Volumetric Microsampling of Capillary Blood Spot vs Whole Blood Sampling for Therapeutic Drug Monitoring of Tacrolimus and Cyclosporin A: Accuracy and Patient Satisfaction

Abstract

Background

Immunosuppressant therapeutic drug monitoring (TDM) usually requires outpatient travel to hospitals or phlebotomy sites for venous blood collection; however Mitra^® Microsampling Device (MSD) sampling could allow self-collection and shipping of samples to a laboratory for analysis. This study examined the feasibility of using volumetric microsampling by MSD for TDM of tacrolimus (TaC) and cyclosporin A (CsA) in transplant patients, along with their feedback on the process.

Methods

MSD was used to collect TaC and CsA from venous (VB) or capillary (CB) blood. The MSDs were rehydrated, extracted, and analyzed using on-line solid phase extraction coupled to tandem mass spectrometry (SPE-MS/MS). We report an abbreviated method validation of the MSD including: accuracy, precision, linearity, carry-over, and stability using residual venous whole blood (VB) samples. Subsequent clinical validation compared serially collected MSD + CB against VB (200 µL) from transplant patients.

Results

Accuracy comparing VB vs. MSD+VB showed high clinical concordance (TaC = 89% and CsA = 98%). Inter- and intra-precision was ≤11.5 %CV for TaC and CsA. Samples were stable for up to 7 days at room temperature with an average difference of <10%. Clinical validation with MSD+CB correlated well with VB for CsA (slope = 0.95, r² = 0.88, n = 47) and TaC (slope = 0.98, r² = 0.82, n = 49). CB vs. VB gave concordance of 94% for CsA and 79% for TaC. A satisfaction survey showed 82% of patients preferred having the capillary collection option.

Conclusion

Transplant patients favored having the ability to collect capillary samples at home for TaC/CsA monitoring. Our results demonstrate good concordance between MSD+CB and VB for TaC and CsA TDM, but additional studies are warranted.

Mitra^®, microsampling, dried blood spot, tacrolimus, cyclosporine, therapeutic drug monitoring (TDM), Neoteryx^®, LC-MS/MS, SPE-MS/MS, patient satisfaction

Impact Statement

This article illustrates accuracy and patient satisfaction in therapeutic drug monitoring (TDM) of tacrolimus and cyclosporine using 20 µL of capillary dried blood (CDB) collected from a volumetric micro-sampling device. We advance the field of TDM by reporting a technical method for extracting CDB collected using a MSD, followed by rapid analysis with SPE-MS/MS (<30 min for a 96-well plate) to quantitate tacrolimus and cyclosporine. The use of CDB for immunosuppresant TDM has the potential to benefit pediatric allogeneic transplant patients and facilitate home self-collection for allogeneic transplant out-patients who usually have to travel to outpatient phlebotomy sites.

Introduction

Tacrolimus (TaC) and cyclosporine (CsA) are immunosuppressants used in allogeneic solid organ transplants (1,). Both compounds function by inhibiting calcineurin, leading to suppression of T-cell proliferation with attenuation of T-cell mediated mechanisms of acute and chronic graft rejection (1–4,). Addition of calcineurin inhibitors to immunosuppressive maintenance protocols was accompanied with dramatic improvement in long-term graft survival and overall patient outcomes (4, 5,). To date, immunosuppression induction and maintenance protocols are complex, usually taking advantage of synergistic mechanisms from multiple drug classes for optimum immune suppression (e.g., azathioprine, mycophenolic acid, sirolimus, everolimus, TaC, or CsA) (5,). Nonetheless, calcineurin inhibitors are the cornerstones of maintenance immunosuppression therapy, with transplant patients often remaining on CsA or TaC long after other immunosuppresants are withdrawn from the protocol (5).

While calcineurin inhibitors are extensively used, they do have challenges. Both TaC and CsA have narrow therapeutic windows where too little drug may lead to organ rejection, but too high of a concentration leads to adverse drug reactions and organ damage (6,). The target range also changes throughout the life-long maintenance immunosuppression therapy where patient noncompliance can also contribute to subtherapeutic drug levels leading to adverse outcomes (7,). Monitoring drug levels and adjusting the dose compensates for variable inter/intra-individual pharmacokinetics, making it possible to achieve target blood concentrations (2, 6). For this reason, TDM is performed to optimize dose efficacy and confirm patient compliance during the life-long management of transplant patients.

Historically, TDM for calcineurin inhibitors was performed using immunoassays; however, liquid chromatography tandem mass spectrometry (LC-MS/MS) has emerged as the preferred analytical method (8,). LC-MS/MS offers sensitivity and precision rivaling that of immunoassays, along with improved specificity and the ability to multiplex (8,). Multiple studies have reported clinically validated LC-MS/MS assays monitoring immunosuppressant drug regiments for multiple targets in whole blood (9–17,). Regardless of the analytical method used, life-long monitoring of these medications poses a significant TDM burden on transplant patients. First, the amount of blood collected each time is usually ∼4 mL of whole blood. Second, whole blood collection requires venous access which means patients must commute to a phlebotomy site and endure an invasive process to comply with TDM regiments. Furthermore, laboratory assays for these medications are not harmonized, so results for TaC and CsA can vary significantly even when laboratories are using the same type of methodology (i.e., LC-MS/MS) (18,). Previous studies have shown that when the same sample was analyzed for TaC at different labs using LC-MS/MS, results varied from 14 −24 ng/mL. This large variation makes it challenging for clinicians to serially monitor patients and make dosage adjustments unless the patient’s blood is consistently analyzed at the same laboratory (18, 19,). Therefore, patients who relocate or live far away from the original transplant center/laboratory may have specimens drawn and analyzed at different clinical laboratories with results that do not match (18,). Taken together with the life-long logistical burden, the TDM burden can lower patient compliance, leading to negative outcomes. Overall, TDM challenges for transplant patients include: (1,) the need to be at a collection site for timed (trough) blood draws, (2,) venous access, and (3,) the amount of blood collected over time for TDM (11, 20). For these reasons, there is increasing interest for laboratories to help lower this TDM burden.

