Evaluation of a Volumetric Dried Blood Spot Card Using a Gravimetric

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Evaluation of a volumetric dried blood spot card using a gravimetric method and a bioanalytical method with capillary blood from 44 volunteers Gabriel Lenk, Shahid Ullah, Göran Stemme, Olof Beck, and Niclas Roxhed Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02905 • Publication Date (Web): 11 Mar 2019 Downloaded from http://pubs.acs.org on March 13, 2019

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Analytical Chemistry

Evaluation of a Volumetric Dried Blood Spot Card using a Gravimetric Method and a Bioanalytical Method with Capillary Blood from 44 Volunteers Gabriel Lenk,*,† Shahid Ullah,‡ Göran Stemme,† Olof Beck,‡ Niclas Roxhed† † ‡

KTH Royal Institute of Technology Stockholm, Department of Micro and Nanosystems, 10044 Stockholm, Sweden Karolinska University Hospital, Clinical Pharmacology, 14186 Stockholm, Sweden

ABSTRACT: Dried Blood Spot (DBS) sampling is a promising method for collection of microliter blood samples. However, hematocrit-related bias in combination with sub-punch analysis can result in inaccurate quantification of analytes in DBS samples. In this study we use a microfluidic DBS card, designed to automatically collect fixed volume DBS samples irrespective of the blood hematocrit, to measure caffeine concentration in normal finger prick samples obtained from 44 human individuals. Caffeine levels originating from blood drops of unknown volume collected on the volumetric microfluidic DBS card were compared to volume-controlled pipetted DBS samples from the same finger prick. Hematocritindependence and volumetric sampling performance was also verified on caffeine-spiked blood samples in-vitro, using both LC-MS/MS and gravimetric methods, on hematocrits from 2662%. The gravimetric measurements show an excellent metering performance of the microfluidic DBS card, with a mean blood sample volume of 14.25 µl ± 3.0% (n=51). A measured mean bias below 2.9% compared to normal hematocrit (47%) demonstrates that there is no significant hematocrit-induced bias. LC-MS/MS measurements confirm low CV and hematocrit independence of the sampling system and exhibit no substantial mean bias compared to pipetted DBS. Tests with 44 individuals demonstrated applicability of the microfluidic DBS card for direct finger prick blood sampling and measured caffeine concentrations show a good agreement with measurements of pipetted DBS. The presented concept demonstrates a good volumetric performance which can help to improve the accuracy of DBS analysis by analyzing a whole spot, equivalent to a defined volume of liquid blood. (252)

INTRODCUTION Since first developed in the 1960s, Dried Blood Spot sampling and other microsampling technologies have gained interest for many new applications. During the last decades, the number of scientific publications per year in the field of DBS sampling has been growing from approximately 60 in 2000 to more than 400 in 20111. Key factors for a thriving interest in DBS technology in various fields and applications are ethical considerations and logistical advantages that enable new possibilities in bioanalytical procedures which can be beneficial for preclinical and clinical studies, patients, health care providers and laboratories. DBS technology also shows a huge potential from an economical point of view with the cost for collecting a DBS sample being estimated to be only 20-25% of the cost of a conventional venous blood sample2. For patients, study participants and other users of DBS sample collection, minimal invasiveness, small sample

volume and the simplicity of the method, potentially allowing users to take a sample themselves, are the main advantages. Both, the US Department of Transportation and the World Health Organization consider DBS specimen a non-regulated, non-biohazard fright which offers the possibility of sample shipping with the national post3,4. Sample shipment without dry ice packaging is possible since many analytes are stabilized by drying5-6. The ability to ship samples from any sampling site worldwide to any laboratory with conventional mail service has the potential to simplify the process of sample collection and analysis vastly. The combination of self-sampling and simplified sample logistic has the potential to be a viable solution for patient-centric blood sampling to reduce the cost for health care providers2, 7 and simplify sample collection for clinical trials8. For laboratories, the use of DBS samples can help to simplify sample preparation, sample storage and even provide improved sample stability for certain analytes6. Apart from the already established application for newborn

