBS concentration with this ratio
2.2. Sample collection to achieve an estimated Cplasma. This simple ratio-approach has been
used in earlier TDM comparison studies [19,20].
Samples were collected by pediatric nurses at the neuropediatric The second was partly a theoretical approach taking into account
clinic from April 2013 to March 2014. DBS samples were collected at RBC/plasma ratio (= K in the equation below) and patient HCT and
the same occasion as routine TDM samples for plasma and did not re- then converting it based on linear regression (21). The following equa-
quire extra visits to the clinic or additional ngerpricks. All samples tion was used to create a theoretical plasma concentration.
reected trough drug levels. Time for sampling, reported prescriptions
of antiepileptic drug and last intake of drug were recorded. Fingerprick
Theoretical C plasma CDBS =1HCT1K 1
blood (three to ve drops) was collected on lterpaper (Whatman 903
protein saver card, GE Healthcare, Westborough, MA) using a
Microtainer Lansett, 1.8 or 2.0 (Becton, Dickinson and Company, Frank- CDBS is the drug concentration in DBS, HCT is the individual hematocrit
lin Lakes, NJ). Guidelines from CLSI, Procedures and Devices for the Col- value and K is the specic drug RBC concentration/plasma concentra-
lection of Diagnostic Capillary Blood Specimens, was followed [28]. tion ratio derived from in-house in vitro tests, see S3. In calculations,
Hands were always warm before sampling and rst drop of blood was the individual HCT was also replaced with the mean HCT of the patient
wiped away. From the same ngerprick, approximately 300500 L of group for evaluation of individual HCT measurement necessity.

Please cite this article as: C. Linder, et al., Comparison between dried blood spot and plasma sampling for therapeutic drug monitoring of
antiepileptic drugs in children with ep..., Clin Biochem (2016), http://dx.doi.org/10.1016/j.clinbiochem.2016.12.008
 C. Linder et al. / Clinical Biochemistry xxx (2016) xxxxxx 3

