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An Autonomous Microfluidic Device for Generating Volume-defined Dried Plasma Spots (DPS) Janosch Hauser, Gabriel Lenk, Shahid Ullah, Olof Beck, Göran Stemme, and Niclas Roxhed Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 07 May 2019 Downloaded from http://pubs.acs.org on May 7, 2019

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Analytical Chemistry

An Autonomous Microfluidic Device for Generating Volume‐defined Dried Plasma Spots (DPS) Janosch Hauser1,*, Gabriel Lenk1, Shahid Ullah2, Olof Beck2, Göran Stemme1 & Niclas Roxhed1 1Department of Micro and Nanosystems, KTH Royal Institute of Technology, 10044 Stockholm, Sweden 2Karolinska University Hospital, Clinical Pharmacology, 11486 Stockholm, Sweden

Key words: Microsampling, Blood, Plasma, Dried Plasma Spot, DPS, Volumetric, Caffeine, LC‐MS/MS ABSTRACT: Obtaining plasma from a blood sample and preparing it for subsequent analysis is currently a laborious pro‐ cess involving experienced health‐care professionals and centrifugation. We circumvent this by utilizing capillary forces and microfluidic engineering to develop an autonomous plasma sampling device that filters and stores an exact amount of plas‐ ma as a Dried Plasma Spot (DPS) from a whole blood sample in less than six minutes. We tested 24 prototype devices with whole blood from 10 volunteers, various input volumes (40‐80 µL) and different hematocrit levels (39‐45%). The resulting mean plasma volume, assessed gravimetrically, was 11.6 µL with a relative standard deviation similar to manual pipetting (3.0% vs. 1.4%). LC‐MS/MS analysis of caffeine concentrations in the generated DPS (12 duplicates) showed a strong corre‐ lation (R2=0.99) to, but no equivalence with concentrations prepared from corresponding plasma obtained by centrifuga‐ tion. The presented autonomous DPS device may enable patient‐centric plasma sampling through minimally invasive finger‐ pricking and allow to generate volume‐defined DPS for quantitative blood analysis.

INTRODUCTION In recent years Dried Blood Spot (DBS) sampling of capil‐ lary blood has emerged as a promising alternative to tradi‐ tional venous blood sampling with clear merits such as simplified collection, shipment, and storage while being far less invasive.1 However, DBS analysis constitutes the anal‐ ysis of whole blood and not blood plasma, which is the prevailing matrix in laboratory medicine today. Further, whole blood may, due to hemolysis and the release of components from red blood cells, negatively affect the use of spectrophotometric methods2,3 and complicate the anal‐ ysis of certain analytes, such as ferritin4. Blood plasma is usually obtained by separating plasma and red blood cells by centrifugation. This elementary step in blood sample preparation should preferably be made minutes after the sampling event which complicates the logistics of the procedure, takes time and increases costs.5

While simple solutions to perform centrifugation at the point‐of‐care and in resource‐limited settings have been proposed6–8, the necessity for centrifugation of blood sam‐ ples remains a significant hurdle in bringing the sampling event closer to the patient and to allow self‐sampling or sampling by non‐professionals. Alternative methods to obtain blood plasma include blood cell separation techniques based on mechanical filtration9– 12, sedimentation13–15 or a combination of both16–18. Analyte quantification requires an accurate volume definition of the separated blood plasma. Methods for volumetric col‐ lection of dried plasma spots (DPS) by controlling filtration time and/or constricting plasma absorption have been proposed.10,19,20 However, it is challenging to achieve a sufficiently large amount of plasma from a small finger‐ prick blood sample while also ensuring an exact volume, good plasma quality, and hematocrit (HCT) independency.

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Figure 1: a) The top view and cross-section showing the design of the plasma extraction and metering device. The dimensions of the device are 80×20 mm2. b) Cross-sectional schematics illustrating a time sequence of the metering and excess drainage events (-), related to the channel filling level at different time-points. The first filling cycle contains the necessary steps for the generation of a volume-defined DPS. The second cycle shows how the device prevents excessive plasma from reaching the DPS.