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screening, these advantages have been rising the interest and are driving the ongoing development for applying DBS sampling in different areas such as toxicology9, preclinical10-11 and clinical drug development8, therapeutic drug monitoring9, 12-13, and drug14 and sports doping screening15. DBS have also been used to realize large population studies e.g. in the field of medical anthropology16, nutrition17 and others18-19. However, before DBS sampling can be accepted as a viable alternative to venous blood sampling with performance good enough to obtain regulatory acceptance, a few pressing issues that currently limit the use of DBS for new applications need to be solved. Traditionally, a fraction of the spotted and dried blood sample is extracted by punching out a disk with a defined diameter correlating to a certain volume that is analyzed. The two main issues with this approach have been the blood hematocrit, which affects the volume of blood absorbed in the punched-out disc20 and spot inhomogeneities which can cause variations in concentration depending on the punch location of a sample within the spot21-22. Also, DBS sample quality, which is normally not a concern in studies based on venous blood where samples are carefully pipetted from a sufficient blood reservoir, is often much more demanding in studies with DBS collected from patients23. With DBS collected directly from a finger prick the yield of successful DBS samples is reported to be anything from 70%23, 75-90%18 up to 98%17. In recent years there have been a few concepts developed to overcome the issues associated with DBS sample analysis such as special paper delivering DBS spot sizes independent from hematocrit24, volumetric absorptive microsampling (VAMS)25, microfluidic DBS card holders26, glass capillary based sampling systems27 and microfluidic DBS chips28-30. Even though differently realized, all these approaches aim at better volume control of the sample that is dried and analyzed, something that is crucial for accurate quantification. VAMS sampling is the only concept of the above that has developed to the extent that various patient studies have been published including sample collection in a clinic31 or in a self-sampling scenario32 which is highly relevant when considering patient-centric blood sampling. However also VAMS technology has to be used carefully according to the instructions to prevent unacceptable bias25 and requires the use of a suitable extraction method to achieve a consistent and hematocrit independent extraction recovery33. This paper presents a detailed evaluation of the previously presented concept29 based on microfluidic volume metering. A commercially available version of the concept has been developed and has been evaluated by different methods. One study evaluates the volumetric performance of the microfluidic DBS card across a range of hematocrit levels based on a radiolabeled analyte28 and another study has presented the use of the microfluidic DBS card for the quantification of PEth30, a biomarker for alcohol abuse. A recent study has performed quantification of caffeine in venous patient samples covering a hematocrit range of 18% to 55% and systematically evaluating the volumetric performance in relation to the hematocrit and the input volume of 25-50 µl34. While the commercially available version of the microfluidic DBS card uses an

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automated manufacturing process, this study describes the evaluation of the underlying concept based on manually built protypes. The volumetric performance of the prototypes was evaluated using a gravimetric method and using a bioanalytical method with caffeine as study compound. The study presents for the first time bioanalytical results based on authentically collected capillary DBS using the microfluidic metering principle. Caffeine levels in volumetric DBS from volume undefined finger prick samples involving 44 healthy volunteers were used to assess the volumetric performance of microfluidic DBS from finger pricks and test the robustness of the concept in use with untreated human whole-blood. The latter is an essential requirement for the implementation of microfluidic DBS in clinical trials and patient-centric blood sampling. MATERIALS AND METHODS Chemicals, reagents and stock solutions. Reference standards including caffeine and its isotope labelled analogue used as internal standard (Cerilliant Corp., Round Rock, USA) were purchased from Sigma-Aldrich Sweden AB (Stockholm, Sweden). Stock and working solutions were prepared in methanol and stored at –20 ℃. All other chemicals including LC-MS grade methanol (Fisher Scientific AB, Gothenburg, Sweden) and ammonia (25%) (Merck KGaA, Darmstadt, Germany) were of highest analytical grade. Milli-Q DI water was prepared in-house with ultra-pure quality (>18 MΩ/cm). Standard blood sample preparation. Fresh citrated blood from a caffeine-abstinent healthy male volunteer (at least four days avoiding caffeine containing products) was collected by venous puncture and hematocrit was measured. Four 5-ml aliquots were centrifuged for 10 minutes at 2400 g to separate the blood cells from the blood plasma. A defined amount of blood plasma was removed or added from the centrifuged blood to reach a specific hematocrit of 26%, 37%, 47%, and 62% covering nearly the full range of physiological possible hematocrit concentrations. Samples were carefully remixed on a Gyromini 3D Rocker (LabNet International Inc., Edison, USA) and hematocrit in all samples was confirmed using a XP-300 hematology analyzer (Sysmex Europe GmbH, Norderstedt, Germany). A stock solution of 2.39 mg/ml caffeine in methanol was diluted to working solutions of 4 and 80 µg/ml. Three 1-ml aliquots at each hematocrit were prepared and two of the aliquots were spiked with 25 µl of the respective caffeine working solutions to obtain control target concentrations of 100 ng/ml and 2000 ng/ml. Standards and controls were prepared between 100–10000 ng/ml by pipetting 15 µl on pre-cut PVA-paper discs with an air displacement pipette. Calibrators were prepared using blood with 47% hematocrit. Microfluidic DBS card fabrication. The microfluidic DBS cards as illustrated in Figure 1 were prepared similar to previously described DBS chips29 with several adjustments made to improve the performance and function with whole blood over a wide range of hematocrits. While DBS chips were built for a single volumetric DBS, the microfluidic DBS card comprises up to six volume metering units. The form resembles that of traditional DBS cards and potentially allows for an