This theoretical plasma value was used to plot theoretical plasma 3.3. Plasma and DBS comparisons with Passing and Bablok regression and
concentrations (x-values) versus measured plasma concentrations Bland-Altman plots
(y-values). The linear regression equation was used to calculate an es-
timated theoretical plasma concentration from the DBS concentration. Passing and Bablok regression were constructed for regression anal-
ysis between plasma and DBS concentrations, Fig. 1. DBS concentrations
showed strong correlations (N0.89) compared to capillary plasma con-
Estimated Theoretical C plasma m  CDBS =1HCT  K c 2 centrations (Fig. 1). Passing and Bablok analysis revealed a proportional
bias for VPA since the slope was 0.67 (95% CI 0.58 to 0.76) and the CI did
not include 1. Also for CBZ a small proportional bias could be seen since
where m is the slope and c is the intercept from the regression of the slope was 1.32 and the 95% CI was 1.01 to 1.45, just outside the
theoretical plasma concentration plotted against plasma concentra- limits of including 1, see Fig. 1. This proportional bias suggests the
tions, CDBS is the concentration in DBS, HCT is the individual hematocrit need to use a conversion factor in VPA and CBZ. No proportional bias
value and K is the specic drug RBC/p ratio derived from in vitro exper- was detected in LTG. No constant bias was found for any of the drugs.
iments (S3). Bland-Altman plots for VPA and CBZ were analyzed with the ratio
The ratio-approach and the theoretical approach of calculating esti- approach converted concentrations, see Fig. 2. The differences in VPA
mated plasma concentrations from DBS concentrations were compared were evenly distributed on both sides of the mean with a tendency of
and evaluated. If both approaches showed similar results, the ratio ap- higher scatter in the upper concentration range. Also in CBZ the differ-
proach was chosen. ences were evenly spread except for one patient with a concentration
of 7.1 g/mL measured in plasma and 5.5 g/mL with DBS, Fig. 2. LTG
concentrations were not corrected and the plot revealed some uneven
2.6. Clinical evaluation of DBS versus plasma concentrations
distribution around the mean. LTG mean concentrations were 6% higher
for DBS compared to plasma. One patient outside the 95% CI had a
Based on results of suggested conversion to estimated plasma con-
concentration of 17.2 g/mL measured in plasma and 14.9 g/mL in
centrations or no conversion, the recommended approach for evaluat-
DBS, Fig. 2.
ing DBS concentrations were used in the comparison (Section 2.5).
Every single DBS sample concentration was compared to the plasma
3.4. Results using different conversion approaches
concentration. Each concentration was categorized into one of three
groups depending on recommended therapeutic ranges for the respec-
In-house in vitro estimation of RBC/plasma ratio was made at three
tive drugs [3]. The categories were; risk of adverse effect, within thera-
different HCT levels, 0.30, 0.43 and 0.55 L/L. This experiment was per-
peutic range and low. The clinical implications of each concentration
formed to cover possible variations in RBC/plasma ratio for these
were assessed by the responsible pediatric neurologist.
drugs at different concentrations and at different HCT levels. RBC/plas-
ma ratio (K-values), was found to vary depending on concentration