We recently demonstrated a passive microfluidic filtration technique that utilizes capillary forces to obtain blood plasma at high yields directly from finger‐prick blood samples within minutes.21 An autonomous microfluidic metering principle to obtain an exact volume‐defined DBS was also demonstrated22 which effectively eliminates criti‐ cal issues related to hematocrit23,24 and spot‐drying25 bias‐ es. Here we combine these technologies to obtain volume‐ defined Dried Plasma Spots (DPS). Analogous to the previ‐ ous volume‐metered DBS sampling concept24, the plasma spot is volume‐defined independent of blood hematocrit – allowing analyte quantification – and shares the same merits as DBS samples in terms of simplicity, stability and sample transportation easiness. We now demonstrate and characterize a fully autonomous sampling device that gen‐ erates a DPS of 11.6 µL from a blood droplet of undefined

volume (40‐80 µL) and with hematocrit values between 35‐55%, within less than 6 minutes. We measure caffeine concentrations in the obtained DPS and corresponding centrifuged plasma samples using LC‐MS/MS (liquid chromatography ‐ tandem mass spectrometry) and com‐ pare the results. DEVICE DESIGN The presented device is designed to generate volume‐ defined DPS, based on the volume of a metering channel. Figure 1a shows a schematic of the device design, which consists of a filtration membrane in connection to a meter‐ ing channel, an excess drainage valve, and a DPS paper matrix to store the volume‐metered plasma. A precise and user‐independent volume‐metering is enabled by an air pinch‐off structure, which is realized as a vented geomet‐ rical constriction below the filtration membrane, providing

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a controlled air inflow when the high capillary force of the DPS paper matrix acts on plasma in the metering channel. Excessive plasma volume, potentially abundant depending on input volume variations or inter‐individual differences in blood hematocrit, is removed through an excess drain‐ age valve. This valve consists of a porous plug in the meter‐ ing channel, a water‐soluble Polyvinylalcohol (PVA) film and an absorbent paper. Figure 1b presents the conceptual sequence of events for volume metering and excess drainage. Blood plasma is passively filtered, using our previously demonstrated plasma filtration principle21, when blood is applied to the filtration membrane and fills the metering channel, driven by capillary forces (Figure 1b ). When the plasma menis‐ cus reaches the porous plug of the excess drainage struc‐ ture for the first time (first filling cycle), the PVA film pre‐ vents the plasma from being drained into the absorbent paper. Therefore, the plasma meniscus can reach the DPS paper at the end of the metering channel. The high capil‐ lary force of the DPS paper leads to a fast absorption of the plasma volume contained in the metering channel, gener‐ ating a volume‐defined DPS. The volume of the transferred plasma is defined by the dimensions of the metering chan‐ nel and the controlled air inflow at the inlet of the meter‐ ing channel enabled by the air pinch‐off structure (Figure 1b ). After the successful generation of the volume‐ metered DPS the PVA film at the excess drainage needs to dissolve to prevent excessive plasma from reaching the DPS paper (second filling cycle). The filled cavities around the porous plug, filled during the first filling cycle, dissolve the PVA in the excess drainage, thereby opening the excess drainage valve (Figure 1b ). Plasma reaching the porous plug a second time is therefore immediately drained into the excess absorbent paper, protecting the volume‐defined DPS from excessive plasma (Figure 1b ). EXPERIMENTAL Materials The device is fabricated from hydrophilic plastic sheets (Type C laser printing transparency, Xerox, Elmstock, UK), adhesive tape 1 (64620, Tesa, Norderstedt, Germany) and adhesive tape 2 (300LSE, 3M, VWR, Spånga, Sweden). A filtration membrane (IPOC “SGR”, Toronto, Canada) is used as blood filter. Ahlstrom Grade 222 (Mt Holly Springs, USA) is used as the DPS paper and the absorbent paper. The porous plug is cut from Ahlstrom Grade 319 paper (Mt Holly Springs, USA). The PVA films are fabricated from granular Polyvinyl alcohol (360627, Sigma‐Aldrich, St. Louis, USA). Reference standards, including caffeine and its deuterated 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 LC‐MS grade methanol (Fisher Scientific AB, Gothenburg, Sweden) and stored at −20 ˚C. All other chemicals, including ammonia (25%) (Merck KGaA, Darm‐ stadt, Germany), were of highest analytical grade. Milli‐Q DI water was prepared in‐house with ultra‐pure quality (>18 MΩ·cm). Device fabrication The devices were fabricated in lamination technology as described in our earlier publication.21 The different parts of the device are indicated in Figure 1. The metering chan‐ nel was fabricated from 100 µm thick hydrophilic plastic sheets and adhesive tape 1 (170 µm), which defines the height of the channel. The lower level with the vented cavity was fabricated from hydrophilic sheets (100 µm) and adhesive tape 2 (50 µm). The plastic sheets and adhe‐ sive tape were structured using a cutting plotter (CE6000, Graphtec America Inc., Irvine, USA), manually assembled using alignment pins, and laminated with a thermal pouch laminator (Heat Seal Pro H600, GBC, Northbrook, USA). The filtration membrane and paper material were struc‐ tured using a laser cutter (VLS 2.30, Universal Laser Sys‐ tems, Vienna, Austria). Filtration membranes were cut to a size of 12×12 mm2 and the edges were sealed by dipping them into molten wax, which was printed onto a transpar‐ ency by a wax printer. The filtration membrane was at‐ tached to the device using the adhesive tape 2. The porous plug was laser‐cut to a disc with a diameter of 1.6 mm to fit the opening in the metering channel. The DPS paper with an initial thickness of 830 µm was cut to 5×10 mm2 and partially laser ablated to fit the channel height of 170 µm. The PVA film was fabricated from aqueous solution of granular Polyvinyl alcohol using a thin‐film applicator (4340, Elcometer, Manchester, UK) resulting in PVA sheets with a film thickness of around 10 µm. Blood samples To characterize the volume metering performance of the device with whole blood, venous EDTA (Ethylenedia‐ minetetraacetic acid) treated blood from 10 coffee drink‐ ing volunteers was collected at the day of the experiments by venous puncture. Six volunteers were female and four were male covering an age range of 25–56 years. The ex‐ periments were approved by the regional ethics committee (EPN Stockholm, Dnr 2015/867‐31/1). Hematocrit in all blood samples was determined using a XN‐1000 (Sysmex, Chuo‐ku, Japan) hematology analyzer and the range of blood hematocrit levels measured was between 39.4% and 44.8%. For the evaluation of the hematocrit‐dependent filtration performance, one blood specimen was split up into three 1‐ml aliquots and centrifuged at 2000 g for 10 minutes. Plasma was either removed or added to achieve target concentrations of 35%, 45% and 55% HCT. The hematocrit level of these samples was estimated based