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automated extraction of the sample. The DBS paper which in the DBS-chip design was sandwiched in between multiple laminated plastic layers is now replaced by a pre-cut paper disk attached to the backside of the card which allows removal of the disk without the need for punching (see Figure 1C and Figure 2). To allow a good timing control of the valves across a whole range of blood hematocrits, several adjustments were made. The volume of the microchannel was increased allowing for more reliable PVA valve dissolving. A different PVA valve material with a lower molecular weight was used to achieve better control of dissolving times and allow dissolution at high blood hematocrit levels. Lower molecular weight of the dissolvable PVA valves enabled the use of thicker PVA valve films (8 µm and 10 µm) which simplifies fabrication and yields better control of dissolution times. A more detailed description of the manufacturing process can be found in the supporting information.

due to evaporation of water from the blood spots was considered and corrected for when analyzing the results. The average loss of mass for detaching the disk from the adhesive backside of the microfluidic DBS card was determined to be 0.39 mg. The mass loss due to evaporation was determined by recording evaporation curves from DBS discs containing 15 µl of blood. The time needed to complete a gravimetric measurement was 60 seconds for hematocrits 26%, 37%, 47% and 90 seconds for hematocrit 62% corresponding to evaporation correction values of 0.42 µl and 0.62 µl respectively. The final volumes were calculated from the weight difference of the disk before spotting and after spotting using the described correction values as well as the specific gravity of blood at hematocrits of 26%, 37%, 47% and 62% assuming an average protein content35. For comparison, 15 µl reference samples at each hematocrit were pipetted on pre-cut DBS disks with a calibrated Thermo Fischer air displacement pipette (Finnpipette 2-20 µl, Thermo Fisher Scientific, Waltham, USA). The mass of the pipetted blood was weighed directly on the high precision scale and used to calculate the pipetted sample volume at each hematocrit.

Figure 2. Bottom view of the microfluidic DBS card showing the pre-cut sample pads for the gravimetric measurements. (A) shows a blank disk while (B-D) show how blood at different hematocrit (but same volume) has spread differently on the sample pad.

Figure 1. (A) The microfluidic DBS sample collection card containing six metering units for separate collection of 14.25 µl aliquots. (B) Detailed view of a metering unit with the functional elements. (C) Schematic cross section of a metering unit with all components and functional layers.

Gravimetric measurements. To measure the blood volume metered with the card, 40 µl of blood was applied to the microfluidic DBS card and a differential gravimetric method was used to calculate the resulting spotted volume. The mass of each pre-cut DBS disk was weighed on a high precision scale (AT261 Delta Range, Mettler Toledo Inc., Zurich, Switzerland) before being attached at the outlet structure. After the metered volume was transferred to the DBS disk, the DBS disk was immediately removed from the adhesive backside of the card and weighed again. For accurate results, the mass loss during separation of the disk from the adhesive backside of the card as well the mass loss