3. Results and HCT which made it complicated to choose a K-value for the theoret-
ical approach. Average K-values derived from three concentration
3.1. Patient characteristics ranges at HCT 0.43 were used in the Eqs.s (1) and (2) (theoretical ap-
proach). Results from the experiment are shown in S3.
Forty-six neuropediatric patients aged 2 to 18 years old with a mean RBC/plasma ratios at HCT 0.43 L/L in CBZ was 0.88, in LTG 1.15 and in
age of 9 years on CBZ, LTG or VPA treatment or on combination therapy, VPA 0.06, see S3. CBZ DBS concentrations were on average 18% higher
were included, see Table 1 and S1 for epilepsy characteristics. In the pa- than in plasma (unconverted Bland-Altman plots, S5). For CBZ, conver-
tient group 67% of the patients had concomitant diagnoses such as cere- sion with a factor of 0.84 from the ratio-approach (range 0.661.06) 82%
bral palsy and mental retardation, autism and mental retardation, of the samples were within the 20% EMA limit. Conversions by the
ADHD/ADD (S2). Collected samples from these patients are summa- theoretical approach also reached 82% within 20% (S4, S6, S7).
rized in Table 2. The mean LTG ratio between plasma and DBS concentrations was
close to 1 (0.96) implying that a correction factor was not needed. A
total of 60% of unconverted LTG concentrations were within the 20%
3.2. Sample collection limit, i.e. the EMA method comparison criterion was not accepted for
LTG, supplementary S7. Samples in the lower measuring range had a
A total of 68 paired DBS-plasma samples from 46 patients were col- high scatter which decreased when reanalyzing plasma samples with
lected, generating in total 83 pairs of concentrations, due to that several an LC-MS/MS method for LTG in plasma. Only 18 samples were com-
patients were on polytherapy. 67 samples for HCT determinations were pared with the LC-MS/MS method due to shortage of plasma for two
collected. One VPA sample was excluded from the study due to lack of samples. In this comparison 78% of the samples were within the
preanalytical documentation. Spots with small volumes (b 15 L) were 20%, supplementary S7. LTG corrected with the theoretical approach
found in 12 samples and were not included in the results since they resulted in 75% of the samples within 20%, but the conversion made
were outside the criteria for the bioanalytical method. Samples lacking the bias higher at lower concentrations, (Passing & Bablok and Bland-
HCT values were included in the results and an average HCT was applied Altman plots theoretical approach in S4, S6 and data from comparison
for these samples in calculations, Table 2. in S7).

Table 1
Patient characteristics in study population (n = 46).

Drug therapy for epilepsy

Patient characteristics Valproic acid (VPA) Carbamazepine (CBZ) Lamotrigine (LTG) Combinations

Female/male (n = 17/29) 6/10 4/12 6/1 VPA + CBZ: 0/1

VPA + LTG: 1/5
Age (mean, range) 9 (318) 9 (217) 13 (118) VPA + CBZ: 13
VPA + LTG: 9 (217)

Please cite this article as: C. Linder, et al., Comparison between dried blood spot and plasma sampling for therapeutic drug monitoring of
antiepileptic drugs in children with ep..., Clin Biochem (2016), http://dx.doi.org/10.1016/j.clinbiochem.2016.12.008
4 C. Linder et al. / Clinical Biochemistry xxx (2016) xxxxxx

Table 2
Description of collected samples from study population (n = 46).