on the hemoglobin (Hb) concentration measured with a hematology analyzer (HB201+, HemoCue AB, Ängelholm, Sweden) and the correlation between Hb and HCT.26 Dur‐ ing the experimental work all blood samples were stored at room temperature on a Gyromini 3D Rocker, gently mixing the samples. Blue Microtainer® contact activated safety lancets (BD, Franklin Lakes, USA), with a 1.5 mm blade and a nominal penetration depth of 2 mm, were used for finger prick sampling. Device operation kinetics To demonstrate the device operation kinetics dependent on hematocrit levels, 55 µL of blood with hematocrit val‐ ues of 35%, 45% and 55% were pipetted on the filtration membrane and the device performance was recorded using a camera. The video and modelling software Tracker (https://physlets.org/tracker) was used to read out filling levels throughout the filtration, metering and excess drainage procedure. Volume metering A gravimetric method was used to evaluate the volume precision of plasma collected by the plasma metering de‐ vice. A differential measurement weighing the dry DPS paper before the device assembly and directly after the volume metering event allows to calculate the plasma sample weight. This weight was used to calculate the plasma volume of the volume‐defined DPS, based on a plasma density of 1.025 g/ml.27 To evaluate the potential impact of applied volume on the metered plasma volume, input volumes between 40‐80 µL (10 µL increments) were tested. For each volume, blood samples from two volun‐ teers (one with a higher and one with a lower HCT) were tested, each in duplicate. For finger‐prick samples, a single drop of blood with unknown volume was applied to the filtration membrane and the volume was measured in the same way as for venous blood samples. The scale used was a Mettler Toledo Delta Range AT261 (Columbus, USA) precision scale with 0.01 mg resolution. LC‐MS/MS caffeine measurement To investigate the correlation between analytes in mem‐ brane filtered plasma and centrifuged plasma, the samples obtained from the volume metering experiments were used to measure caffeine concentrations by LC‐MS/MS. Caffeine is a commonly used drug model in studies of DBS devices28–30, because of its widespread consumption among volunteers, providing easily available positive sam‐ ples30. Left over venous blood samples from the volunteers were centrifuged for 10 minutes at 2000 g in a centrifuge (3‐18KS, Sigma, Osterode, Germany) and reference sam‐ ples were prepared in duplicates by pipetting 12 µL onto pre‐cut DPS paper identical to the ones used for absorption of the filtered plasma. In total, 48 DPS were generated, corresponding to 24 sets of duplicates, of which 12 sets