Volumetric sampling precision and hematocrit dependent sampling performance. For DBS analysis of the entire blood spot, the volume of the spot is directly correlated to the analyzed concentration. Hence, having an accurate DBS volume is of utmost importance for an accurate concentration measurement. This contrasts with partial punch analysis where the volume is estimated based on the punch size. To evaluate the performance of the microfluidic DBS card as compared to pipette DBS, the above described gravimetric method was used. For the microfluidic DBS card nine samples at each hematocrit level were evaluated for hematocrit levels of 26%, 37%, and 62%, as well as 24 samples at a hematocrit of 47%. For the pipetted DBS samples, nine samples at 26%, 37%, 47% and 62% hematocrit were evaluated. LC-MS/MS measurements and sample preparation. To demonstrate sample integrity after being in contact with the microfluidic DBS card and to verify accurate LC-MS/MS measurements of DBS samples over the full range of possible hematocrit, microfluidic DBS card samples were collected in triplicate with blood at four hematocrit levels with two different concentrations of caffeine. Samples were collected by pipetting 40 µl of the respective blood sample to the inlet of the device to perform microfluidic volume metering to the sample pad. Reference samples at all four hematocrit levels and two caffeine concentrations were



prepared in triplicate by pipetting 15 µl on pre-cut PVApaper disks. After drying, all pre-cut disks were transferred to Eppendorf tubes and 200 µl of methanol containing 5 pg/µl of caffeine internal standard were added to fully emerge the spot into the solvent. All samples were vortexed, stored overnight in a refrigerator and vortexed again before approximately 150 µl of the extract was transferred to glass lab tubes for evaporation. After evaporation all samples were reconstituted with 100 µl of mobile phase A and transferred to vials for injection of 2 µl into the LC-MS/MS system. The mass spectrometer was a TSQ Quantiva triple quadrupole coupled to a Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Waltham, USA). The instrument was operated in electrospray positive ionization mode with a spray voltage of 3.6 kV. The achieved product ions were m/z 195 > 138, 110 for caffeine and m/z 198 > 140 for caffeine internal standard. The LC separation was performed on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 µm) (Waters Co, Milford, USA. The column temperature was set to 50 °C. The mobile phase flow rate was 500 µl/min, operating in a gradient mode with initial mobile phase after injection being 95% A for 0.5 min and a linear gradient starting at 0.5 min to 95% B at 2.5 min. Mobile phase A and B consist of water and methanol respectively, with both containing 0.1% ammonia. Method verification. For recovery evaluation, pre-cut PVA-paper discs with attached dissolvable layers were used and volumes were spotted with an air displacement pipette. Blank blood and blood with a caffeine concentration of 100 ng/ml were spotted in quintuple on precut 7 mm PVApaper dummies. The blank blood extracts were spiked post extraction to achieve a comparable caffeine concentration. For matrix effect evaluation a blank blood extract was spiked post extraction with 15 µl saline solution containing 100 ng/ml caffeine. For reference a duplicate sample without blank blood matrix was prepared. The limit of detection was estimated from blank blood spiked at 10 ng/ml and 1 ng/ml caffeine concentrations (the signal to noise was more than 3 at 10 ng/ml). The intra-day imprecision in quantification was studied at 7000 ng/ml concentration level. Extracts were always run directly and the stability of caffeine has not been evaluated, however Velghe et al. also demonstrate stability of caffeine up to three month under different storage conditions9. It should be noted that the method is not fully validated and can thus not be used for quantification of caffeine. It is intended for comparative purposes only where the same methodology has been used for the comparison of two types of DBS: pipetted whole spot and microfluidic whole spots. A full method validation based on samples collected with the microfluidic DBS card as performed by Velghe et al.9 would have been preferred but was not possible due to the limited availability of sample collection devices. Collection of volunteers DBS samples. Collection of high quality DBS samples can be difficult and there is several mistakes that could occur which may result in low quality DBS samples that are unusable for analysis. To evaluate the robustness of the concept with fresh and untreated blood from a finger prick not containing anticoagulant, and to evaluate the feasibility of the concept for the application of blood directly from the fingertip to the