Valproic acid Carbamazepine Lamotrigine

Number of collected samples (n = 83)a 42 20 21

Excluded, sample volume too small (n = 13) 9b 3 1
Number analyzed (n = 70) 33 17 20
Number of HCT values 33 16 18
Hematocrit (%, mean and range) 37 (3242) 37 (3542) 37 (3440)
Plasma levels (g/mL, mean and range) 73 (27111) 5.7 (1.910.4) 6.7 (1.517.2)
DBS levels (g/mL, mean and range) 47 (1771) 6.9 (2.813.6) 7.4 (1.115.0)
Estimated plasma levels (g/mL, mean and range)c 74 (27113) 5.7(2.311.3) Not corrected
a
11 patients of the total 46 contributed with several samples throughout the study period.
b
One VPA sample was excluded due to lack of documentation.
c
DBS concentrations were multiplied with an average ratio between measured plasma concentrations and DBS concentrations.

Uncorrected VPA DBS concentrations were on average 35% lower range and low, S8. Recommended therapeutic ranges from Patsalos et
than plasma concentrations, showed as Bland-Altman plot in supple- al. [3] were 412 g/mL for CBZ, 113 g/mL for LTG, and 50100 g/mL
mentary S5. With the ratio-approach, a conversion factor of 1.58 for VPA. For LTG the decision was to use a smaller recommended range
(range 1.272.16) was calculated, resulting in 97% of estimated VPA of 210 g/mL as concentrations below 2 g/mL for LTG may indicate
within 20% in the cross validation (comparison data in S7). With the compliance problem or being in risk of low or no effect. The upper
use of the theoretical approach, a conversion including the HCT and limit of 13 g/mL was lowered to 10 g/mL due to scatter in the compar-
the drug RBC/p ratio with a K of 0.06 in the equation, the same 97% with- ison of plasma and DBS concentrations to be sure of not missing any
in 20% was reached in the EMA cross validation, see supplementary patient who might be in risk of adverse effects for TDM decisions from
S3, S4, S6 and S7. DBS.
Nine pairs of samples differed in the way they were categorized, see
3.5. Individual HCT measurements versus mean HCT Table 3 and S8. Clinical decisions made by the pediatric neurologist on
drug treatment from these concentrations were the same as clinical de-
Estimated theoretical plasma concentrations calculated with a mean cisions based on the plasma concentrations since the absolute differ-
HCT from the population (0.37) compared to theoretical plasma con- ences between these samples were small, see S8.
centrations calculated with individual HCT showed no signicant differ-
ences for CBZ and LTG, the biggest difference was 2.2%, see S7. For VPA 4. Discussion
differences in HCT had greater impact and the biggest difference was
11.2% resulting in an estimated concentration (theoretical approach) 4.1. Sample collection and bioanalysis
of 32.6 g/mL with individual HCT and 36.5 g/mL with the average HCT.
This difference did not affect the clinical decision, Supplementary S7. Small volume spots may inuence accuracy according to the validat-
ed analytical method [29] and was found to be a signicant problem
3.6. Clinical evaluation of DBS versus plasma concentrations since 12 of 83 samples generated small volume spots. This stresses the
importance of training and education for patients and their guardians
Unconverted LTG DBS concentrations and ratio-approach converted as well as nurses when applying DBS sampling at home or at the clinic
CBZ and VPA DBS concentrations were used for clinical evaluation. [10,31]. In relation to this, the project also included the production of
Every single converted (CBZ and VPA) or unconverted (LTG) DBS con- text-based and audiovisual learning material that is now used in part
centration as well as every single plasma concentration were put into two of this study where guardians are collecting samples from their
three different categories, risk of adverse effects, within therapeutic children [32]. A future improvement to avoid sampling errors is to use

Fig. 1. Scatter plots with Passing and Bablok t. Correlations of plasma measured with routine method and DBS measured with an LC-MS/MS method for lamotrigine, carbamazepine and
valproic acid. Dotted lines are identity lines, continuous lines representing Passing & Bablok regression. R2 values from simple linear regression and Passing & Bablok equation for
carbamazepine: R2 = 0.892 (n = 17) y = 1.32x 0.44 with a 95% CI for slope: 1.01 to 1.45,intercept: 1.29 to 0.78, lamotrigine: R2 = 0.978 (n = 20) y = 1.18x 0.39 with a 95%
CI for slope: 0.32 to 1.32, intercept: 1.01 to 0.96 for valproic acid R2 = 0.899 (n = 33) y = 0.67x 1. 55 with a 95% CI for slope: 0.58 to 0.76, intercept: 7.70 to 4.16.