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were generated by pipetting 12 µL of centrifuged plasma and 12 sets were generated by plasma filtration using the device. All 48 samples were dried for 2 hours before being transferred to Eppendorf tubes. To also evaluate the feasi‐ bility of the plasma metering concept for the direct appli‐ cation of finger‐prick blood, two volunteers applied an undefined amount of blood directly from the fingertip to the filtration membrane. As reference blood, blood sam‐ ples of approximately 250 µL from the same finger‐prick were collected into EDTA‐primed blood collection tubes (Microtainer®, BD, VWR, Spånga, Sweden). These tubes were centrifuged for 10 minutes at 2000 g to obtain centri‐ fuged reference plasma. The reference sample spots were prepared by pipetting 12 µL plasma onto pre‐cut DPS pa‐ pers. The dried plasma samples were analyzed using a TSQ Quantiva triple quadrupole mass spectrometer 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 monitored ion transitions were m/z 195 > 138, 110 for caffeine and m/z 198 > 140 for caffeine‐13C3 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 a total run time of 3.2 min. Mobile phase A and B consisted of water and methanol respectively, both containing 0.1% ammonia. The gradient was 0‐0.5 min 5% B, 0.5‐2.0 min 95% B, 2.0‐ 2.5 min 95% B, 2.5‐3.2 min 5% B. Calibrators covered the concentration range 0.01–10 µg/mL at seven levels and were prepared in duplicate by spiking blank centrifuged EDTA‐plasma and pipetting 12 µl of spiked plasma to ref‐ erence DPS paper. The calibration line fitted a 1/x weighted linear model with R2=0.999 and for a QC sample at 0.4 µg/ml the imprecision (%CV) and bias were 6.3% and 5.4%, respectively. Here, a limited method validation was done because of the comparative purpose of the measurements. For sample analysis, the pre‐cut DPS paper containing the dried plasma samples were transferred to Eppendorf tubes and 200 μl of methanol containing 15 pg/μl of internal standard were added to fully immerse the DPS paper in the solvent. All samples were gently shaken, centrifuged, stored overnight in a refrigerator and shaken 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 a 10% methanol solution and transferred to vials for injec‐ tion into the LC‐MS/MS system. The injection volume was 2 µl. RESULTS AND DISCUSSION


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Device operation In Figure 2 we present a picture series of the device opera‐ tion in a top view and a close‐up view from below the fil‐ tration membrane. The sequence is divided into a first filling cycle (a‐d) with the plasma extraction and the vol‐ ume metering event and a second filling cycle (e‐f) where the channel refills with excess plasma and this plasma is drained. The first cycle starts when blood is applied to the filtration membrane and plasma starts filling the metering channel by capillary action. The metering channel is emptied once the DPS paper absorbs the plasma volume. Thereby, the plasma volume is defined by the channel dimensions and the controlled air pinch‐off intersecting the filtered plasma at the channel inlet (fig 2c). By the time the metering channel refills the PVA film has dissolved, opening the excess drainage and protecting the volume‐defined DPS from excessive plasma. An air bubble, potentially intro‐ duced when the channel refills (fig 2e), does not influence the precise volume‐metering.

Figure 3 shows measurements of the channel filling level during plasma extraction of a single device (n=1) per hem‐ atocrit level (35%, 45%, 55%). The filling profile resem‐ bles the conceptual filling graph from Figure 1b, here with 55 µL blood samples. The results show that the plasma extraction kinetics have significantly different slopes de‐ pendent on HCT levels, filtering 12 µL (100% filling) in 2‐6 minutes, where blood with lower HCT is filtered faster. This is in agreement with our previously published re‐ sults.21 In contrast to the plasma extraction kinetics the channel emptying kinetics have almost identical slopes, which indicates that the volume‐metering event is HCT independent, storing the same volume of plasma in the DPS paper irrespective of HCT. The reason for this is that when the plasma meniscus in the metering channel reach‐ es the DPS paper (100% filling), the capillary force in the paper outweighs the capillary force of the microchannel, and absorbs the plasma in the channel (0% filling). For blood samples with 35% and 45% HCT not all blood has been filtrated and a second filling cycle starts as filtration continues. For HCT 55% the blood reservoir is already depleted, and no refilling occurs. Between the first and the second filling cycle, plasma connected to the excess drain‐ age valve dissolves the PVA film and opens the drainage valve. As a result, all plasma in the second filling cycle is immediately absorbed by the waste paper when reaching the position of the drainage valve (85% filling). This effi‐ ciently prevents the absorption of additional plasma at the DPS paper, thereby protecting the volume‐defined DPS from overfilling with excess plasma. This shows that the excess drainage structure effectively compensates for variations in abundant plasma caused by different hemato‐ crit levels. In contrast to other plasma volume‐metering approaches where authors reported an unacceptable HCT bias20, the presented concept can be used to collect volume‐metered DPS while avoiding any HCT bias within the range of 35% to 55%.