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device, the microfluidic DBS card was tested with 44 human volunteers. The study participants were between 19 and 74 years old with 26 participants being male and 18 females. The study was approved by the regional ethical board (EPN Stockholm, Dnr 2015/867-31/1) and all volunteers were informed, and written consent was obtained. Because sample collection took place during winter time in a public area inside a building and there was no access to warm water during sample collection, volunteers were asked to use a single-use heating pad to improve blood circulation in the hands. For collection of the blood sample, a nurse performed a finger prick using a blue BD Microtainer® contact activated safety lancet (BD, Franklin Lakes, USA), with 1.5 mm blade and a nominal penetration depth of 2 mm. The nurse had a single attempt per volunteer to collect a microfluidic DBS sample by touching the inlet port of the DBS card with the blood drop from the persons finger (see Figure 3). The sampling procedure was observed and notes about the device performance were taken. As reference, liquid blood from the same finger prick wound was collected in an EDTA treated BD Microtainer® blood collection tube (BD, Franklin Lakes, USA) which was used to immediately create a 15 µl pipetted reference spot, on a PVA-paper disc. Both DBS samples, the microfluidic spot and the pipetted reference spot were analyzed using the above described LC-MS/MS method to quantify the blood caffeine concentration of each individual.

Figure 3. The procedure of microfluidic DBS collection of capillary blood samples from finger pricks: Upon application of a blood drop (A-B), the card automatically removes excess blood (C) and subsequently transfers the channel volume to the sample pad (D-E).

RESULTS AND DISCUSSION Microfluidic DBS card function. For all experiments, notes about the device performance were taken. For the control samples involving four different hematocrit levels and nine samples per hematocrit level, 31 out of 36 samples were successful (86% success rate) while five samples were malfunctioning, and the sample collection was repeated immediately on a new channel. For the evaluation of volumetric sampling precision at a hematocrit of 47%, 15 out of 18 samples were successful (83% success rate) while the remaining three samples were not considered for the sample analysis. It was observed that device failure is most likely due to insufficient process control during the manual fabrication process of the dissolvable PVA films. Volumetric sampling precision. The volume collected with the microfluidic DBS cards determined by gravimetric measurements was 14.1 µl ± 2.8% (n=24) for blood at a


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hematocrit of 47%. Including all samples in the analysis irrespective of hematocrit the mean volume measured was 14.3 µl ± 3.0% (n=51). Pipetted reference spots with a volume of 15 µl and different hematocrit concentrations had a measured mean volume of 14.5 µl ± 5.1% (n=36). This demonstrates a good volumetric precision for the microfluidic DBS card with a performance comparable to pipetting of blood with an air displacement pipette. The high precision of the microfluidic DBS is remarkable considering that the accuracy of gravimetric measurement itself is likely to be better for pipetted DBS than for microfluidic DBS. This is the case because microfluidic DBS per se must be measured differentially and get corrected for evaporation and detach loss while pipetted DBS can be measured directly on the scale. The results on both methods of collecting a volumetric DBS sample are in good agreement with previous results by Spooner et al. which found a 2-5% precision for microfluidic DBS and a 2-7% precision for pipetted DBS. Hematocrit dependent sampling volume. The results of the gravimetric measurements at different hematocrits are shown in Figure 4 and in Table 1 and 2. The mean volume of nine microfluidic DBS samples collected at each hematocrit level has a mean bias smaller than 3% in comparison to the normal hematocrit level of 47%. This demonstrates that the volume collected with the microfluidic DBS card does not show a positive mean bias with increasing hematocrit as reported for traditional subpunch approaches where a positive bias with increasing hematocrit can typically be observed20-21. The CV measured at each individual hematocrit level is between 2.4% and 3.5% (n=9) which is similar to the overall CV irrespective of hematocrit (3.0%, n=51). The mean volume of nine pipetted reference samples at each hematocrit level were measured to have a mean bias between -1.7% and -3.8% compared to blood at normal hematocrit, while showing relative CV of 3.4% to 5.7%. This shows that the microfluidic DBS card also has an accuracy and precision comparable to that of an air displacement pipette when used at different hematocrit levels. Blood residues that are visible after emptying, located along the edges of the microchannel, did not seem to vary significantly or be dependent on the hematocrit. Table 1. Gravimetric evaluation of hematocritdependent sample volumes of the microfluidic DBS card. Hematocrit

Mean

CV

Mean bias

[%]

[µl]

[%]

[%]

26 (n=9)

14.5

2.4

2.8

37 (n=9)

14.5

3.5

2.8

47 (n=24)

14.1

2.8

0.0

62 (n=9)