Please cite this article as: C. Linder, et al., Comparison between dried blood spot and plasma sampling for therapeutic drug monitoring of
antiepileptic drugs in children with ep..., Clin Biochem (2016), http://dx.doi.org/10.1016/j.clinbiochem.2016.12.008
 C. Linder et al. / Clinical Biochemistry xxx (2016) xxxxxx 5

Fig. 2. Bland-Altman plots. Bland-Altman plots for carbamazepine and valproic acid; x-axis shows the mean concentrations of the two methods while y-axis shows differences between
ratio approach estimated plasma concentrations from DBS and analyzed plasma concentrations. For lamotrigine unconverted DBS and plasma concentrations are used. Blue bold line
representing the mean difference between methods, blue dotted lines are 95% CI of the mean and red dotted lines are 95% CI. Mean bias for CBZ is 0.087 and SD is 0.68 CI 1.25 to
1.42. Mean bias for LTG is 0.47 and SD is 1.29 95% CI 2.06 to 3.00. Mean bias for VPA is 0.001 and SD is 8.57 95% CI 16.8 to 16.8. (For interpretation of the references to colour in
this gure legend, the reader is referred to the web version of this article.)

lter paper devices where the volume is automatically measured [11, decision to keep the concentrations unconverted was made since the re-
12]. Multiple reaction monitoring LC-MS/MS analysis makes it possible gression analysis did not show proportional bias. Also, the theoretical
to add more drugs to the method so that several AEDs can be included. approach resulted in larger % differences for some samples, see S7.
Levetiracetam has now been added to the validated method by Linder et Replacing the individual HCT with a mean HCT in the theoretical ap-
al. [29]. proach did not affect the clinical decision for CBZ, LTG or VPA. The rec-
ommendation is to use the ratio approach for conversion to estimated
4.2. Conversion of DBS to estimated plasma values plasma concentrations, where no HCT is included in the conversion.
However, extreme individual HCT will generate bias on measured con-
For CBZ the slope of 1.32 indicated a conversion factor and it was centrations. The conclusion is that patients with expected HCT within
needed to fulll EMA criteria. Published methods on clinical validation bioanalytical validated ranges could be suitable for home sampling. A
of CBZ reported divergent results when comparing plasma and DBS con- way to control that the HCT is within validated ranges is to measure it
centrations. One study used ngerprick capillary samples and presented from the same DBS [33,34].
a slope of 1.32 which is the same as in this study [25]. Other studies
compared DBS samples taken from venous blood and test tubes, or it 4.3. Clinical application of DBS in TDM in children
was not clear how the DBS were sampled. No signicant difference be-
tween plasma and DBS samples (slopes of 0.97 to 1.13) has also been re- Earlier comparisons between plasma and DBS for these drugs were
ported [21,26]. The RBC/plasma ratio in capillary blood might be slightly often based on venous and not capillary DBS samples and do not pro-
different than in venous blood, resulting in higher CBZ concentrations in pose recommendations for the use of DBS concentrations in TDM [25,
capillary DBS than with venous DBS. For this reason it is important to in- 26]. Our aim is to suggest how the TDM laboratory can make decisions
vestigate the relationship between capillary blood and plasma since when both plasma and DBS concentrations are used. An important ad-
capillary blood is collected in home sampling. 17 samples are not vantage with this study is that it is performed on capillary DBS, which
enough to conclude a difference between DBS capillary and venous is the true matrix for home sampling.
samples and more data will be gathered in the ongoing part two of Despite the matrix differences in DBS compared to plasma the clini-
this study to evaluate this nding. The two different methods of conver- cal decision can be made directly from DBS (LTG) or after conversion to
sion showed similar results; hence the ratio approach was used. Since estimated plasma concentrations (CBZ, VPA) and used in the future as
venous blood collection is the traditional way of collecting samples an alternative to traditional sampling for these children. The result
from adults it is also important to study the relationship between ve- from the cross validation showed that there were concentrations that
nous DBS and capillary DBS from adults before DBS can be offered as differed more than 20% and in some individuals, especially for LTG,
home sampling for these patients. even N 30% difference (S7). For TDM of antiepileptic drugs the ranges
A conversion factor in VPA was derived from the ratio between mea- are recommended ranges. Every patient should have an individual
sured plasma and DBS concentrations (n = 33). We used the ratio ap- dose that is suitable depending on type of epilepsy and combinations
proach with a correction factor of 1.58 for these patients since it was of other drugs. If a plasma or DBS concentration will be in a range
comparable with results from theoretical approach. Conversion factors around the critical concentration where there is risk of adverse effects,
of 1.88 and 1.96 have been reported by others [20,21]. For LTG, the the clinician will always contact the patient for an evaluation and health
status. Unexpected deviations from earlier concentration levels or levels
being close to be at risk of serious adverse effects should always result in
Table 3
Comparison of plasma and DBS concentrations in relation to therapeutic ranges. considering a new sample for verication, and in the case of DBS, a new
sample in plasma is recommended.
CBZ n = 17 LTG n = 20 VPA n = 33
We conclude that the differences noticed for CBZ and VPA are ac-
Categories Plasma DBS Plasma DBS Plasma DBS ceptable from a clinical point of view. The differences in LTG are large
Low 3 2 4 4 4 6 in some of the compared concentrations and the decision is to be careful
Within range 14 15 13 11 25 21 with LTG and all concentrations above 10 g/mL should be considered as
Risk of adverse effects 0 0 3 5 4 6 being in risk of adverse effects. When each pair of DBS concentrations
Different categories 1 2 6
and plasma concentrations were evaluated no patient were identied