Figure 2: Picture series taken from a video during device operation with human whole blood. The frames show the generation of a volume-defined DPS (first cycle, a-d) and the excess drainage event (second cycle, e-f) in a top view, and a close-up view from below the filtration membrane. For visibility the plasma is dyed with a color pigment added to the filtration membrane. For pictures from below the membrane, the plasma is digitally enhanced for better visibility.

Device operation kinetics Figure 3: Measurements showing the operation kinetics of the



microfluidic DPS device at three different blood hematocrit values (35%, 45%, 55%). The filtration time is hematocrit dependent and ranges from 2 minutes (HCT 35%) to 6 minutes (HCT 55%). The graph shows how plasma filtration fills the microchannel before the sample pad causes emptying of the channel. For medium and low hematocrit refilling with excessive plasma occurs which is drained by the excess drainage valve.

Volume metering Figure 4 shows the results from the gravimetric measure‐ ment of the metered plasma spots. The measurements demonstrate that the microfluidic plasma metering device precisely transfers 11.6 µL ± 0.3 µL, corresponding to a CV of 3% (n=24). A variation in input volume between 40‐ 80 µL does not affect the transferred metering volume which could potentially eliminate the volume‐dependent bias for analytical assays reported by others19. Pipetted reference samples of plasma show a gravimetrical meas‐ ured volume of 11.7 µL ± 0.2 µL, corresponding to a CV of 1.4% (n=24), indicating a similar precision to the microflu‐ idic plasma metering device. All four DPS samples obtained by finger‐pricking show gravimetrically evaluated volumes within the range of the venous DPS samples. Potential errors in the gravimetric determination of plasma volumes obtained with the device might be caused by loss of paper material upon removal of the DPS paper and/or evapora‐ tion during the filling of the DPS paper. However, the de‐ vice design enabled a smooth removal of the DPS paper with no observed traces of paper material and thus an insignificant influence on the error. The influence of evap‐ oration on a similar porous material has previously been reported to be approximately 0.3 μL/min for water.21 The maximum time needed to fill the DPS paper with the me‐ tered plasma volume was approximately 20 s (cf. Figure 3). Hence, the resulting maximum error due to evaporation is estimated to be around 0.1 µL which is well within the deviation of the gravimetric measurement results.

Figure 4: The results of the gravimetric measurements of 24 devices showing the metered plasma volume from whole blood samples from 10 different donors with different HCT values,

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blood sampling method and input volume. The average metered volume is 11.6 µL with a CV of 3%.

LC‐MS/MS caffeine measurement Both pipetted reference spots from centrifuged plasma and membrane filtered volumetric plasma spots were extract‐ ed and analyzed with the described LC‐MS/MS method. Since the overall goal of this study is to validate the accu‐ racy of the microfluidic plasma sampling device, the results were used as obtained from the measurements and not corrected for differences in volumes. To correlate the fil‐ trated DPS samples to the centrifuged DPS samples, the results from the 24 DPS (12 duplicates) generated by the membrane filtration devices, were compared with the corresponding mean reference concentrations. For refer‐ ence plasma concentrations of venous and finger‐prick blood samples, the mean concentration was calculated from duplicate centrifuged plasma samples. The results are presented in Figure 5 and show a strong linear correlation (R2=0.99) between centrifuged and membrane filtered DPS with a minimal intercept. When comparing the caffeine concentration of the individual filtration devices to their corresponding reference concentration in centrifuged plasma, the resulting average ratio was 0.664 ± 0.039, corresponding to a CV of 6% (n=23). This is in good ac‐ cordance with the linear fit presented in Figure 5 and un‐ derlines the strong linear correlation between caffeine concentrations in filtered and centrifuged plasma. To as‐ sess the reproducibility of the devices, we compared the caffeine concentrations obtained from the 12 sets of dupli‐ cates generated by the plasma filtration devices. Between the duplicates, the results lay within 1‐5% of their respec‐ tive mean. Corrected for differences in volume (cf. Figure 4) the measured caffeine concentration of the duplicates lies within 0‐4% of their respective mean. Hence, the varia‐ tion in the LC‐MS/MS measurement is considerably larger than the imprecision of the volume metering. The regres‐ sion slope in Figure 5 shows that on average membrane filtered plasma yields a 36% lower caffeine concentration than the centrifuged reference plasma. Loss of protein‐ bound caffeine in the filtration membrane and/or the mi‐ crochannel could be reasons for the lower caffeine concen‐ tration in filtered plasma samples. Our previous results have shown that the total protein recovery from mem‐ brane filtered plasma is approximately 73 ± 8% as com‐ pared to centrifuged plasma21. The fraction of caffeine bound to plasma proteins has been reported to be approx‐ imately 35%, independent of the individual donor31. This could indicate that the filtered plasma relates to the free fraction of caffeine. Further investigation of the loss mech‐ anisms in the membrane, including unspecific binding of caffeine and plasma proteins, could contribute to better understanding the exact features of the membrane filtrat‐ ed plasma samples. However, the strong correlation of