14.1

2.5

0.1

Table 2. Gravimetric evaluation of hematocritdependent sample volumes of pipetted DBS samples. Hematocrit

Mean

CV

Mean bias

[%]

[µl]

[%]

[%]

26 (n=9)

14.5

5.7

-2.0

37 (n=9)

14.6

3.4

-1.7

47 (n=9)

14.8

4.7

0.0

62 (n=9)

14.2

5.4

-3.8

Figure 4. Box and Whisker plot illustrating the volumetric performance when spotting DBS using either the microfluidic DBS card or a conventional air displacement pipette for different hematocrit between 26% and 62%. The spotted volume was evaluated using a gravimetric method for both devices. Boxes represent the interquartile range and whiskers the 1.5×interquartile range (median excluded). Horizontal lines display the median and crosses the mean values. Outliers are marked as closed circles. Dashed lines illustrate ±10% around the 14.25 µl overall mean (irrespective of hematocrit) of the microfluidic DBS card (red dashed line).

Mass spectrometry measurements. A 7-point calibration line was set up between 100 ng/ml and 10000 ng/ml with R2=0.999 using a weighted 1/x linear model. When the calibrator values were back-calculated they were all within 5% accuracy. The intra-day imprecision and accuracy in quantification at the 7000 ng/mL QC were 6.6% and 101% (n=9). The LC-MS/MS measurements support the gravimetric data, indicating a good volumetric performance of the microfluidic DBS card, independent of the blood hematocrit and comparable to the performance of pipetting (see Table 3). The control samples of the microfluidic DBS show good performance with a coefficient of variation (CV) between 1.4% and 6.4%, while the pipetted reference samples show in 6 out of 8 cases a slightly higher CV with individual CVs between 0.4% and 10.1%. This reflects the results of the gravimetric measurement showing a better volumetric performance for the microfluidic DBS than for the pipetted DBS at three out of four hematocrit levels. No indications for hematocrit induced bias can be observed. For direct comparison of the microfluidic DBS (average 14.3 µl) and the pipetted reference DBS (average 14.5 µl), each LC-MS/MS measurement is normalized to 15 µl, using the gravimetric measurements of the individual samples (see Table S1 and S2). This normalization was necessary since calibrators were prepared by pipetting 15 µl blood volumes to pre-cut DBS paper disks, but pipetted reference spots were characterized to contain on average 14.5 µl and microfluidic DBS were characterized to contain on average 14.3 µl. A more detailed description of the normalization calculations can be found in the supplementary information. After the normalization the mean of triplicate measurements of microfluidic DBS and pipetted DBS,



respectively, is compared to the nominal concentration. For low and medium control samples, the mean bias is below 6.3% for microfluidic DBS with one exception (Control 1, HCT 37) where the mean bias is 17.7%. For the corresponding pipetted DBS samples the mean bias at low and medium concentration is below 3.3%. This shows that both microfluidic DBS and pipetted DBS yield accurate results independent of the blood hematocrit. The trend of positive mean bias with increasing hematocrit observed by others21 and typically associated with DBS analysis cannot be observed in these measurements. No trend in differences of mean bias between pipetted DBS and microfluidic DBS can be observed which indicates good sample integrity for microfluidic DBS. These findings are in good agreement with two other studies based on the microfluidic metering principle by Spooner et al.28 and Velghe et al.9 both

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concluding hematocrit independence for the microfluidic DBS device. Spooners study describes a hematocrit dependent mean bias between -3.5% and 3.9% from the nominal volume and a CV between 1.7% and 4.9 % which is similar to the results presented in this study. Velghe et al. present a fully validated method for the analysis of caffeine and paraxanthine on the microfluidic DBS card. The study involving 133 patient samples for comparing analyte concentrations in liquid blood and microfluidic DBS with a hematocrit range from 18 to 55 concludes hematocrit independence for microfluidic DBS with all pre-set acceptance criteria being met. The results demonstrate that the microfluidic DBS card has the potential to improve the accuracy of traditional DBS analysis by performing whole spot analysis of a well-defined and hematocrit independent dried blood volume.