Please cite this article as: C. Linder, et al., Comparison between dried blood spot and plasma sampling for therapeutic drug monitoring of
antiepileptic drugs in children with ep..., Clin Biochem (2016), http://dx.doi.org/10.1016/j.clinbiochem.2016.12.008
6 C. Linder et al. / Clinical Biochemistry xxx (2016) xxxxxx

to have a different recommendation of dose adjustment, even though Appendix A. Supplementary data
some fell into different categories. On the other hand DBS is a new ma-
trix and more data is needed to be sure of how much DBS concentra- Supplementary data to this article can be found online at http://dx.
tions may differ from plasma concentrations when using a traditional doi.org/10.1016/j.clinbiochem.2016.12.008.
lter paper for collection.
Although the population studied is small, we consider it large
enough to go on with the next part of the study where guardians are ed- References
ucated to collect DBS samples from their children or older children col-
lect the samples themselves. The data gathered will be added to these [1] R. Ackers, M.L. Murray, F.M.C. Besag, I.C.K. Wong, Prioritizing children's medicines
for research: a pharmacoepidemiological study of antiepileptic drugs, Br. J. Clin.
DBS data and in this way enlarge the study population. Preliminary re- Pharmacol. 63 (6) (2007) 689697.
sults show that guardians can collect qualitative DBS samples from [2] P. Mattsson, T. Tomson, K. Edebol Eeg-Olofsson, L. Brnnstrm, G. Ringbck Weitoft,
their children. Using DBS in a home based setting may facilitate for chil- Association between sociodemographic status and antiepileptic drug prescriptions
in children with epilepsy, Epilepsia 53 (12) (Dec 2012) 21492155 Available
dren who also have concomitant neurological and/or cognitive disabil- from: http://www.ncbi.nlm.nih.gov/pubmed/23061699.
ities and their guardians [3] P.N. Patsalos, D.J. Berry, B.F.D. Bourgeois, J.C. Cloyd, T.A. Glauser, S.I. Johannessen,
The calculation of theoretical plasma concentrations aims at apply- et al., Antiepileptic drugs best practice guidelines for therapeutic drug monitoring:
a position paper by the subcommission on therapeutic drug monitoring, ILAE Com-
ing therapeutic ranges and decision limits based on plasma concentra-
mission on Therapeutic Strategies, Epilepsia 49 (7) (Jul 2008) 12391276 Available
tions on DBS data. Still, there is an added uncertainty in treating the from: http://www.ncbi.nlm.nih.gov/pubmed/18397299.
calculated theoretical plasma values as normal plasma values in a [4] A. Verrotti, G. Loiacono, G. Coppola, A. Spalice, A. Mohn, F. Chiarelli, Pharmacother-
TDM context. apy for children and adolescents with epilepsy, Expert. Opin. Pharmacother. 12 (2)
(Feb 2011) 175194 Available from: http://www.ncbi.nlm.nih.gov/pubmed/
The obvious alternative to DBS estimated plasma concentrations 21208135.
from DBS would be to construct therapeutic ranges and decision limits [5] G. Lawson, S. Tanna, Self-sampling and quantitative analysis of DBS: can it shift the
for uncorrected DBS data instead of taking the detour over plasma con- balance in over-burdened healthcare systems? Bioanalysis 7 (16) (2015)
19631966.
centrations. This could be done but calls for much more DBS data than is [6] A.J. Wilhelm, J.C.G. den Burger, E.L. Swart, Therapeutic Drug Monitoring by Dried
present at this stage as the power of clinical data from plasma over DBS Blood Spot: Progress to Date and Future Directions, Clin. Pharmacokinet. 53 (11)
is indisputable. The need for a straightforward evaluation and decision (Sep 10 2014) 961973 Available from: http://www.ncbi.nlm.nih.gov/pubmed/
25204403.
making in the clinical situation also supports the notion of translatabil- [7] Li W, Tse FLS. Dried blood spot sampling in combination with LC-MS/MS for quan-
ity between plasma and DBS concentrations as the main clinical scenar- titative analysis of small molecules. Biomed. Chromatogr. John Wiley and Sons Ltd
io in the future is that plasma and DBS sampling will be used alternately (Southern Gate, Chichester, West Sussex PO19 8SQ, United Kingdom); Jan 2010;
24(1):4965. Available from: http://www.interscience.wiley.com/cgi-bin/fulltext/
in monitoring the individual patient.
123215196/PDFSTART\nhttp://ovidsp.ovid.com/ovidweb.cgi?T= JS&PAGE=
After nalization of the second part of the study the ratio approach reference&D=emed9&NEWS=N&AN=2010068470
with estimated plasma concentrations for CBZ, VPA and for LTG uncon- [8] P.A. Demirev, Dried blood spots: analysis and applications, Anal. Chem. 85 (2013)
779789.
verted DBS concentrations is suggested to be used in TDM for this pa-
