1. No category

Automated Blood Sampling associated or not with Dried
Blood Spot sample processing provides similar exposure
parameters than classical pharmacokinetic procedures
Nicolas Aubert, Thibault Ameller, Christelle Lefebvre, Stéphanie Laurent, Sébastien Leuillet, Jean-Jacques Legrand
CiToxLAB France, Evreux.
Introduction
Results and discussion
The objective of this study was to evaluate the potential consequences of the sampling process
(automated or manual) and analytical sample preparation (standard plasma or Dried Blood Spot
assay) on pharmacokinetic parameters. Ibuprofen was selected for this assessment because this
compound is compatible with dried blood spotting and has a short half life in rats.
_ SD)
Figure 4: Mean (+
concentrations (vs. time)
in plasma and whole blood
following oral gavage
administration at 25 mg/kg
in rats
70
MS-SB
AS-SB
60
MS-DBS
50
Twenty-four female Sprague-Dawley rats (11 weeks old) with a mean body weight of 247 g on the
day of treatment, were allocated to four groups of 6 animals, and treated once by gavage at the
dose-level of 25 mg/kg with ibuprofen. The test item was administered as a solution in phosphate
buffer saline (PBS) under a constant dose-volume of 5 mL/kg.
After oral gavage, blood was collected at different time-points by either manual collection process
(MS) or automated blood collection (AS), then the blood was processed using standard bioanalytical method (SB) or dried blood spot assay (Figure 1).
Specifically, venous blood samples (300 μL) were taken into tubes containing EDTA (and kept at
+4°C) from all animals at the following time-points: 0.25 15 min, 0.5 30 min, 1, 3, 6 and 9 hours postgavage.
- Manual sampling (MS): Blood was manually sampled from 12 rats by venopuncture of the tail vein
after warming the animals at 37°C during 10 minutes in order to allow tail vein vasodilation. When
sampling from the caudal was difficult, the blood was taken from the retro-orbital sinus of the
animals under a light isoflurane anesthesia.
Automatic sampling (AS): Blood was automatically sampled from 12 rats through a catheter
implanted in the jugular vein and connected to a refrigerated fraction collector.
Oral (gavage)
Treatment
Blood
Sampling
Concentration (Mg/mL)
Materials and Methods
AS-DBS
40
30
20
10
0
0
1
2
3
4
5
6
7
8
9
10
Time (hour)
Blood
Sampling
tmaxa
(h)
Blood
Processing
λZ
(1/h)
Cmax
(μg/mL)
AUC0-t
(μh/mL)
t1/2
(h)
AUC0-∞
(μh/mL)
MS
SB
1.0
35.1
13.5
0.227
0.099
3.7
2.0
157.3
73.2
202.9
91.9
AS
SB
1.0
35.5
11.7
0.273
0.096
3.0
1.7
121.0
20.8
149.5
33.4
MS
DBS
0.8
34.8
20.3 0.222
0.053
3.3
1.0
101.6
27.4
119.2
24.3
AS
DBS
0.9
27.1
8.0
0.101
2.1
0.6
83.8
19.1
89.5
19.6
TABLE 1: The pharmacokinetic parameters (mean
0.359
SD) using winnonlin software
AS: Automated blood sampling, MS: Manual sampling, DBS: Dried blood spot, SB: Standard bioanalysis
a: value corresponds to the calculation of the median. *: statistically significant (p-value <0.05).
Manual sampling
(MS)
(12)
Dried spotting
(6)
Dried
Blood Spotting
(DBS)
Automated sampling
(AS) (ABS 110)
(12)
Standard
Bioanalysis (SB)
(6)
Standard
Bioanalysis (SB)
(6)
Dried Plasma
Spotting
(DPS)
Dried spotting
(6)
Dried
Blood Spotting
(DBS)
Dried Plasma
Spotting
(DPS)
For each tested matrix (plasma or blood), a similar shape of the test item levels vs. time curves was
observed whatever the sampling collection (automated or manual) and preparation (Dried Blood
Spot or standard bioanalysis). No significant differences were noted between the main calculated
PK parameters (AUC0-t, Cmax, Tmax and t1/2).
The extrapolated areas using the half-life values to obtain AUC0-∞ values were sometimes much
higher (>20%) than the corresponding AUC0-t values. The AUC0-∞ values were therefore considered
as less reliable, and consequently the observed differences were not considered as related to the
sample collection and/or preparation processes.
Pharmacokinetic
Modeling
Statistical
analysis
Figure 5: Mean ( SD)
concentrations (vs. time)
in plasma and whole blood
using DBS cards following
oral gavage administration
at 25 mg/kg in rats
70
MS-DPS
60
AS-DPS
MS-DBS
Figure 1: Study design (x: number of animals)
50
The automated blood sampler (Instech’s model ABS110 –
FIGURE 2) can be useful in drug metabolism and
pharmacokinetics studies by eliminating labor-intensive manual
withdrawals of laboratory animals, and consequently, reducing
the stress on animals caused by frequent handling. The sampler
withdraws blood from a catethered freely-moving animal according to programmed schedule. The sampling schedules, volumes
and withdrawal rates are controlled and monitored from a
computer. The main advantages of ABS include also : undiluted
samples (verified by in-line sensors), the shortest collection
path (to minimize the chance of cross-over) and reduced volume
of wasted blood per sample (3 μL, to minimize hemodilution).