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Table 3. Results of the LC-MS/MS measurement for microfluidic DBS and pipetted reference DBS at four different hematocrit levels and two different spiking concentrations. Control 1

Control 2

HCT

µFlu

Pipette

µFlu

Pipette

26%

[ng/ml]

[ng/ml]

[ng/ml]

[ng/ml]

104

104

1920

1981

95.1

82.2

1983

1797

102

101

1980

1934

Mean

100

95.7

1961

1904

SD

3.8

9.6

29.1

78.0

CV [%]

3.8

10.1

1.5

4.1

109

93.5

1936

1857

127

94.1

1854

1960

119

104

1962

1963

Mean

118

97.1

1917

1927

SD

7.4

4.6

46.3

49.5

CV [%]

6.3

4.7

2.4

2.6

99.7

105

1903

1988

90.9

102

1921

2003

37%

47%

103

88.6

1965

2005

Mean

98.0

98.6

1929.7

1999

SD

5.2

7.1

26.2

7.5

CV [%]

5.3

7.2

1.4

0.4

97.5

93.5

1841

2008

97.1

86.5

1903

2088

62%

102

103

1845

1831

Mean

98.8

94.3

1863

1976

SD

2.2

6.7

28.4

107.1

CV [%]

2.2

7.1

1.5

5.4

Recovery and Matrix effects. Comparison of extracts from caffeine spiked post extraction with caffeine spiked blood extracts at 47% hematocrit indicate that caffeine can be quantitatively recovered from the volumetric DBS spots. The recovery was calculated to be 101% with a CV of 2.9 % (n=5). Although a hematocrit dependent recovery evaluation has not been performed, the results presented in Table 3 and Table S 2 indicate a hematocrit independent recovery of caffeine from the DBS. If the recovery was hematocrit dependent the extraction of volume defined DBS would result in a hematocrit dependent mean bias. The matrix effect evaluation indicates that there is a negative matrix effect of -37% for extracted samples as compared to samples spiked directly in extraction solvent. Collection of volunteers DBS samples. The aim of the volunteer study was to validate feasibility of the developed concept under realistic sampling conditions as compared to

laboratory testing with a pipette. The two main differences between laboratory testing and finger prick testing is the procedure of the sampling event and the quality of the whole blood used. While testing in a laboratory allows to apply a sufficient drop of venous blood (at least 30 µl) with a pipette precisely to the inlet area of the device, finger prick sampling is more challenging with an undefined amount of blood that has to be applied to the inlet area of the sampling card. Furthermore, for laboratory testing, the blood is usually prepared with an anticoagulant which eliminates time dependent coagulation of blood. For fresh blood from finger prick wounds the uninhibited initiation of the coagulation cascade may cause blood at different coagulation states being applied to the device. This poses a challenge for a sampling system based on microfluidic capillary flow and pre-programmed events.



The microfluidic sampling performance of the devices was evaluated by sorting devices into three different categories: successful metering and transfer of volumes, incomplete channel filling and incomplete channel emptying. 34 out of 44 devices performed successful metering and transfer of the blood sample corresponding to a success rate of 77%. For 7 out of 44 devices (16%) the device failure was due to incomplete channel filling which can be correlated to a too low volume of blood applied to the microfluidic DBS card. Obtaining sufficient blood flow of capillary blood from finger pricks can be a challenge for DBS sample collection. The blood flow can be improved by several measures such as the correct choice of lancet, warming up the hands prior to the use of the lancet, and applying the correct technique during pricking and blood extraction. While blue BD Microtainer® lancets were observed to be a good choice for good capillary bleeding and were used during sample collection, both the training of the sampling personnel and sampling situation were not optimized. The sampling personnel taking the blood samples was not specifically trained in capillary blood sampling and did not have any previous experience in use of the microfluidic DBS card. The sampling location was in a public building with volunteers sometimes entering the building from outside during winter time which could also have a negative effect on the bleeding ability because of vasoconstriction due to low temperatures. The remaining three devices (7%) did not transfer the channel volume completely to the paper. This was caused by bubble formation at the outlet which was identified to be related to insufficient PVA-paper bond. The success rate of microfluidic DBS of 77% is similar to the success rate reported by others for the collection of un-metered DBS18, 23. The criteria for successful sample collection may vary for different studies and different collection sites while for the microfluidic DBS the transfer of the metered volume can be easily recognized and is a clear indicator that a quality DBS sample was collected. This inherent feature may help to educate users and have a positive impact on the success rate as users gain more experience. Of the 34 successfully collected samples, 30 samples could be used for quantification while four samples did not contain a sufficient amount of caffeine. The caffeine concentration of the 30 microfluidic samples ranged from 131 ng/ml up to 8093 ng/ml. Good linear agreement (R2=0.97) between caffeine levels in 14.5 µl pipetted reference DBS samples and microfluidic DBS samples normalized to 14.5 µl was achieved (see Figure 5) with a slope of 0.999 (95% CI; [0.932 to 1.065]) and an intercept of -95.7 (95% CI; [-289.5 to 98.1]). All measurements of microfluidic DBS samples were within ±20% of the pipetted reference measurements. Similar linear agreement (R2=0.99) between the concentration of PEth 16:0/18:1 measured in microfluidic DBS and liquid whole blood has been described by Beck et al.30. The results also support the findings by Velghe et al. who described no effect of the input volume on the measured concentration in microfluidic DBS and confirms their preliminary tests of capillary blood sampling with four healthy volunteers indicating that the