[9] A. Sharma, S. Jaiswal, M. Shukla, J. Lal, Dried blood spots: concepts, present status,
tient group. It is of importance that DBS home sampling will be and future perspectives in bioanalysis, Drug Test. Anal. 6 (5) (May 2014) 399414
offered as a service to this patient group and that there is a possibility Available from. (http://www.ncbi.nlm.nih.gov/pubmed/24692095).
to choose traditional collection at a clinic. DBS collection will not be [10] M. Wagner, D. Tonoli, E. Varesio, G. Hopfgartner, The use of mass spectrometry to
analyze dried blood spots, Mass Spectrom. Rev. (Sep 22 2014) Available from:
the most convenient solution for all patients. http://www.ncbi.nlm.nih.gov/pubmed/25252132.
[11] P. Denniff, N. Spooner, Volumetric absorptive microsampling: a dried sample collec-
5. Conclusion tion technique for quantitative bioanalysis, Anal. Chem. 86 (2014) 84898495.
[12] G. Lenk, S. Sandkvist, A. Pohanka, G. Stemme, O. Beck, N. Roxhed, A disposable sam-
pling device to collect volume-measured DBS directly from a ngerprick onto DBS
DBS has a great potential as a valuable tool in the treatment and paper, Bioanalysis 7 (16) (2015) 20852094.
evaluation of epilepsy in children especially among children with dis- [13] L.A. Leuthold, O. Heudi, J. Dglon, M. Raccuglia, M. Augsburger, F. Picard, et al., New
microuidic-based sampling procedure for overcoming the hematocrit problem as-
abilities, for whom a visit to clinic is an effort. This clinical validation sociated with dried blood spot analysis, Anal. Chem. 87 (4) (2015) 20682071.
shows that the time is right to go on and investigate whether it is feasi- [14] R. Sturm, J. Henion, R. Abbott, P. Wang, Novel membrane devices and their potential
ble for this patient group to collect DBS samples in their home environ- utility in blood sample collection prior to analysis of dried plasma spots, Bioanalysis
7 (16) (2015) 19872002.
ment. The need to monitor other AEDs can be met by expanding the [15] G. Emmons, M. Rowland, Pharmacokinetic considerations as to when to use dried
method. blood spot sampling, Bioanalysis 2 (11) (2010) 17911796.
With careful TDM of these patients, DBS is suggested to be used as an [16] P.H. Hinderling, Red blood cells: a neglected compartment in pharmacokinetics and
pharmacodynamics, Pharmacol. Rev. 49 (3) (1997) 279295.
alternative to the traditional venous or capillary sampling at a clinic for
[17] P. Paixo, L.F. Gouveia, M. JAG, Prediction of drug distribution within blood, Eur. J.
patients with normal HCT and no signs of other severe problematic in- Pharm. Sci. 36 (45) (2009) 544554.
teractions between drugs. [18] M.V. Antunes, S. Raymundo, V. de Oliveira, D.E. Staudt, G. Gssling, G.P. Petef, et al.,
Ultra-high performance liquid chromatography tandem mass spectrometric meth-
od for the determination of tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen
Funding and endoxifen in dried blood spots, Talanta 132 (2015) 775784.
[19] C.M. Nijenhuis, A.D.R. Huitema, S. Marchetti, C. Blank, J.B.A.G. Haanen, J.V. van
This study was supported by grants from Swedish Research Council Thienen, et al., The use of dried blood spots for pharmacokinetic monitoring of i
treatment in melanoma patients, J. Clin. Pharmacol. (2016) 16.
(VR-2011/3440) and from Stockholm Healthcare Region (ALF/SLL 2013/ [20] L. Rhoden, M.V. Antunes, P. Hidalgo, C..D. Silva, R. Linden, Simple procedure for de-
0398). termination of valproic acid in dried blood spots by gas chromatographymass
spectrometry, J. Pharm. Biomed. Anal. 96C (Apr 2 2014) 207212 Elsevier B.V. Avail-
able from: http://www.ncbi.nlm.nih.gov/pubmed/24769300.
Disclosure of conict of interests [21] S.T. Kong, S.-H. Lim, W.B. Lee, P.K. Kumar, H.Y.S. Wang, Y.L.S. Ng, et al., Clinical vali-
dation and implications of dried blood spot sampling of carbamazepine, valproic
None of authors has any conict of interest. acid and phenytoin in patients with epilepsy, PLoS One 9 (9) (Jan 2014), e108190.
Available from: http://www.ncbi.nlm.nih.gov/pubmed/25255292.
[22] A. Pohanka, M. Mahindi, M. Masquelier, L.L. Gustafsson, O. Beck, Quantication of
Acknowledgements valproic acid in dried blood spots, Scand. J. Clin. Lab. Invest. 74 (7) (Jul 25 2014)
648652 Available from: http://www.ncbi.nlm.nih.gov/pubmed/25059925.
[23] S. AbuRuz, M. Al-Ghazawi, Y. Al-Hiari, A simple dried blood spot assay for therapeu-
The authors wish to thank research nurse Mirja Neideman who par-
tic drug monitoring of lamotrigine, Chromatographia 71 (1112) (Apr 11 2010)
ticipated in the research and LicMed, Clinical Laboratory Scientist Gerd 10931099 Available from: http://www.springerlink.com/index/10.1365/s10337-
Ackehed for bioanalytical expertise. 010-1569-y.