Figure 2: Automated Blood
Sampler (ABS 110)
3a
3b
Figure 3: Dried Blood Spot
The blood was then processed as follow:
- SB : whatever the collection method, the blood of 6 animals
was centrifuged (3600 g, 10 min, +4°C) and the obtained plasma
was kept at -20°C until analysis.
- DBS : whatever the collection method, the blood of 6 animals
was immediately spotted (4 x 15 μL) on Dried Blood Spot (DBS)
cards, dried for 3 hours and kept at room temperature into
gas-impermeable bag until analysis.
Although this procedure is not recommended by the card
supplier, this was intended for assessing the influence of the
matrix spotted on the obtained pharmacokinetic parameters.
- DPS: whatever the collection method, the remaining blood of
6 animals (following blood spotting) was centrifuged (3600 g,
10 min, +4°C) and the obtained plasma was spotted (4 x 15 μL)
on Dried Blood Spot (DBS) cards, dried for 3 hours and kept at
room temperature into gas-impermeable bag until analysis.
The dried blood spot (DBS) method is an alternative technique that overcomes the drawbacks of standard method. DBS
requires microvolume sampling (only 10 to 20 μl per sample), a
simpler and safer processing and an easier storage and transportation logistics. Using DBS is straightforward: the blood from
the test subject is collected and applied directly onto the card
(Whatman FTA® DMPK Cards) and dried (Figure 3a). For analysis,
a sample disc is punched and the analytes are immediately
extracted using aqueous/organic solvent mixtures (Figure 3b).
The card lyses cells and denatures proteins on contact. It also
inactivates viruses, making handling and shipping of samples
safer. Samples can be shipped and stored at ambient temperature and long term stability has been demonstrated for analytes
and metabolites sensitive to plasma enzymes.
The plasma samples or blood/plasma spots were analyzed using a validated LC-MS/MS method.
The pharmacokinetic evaluation was carried out using a non-compartmental method (WinNonlin®
software) and the following parameters were calculated and/or determined: Cmax (maximum concentration), Tmax (time to maximum concentration), λz (elimination rate constant), t1/2 (terminal half-life),
AUC0-t (area under the curve from 0 hours to the time-point of the last quantifiable concentration)
and AUC0-∞ (area under the curve from time 0 to infinity). The results were submitted to a statistical
analysis (SAS® software).
Concentration (Mg/mL)
AS-DBS
40
30
20
10
0
0
1
2
3
4
5
6
7
8
9
10
Time (hour)
λZ
(1/h)
Blood
Sampling
Blood
Processing
tmaxa
(h)
MS
DPS
0.7
48.5
20.8
0.226
0.066
3.3
1.1
152.3
39.8
180.6
35.5
0.8
34.8
20.3
0.222
0.053
3.3
1.0
101.6
27.4
119.2
24.3
0.36
<0.0001***
MS
DBS
p-value
Cmax
(μg/mL)
0.7993
t1/2
(h)
AUC0-t
(μh/mL)
0.9758
0.0005***
AUC0-∞
(μh/mL)
0.0004***
AS
DPS
1.0
46.6
11.6
0.409
0.118
1.8
0.6
143.7
33.3
149.8
34.4
AS
DBS
0.9
27.1
8.0
0.359
0.101
2.1
0.6
83.8
19.1
89.5
19.6
p-value
0.38
0.0147*
TABLE 2: The pharmacokinetic parameters (mean
0.1725
0.1825
0.0033**
0.0023**
SD) using winnonlin software
AS: Automated blood sampling, MS: Manual sampling, DBS: Dried blood spot, DPS: Dried plasma spot
a: value corresponds to the calculation of the median. *, **, ***: statistically significant (p-value <0.05, 0.01 or 0.001, respectively)
The comparison of the pharmacokinetic parameters between DPS (plasma) vs. DBS (whole blood)
following manual or automated collection processes showed statistically significant differences for
Cmax, AUC0-t and AUC0-∞. As these differences were observed whatever the samples collection process,
they were considered as related to the nature of the matrix (blood or plasma) spotted on the DBS
card.
Conclusion and discussion
Altogether, these results indicate that:
Whatever the bioanalytical method (DBS or Standard), the Automated Blood sampling did not
induce significant differences on the PK parameters when compared to the manual sampling.
Differences were observed between DPS (plasma) vs. DBS (whole blood) processing (whatever
the collection process), suggesting a relationship to the nature of the spotted matrix, but confirming that the DBS card should be used according to specifications with blood only.
Although the automated blood sampling requires catheterization of the animals, the reduction
of handling during the sampling decreases the animal stress and improves the quality and repeatability of the blood samples. Association of automated blood sampling and low volume DBS
analytical method is a real ethical improvement.
Consequently, and taking into account the advantages and disadvantages of each method, these
new processes can be now routinely used at CIT in investigative pharmacokinetic studies using a
limited number of rats and a minimum quantity of product.
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