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caffeine in membrane filtered microfluidic DPS and centri‐ fuged DPS further demonstrates that volume metered DPS could be a valuable sampling method complementing ex‐ isting whole blood microsampling technologies. The com‐ parison of membrane filtered DPS from untreated finger‐ prick blood from 2 volunteers with corresponding centri‐ fuged DPS from EDTA treated finger‐prick blood indicates that the correlation of finger‐prick samples is similar to venous samples.

MS/MS analysis of caffeine concentrations in volume‐ metered DPS showed a strong correlation between results obtained from centrifuged and pipetted DPS with R2=0.99, minimal intercept, and a regression slope of 0.64. Plasma protein binding and unspecific binding of caffeine to the filtration membrane are likely to be two reasons for the reduced caffeine recovery from membrane filtrated plasma samples. However, the strong correlation indicates that volumetric plasma samples could be used for accurate quantification in DPS samples. Application of finger‐prick blood to the microfluidic DPS yielded similar results as the venous microfluidic DPS. In conclusion, the device tackles and presents a promising solution to the challenging re‐ quirements on plasma sampling that are needed for quan‐ titative blood analysis.

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

Author Contributions

Figure 5: The graph shows the correlation of measured caffeine concentrations in plasma samples obtained either by centrifugation or by capillary driven membrane filtration for venous and finger prick blood samples. Data points represent the correlation between the mean of the duplicate reference measurements from centrifuged plasma and individual measurements of membrane filtered and volumetric DPS (12 duplicates; n=23, with one data point missing at the highest concentration). The results show a linear correlation between both plasma extraction methods with minimal intercept and R2=0.99.

CONCLUSIONS A novel capillary driven sampling device for autonomous generation of volumetric DPS was presented. The volume‐ defining concept is based on capillary‐driven filtration through a porous filtration membrane into a microchannel, and absorption of a channel geometry‐defined volume by an absorbent sample pad. The principle does not require any user action, thus eliminating error‐prone manual tim‐ ing or handling steps. An automatic excess drainage mech‐ anism, triggered by a water‐soluble PVA film, prevents overfilling of the DPS paper which enables compatibility of the system with a large range of input volumes and hema‐ tocrit values. Proof‐of‐principle measurements showing the operation kinetics of the device at different hemato‐ crits (35 %, 45 %, 55 %, n=1) indicate that both the vol‐ ume metering and excess drainage can work for blood with different HCT levels. A gravimetric study revealed a sam‐ pling volume of 11.6 µL and a precision of 3% with blood from 10 healthy volunteers (HCT 39–45 %), independent of sampling method and input volume (40–80 µL). LC‐

J.H., G.S. and N.R. conceived and tested the original idea, de‐ signed and developed the sampling device. J.H., G.L. and N.R. planned the experiments. J.H., G.L. and O.B. conducted the volunteer study. S.U. and O.B. performed the LC‐MS/MS analysis. J.H., G.L., G.S. and N.R. wrote the paper. All authors discussed the results, commented on the manuscript and approved the final version of the manuscript.

Notes

The authors declare following competing financial interest: G.L., J.H., G.S., and N.R. are inventors of patent applications on plasma separation devices. G.L., O.B., G.S., and N.R. are found‐ ers of the company Capitainer AB developing microfluidic blood sampling devices.

ACKNOWLEDGMENT This study was supported in parts from grants provided by the Stockholm County Council (ALF 20160517, 20160608 and 20140745), the Swedish Foundation for Strategic Research (SSF) (GMT14‐0071), the European Research Council (727818 xMEMSDBS), and the Foundation Olle Engkvist Byggmästare.

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