Please cite this article as: C. Linder, et al., Comparison between dried blood spot and plasma sampling for therapeutic drug monitoring of
antiepileptic drugs in children with ep..., Clin Biochem (2016), http://dx.doi.org/10.1016/j.clinbiochem.2016.12.008
 C. Linder et al. / Clinical Biochemistry xxx (2016) xxxxxx 7

[24] N.M. Shah, A.F. Hawwa, J.S. Millership, P.S. Collier, J.C. Mcelnay, A simple Clin. Transpl. 20 (2006) 221225 Available from: http://www.ncbi.nlm.nih.gov/
bioanalytical method for the quantication of antiepileptic drugs in dried blood pubmed/16640530.
spots, J. Chromatogr. B 923924 (2013) 6573 Elsevier B.V. Available from: http:// [32] Linder C, Gambell Barroso M. Blodprovtagning p lterpapper, Barn|Lkemedel.
dx.doi.org/10.1016/j.jchromb.2013.02.005. [cited Jun 2 2016]. Available from: https://www.youtube.com/watch?v=
[25] E. Shokry, F. Villanelli, S. Malvagia, A. Rosati, G. Forni, S. Funghini, et al., Therapeutic LpJeyfoWsgA
drug monitoring of carbamazepine and its metabolite in children from dried blood [33] S. Capiau, V.V. Stove, W.E. Lambert, C.P. Stove, Prediction of the hematocrit of dried
spots using liquid chromatography and tandem mass spectrometry, J. Pharm. blood spots via potassium measurement on a routine clinical chemistry analyzer,
Biomed. Anal. 109 (2015) 164170 Elsevier B.V. Available from: http://dx.doi.org/ Anal. Chem. 85 (1) (2013) 404410.
10.1016/j.jpba.2015.02.045. [34] S. Capiau, L.S. Wilk, M.C.G. Aalders, C.P. Stove, A novel, nondestructive, dried blood
[26] G. Martins, S. De Lima, R.Z. Hahn, C. Rama, L. Rhoden, P. Hidalgo, et al., Determinao spot-based hematocrit prediction method using noncontact diffuse reectance
Simultnea de Carbamazepina, fenitona e fenobarbital em sangue seco em papel spectroscopy, Anal. Chem. 88 (12) (Jun 21 2016) 65386546 Available from:
por cromatograa lquida de alta ecincia, Quim Nova 37 (6) (2014) 10671071. http://www.ncbi.nlm.nih.gov/pubmed/27206105.
[27] D. Milosheska, I. Grabnar, T. Vovk, Dried blood spots for monitoring and individual-
ization of antiepileptic drug treatment, Eur. J. Pharm. Sci. 75 (2015) 2539.
[28] CLSI, Procedures and Devices for the Collection of Diagnostic Capillary Blood Speci-
mens; Approved Standard, sixth ed., CLSI document H04-A6, 28, Clin Lab Stand Inst
Doc, 2008 25 (28, no 25):viii. Available from: http://scholar.google.com/scholar? Glossary
hl=en&btnG=Search&q=intitle:
Procedures+and+devices+for+the+collection+of+diagnostic+capillary+bloo- DBS, dried blood spot sampling = collection, transport and storage of capillary blood on cellu-
d+specimens+:+approved+standard#1. lose-based lter papers.:
[29] C. Linder, M. Andersson, K. Wide, O. Beck, A. Pohanka, A LCMS/MS method for ther- TDM, therapeutic drug monitoring = analysis and evaluation of drug concentrations for opti-
apeutic drug monitoring of carbamazepine, lamotrigine and valproic acid in DBS, mization of individual patient drug treatment, for which clinical experience or clinical trials
Bioanalysis 7 (16) (2015) 20312039. have shown improved outcome.:
[30] European Medicines Agency. Guideline on bioanalytical method validation. EMEA/ HCT, hematocrit = the proportion of cells in whole blood by volume.:
CHMP/EWP/192217/2009 Rev.1 Corr. 2011. Available from: www.ema.europa.eu Red blood cell/plasma ratio = concentration of the test compound in red blood cells compared
[31] N. Yonan, R. Martyszczuk, A. Machaal, A. Baynes, B.G. Keevil, Monitoring of cyclo- with plasma.:
sporine levels in transplant recipients using self-administered ngerprick sampling,

Please cite this article as: C. Linder, et al., Comparison between dried blood spot and plasma sampling for therapeutic drug monitoring of
antiepileptic drugs in children with ep..., Clin Biochem (2016), http://dx.doi.org/10.1016/j.clinbiochem.2016.12.008