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use of blood without anticoagulant is no problem for the microfluidic DBS system. In conclusion the correlation between capillary blood samples collected directly on the microfluidic DBS card shows that microfluidic DBS achieve a volumetric performance similar to that of pipetted DBS, even when used for the collection of a finger prick capillary blood sample of unknown volume. CONCLUSION This study presents a microfluidic DBS card for user independent collection of high quality, volumetric DBS samples with the potential to simplify finger prick sampling for both home users and health care professionals. The volumetric performance of the microfluidic DBS card is as least as good as that of a pipette, the standard method of dosing volumes in a laboratory, irrespective of the hematocrit of blood volumes collected. The study also demonstrates microfluidic DBS collection directly from finger pricks with excellent agreement between LC-MS/MS measurements of caffeine in microfluidic DBS and pipetted reference samples obtained from the same finger prick. This clearly shows that the presented microfluidic DBS card can help to improve the accuracy of DBS sample analysis by eliminating the hematocrit-related volumetric bias and by making inhomogeneities within the spot of no concern. At the same time microfluidic DBS sampling maintains the simplicity of traditional DBS sampling. The sample collection process on the microfluidic DBS card is similar to traditional DBS sampling, allowing direct contact between the collection card and the finger and providing a visual feedback to the user on the success of sample collection.

Figure 5. Passing and Bablok regression analysis of caffeine from finger prick capillary blood of volunteers comparing 14.5 µl pipetted DBS to microfluidic DBS normalized to 14.5 µl. Insert shows Bland and Altman plot of the same data set with the limit of agreement (solid lines).

ASSOCIATED CONTENT Supporting Information

8 ACS Paragon Plus Environment

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The Supporting Information is available free of charge on the ACS Publications website. Table S1. Example for the calculation of normalized sample concentrations based on gravimetric evaluated volumes and the LC-MS/MS measured caffeine concentrations. Table S2. Compilation of individual normalized results. The LCMS/MS measurement results of pipetted and microfluidic DBS were normalized to 15 µl using the gravimetric measurements of the individual spotted volume. This allows a direct comparison between LC-MS/MS results of pipetted (14.5 µl) and microfluidic DBS (14.25 µl) despite the different mean volumes. Mean and SD have the unit [ng/ml] while CV and mean bias is displayed in [%] (pdf).

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

Author Contributions G.L., G.S., O.B. and N.R. devised the concept, designed and developed the sampling device. G.L., S.U. and O.B. performed in-vitro characterization. G.L. and S.U. performed the volunteer study. S.U. and O.B. developed the LC-MS/MS method. G.L., G.S. and N.R. wrote the paper. All authors discussed the results and commented on the manuscript.

Notes The authors declare following competing financial interest: Gabriel Lenk, Göran Stemme, Olof Beck and Niclas Roxhed are founders of the company Capitainer AB developing microfluidic blood sampling devices.

ACKNOWLEDGMENT This study was supported by grants provided by the Stockholm County Council (ALF 20160517, 20160608 and 20140745), and European Research Council (727818 xMEMSDBS).

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Evaluation of a Volumetric Dried Blood Spot Card Using a Gravimetric

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