One approach is to offer transplant patients a simple home collection option that requires less blood. In recent years, dried blood spot (DBS) sampling has become a TDM option for enabling patients to self-collect small sample volumes (<100 µL) at home (9–17,). Capillary collections are less invasive and patients can be trained to self-collect samples. In general, DBS are prepared by absorbing blood onto a dry porous medium (commonly filter paper), which is allowed to dry. Drying partially preserves the specimen and inactivates some blood-borne pathogens (21,). Advantages of DBS sampling include: minimal blood volume is collected, simplified transportation and storage without refrigeration, and reduced risk of exposure to biohazards (21,). Since DBS are not classified as hazardous, patients can self-collect at home and mail specimens to a laboratory for analysis. Validation of DBS for TDM applications usually includes method comparison using DBS with VB, followed by clinical validation with CB (10,). This approach is recommended because, unlike venous blood, capillary blood is a mixture of arterial blood, venous blood, and interstitial fluid, a composition which could significantly affect measured drug concentrations (12).

To date, several institutions have validated DBS as alternative sample types for TDM of immunosuppressants (9–12,). The validations involved comparing finger stick and VB results for immunosuppressant TDM on LC-MS/MS platforms (Table 1). One shortcoming of these studies is the use of cellulose blood cards (BC) for preparing DBS. BCs are a nonvolumetric sampling technique associated with significant analytical variation depending on the specimen hematocrit (13,). Nonetheless, Hinchliffe et al. reported the use of blood cards for CsA and TaC quantitation in BC+CB with no significant difference from VB results or corrections for hematocrit (12, 23,). Similarly, Dickerson et al. reported accurate TaC and SiR quantitation in BC+CB with no correction for specimen hematocrit (11,). However, earlier studies from Koster et al. reported accurate TaC and CsA quantitation in BC+CB only for specimens with hematocrit between 23% and 48% (9, 10,). Koster et al. has since shown that CB collected using the MSD gave < 15% bias for TaC and CsA when hematocrit values extended beyond the normal range (i.e., 27% to 60%) (23,). In the end, numerous studies have been published citing methods which can be used to determine the immunosuppressant concentrations and the blood hematocrit from DBS (17, 22, 24, 25,). Taken together, a key limiting factor of DBS preparation using blood cards is the need to determine specimen hematocrit in order to guarantee accuracy of results (26, 27,). Therefore in this study, the Mitra^® MSD was used for TaC and CsA quantitation in whole blood because it uses volumetric absorptive sampling (VAMS^TM) which had previously been shown to have minimal effects on volumetric sampling due to a specimen’s hematocrit (28–30,). Previously, the VAMS has been shown to absorb a fixed volume of blood (e.g., 10 µL) in 2–4 s with less than 5% volume variation across a broad hematocrit range of 20%–70% (29).

Table 1

Clinical validations comparing CDB to VB for TaC and CsA TDM.

Analyte	Linear fit	y-int 95% CI	Slope 95% CI	R2	n	Mean bias (ng/mL)	Mean bias 95% CI	Ref
CsA	y = 0.95x + 13	−3.95 to 23.94	0.84 to 1.05	0.88	47	−9.85	−43 to 24	a
CsA	y = 1.05x −3.64	−10.17 to 2.23	0.97 to 1.15		57	2.6	−0.8 to 5.9	(10)
CsA	y = 0.99x −1.86	−8.31 to 3.64	0.91 to 1.08	0.93	61	−1.8	−4.8 to 1.3	(11)
CsA	y = 1.00x + 0.2	NA	NA		45	2.6	2.2 to 7.5	(13)
CsA	y = 1.01x −9.0	−20.9 to 2.9	0.95 to 1.08		37	−3.6	−121 to 114	(21)
TaC	y = 0.98x + 0.39	−0.15 to 1.83	0.84 to 1.11	0.82	49	−0.88	−3.40 to 1.64	a
TaC	y = 1.04x −0.25	−0.73 to 0.16	0.96 to 1.12	NA	85	−0.01	−0.17 to 0.15	(10)
TaC	y = 1.00x −0.23	−0.69 to 0.30	0.91 to 1.08	0.93	106	−0.28	−0.45 to 0.12	(11)
TaC	y = 1.12x −1.33	NA	NA	0.92	26	0.6	−1.14 to 2.34	(12)
TaC	y = 1.05x −0.29	−1.00 to 0.23	0.96 to 1.17	NA	24	NA	NA	(22)
TaC	y = 0.92x −0.15	NA	NA	NA	42	0.7	1.1 to 0.3	(13)
TaC	y = 0.96x + 2.72	NA	NA	0.74	18	2	NA	(8)

a

This article.

In this pilot study, the analyte recovery from the MSD was assessed by comparing the accuracy of the immunosuppressant results from the VAMS extraction to a fully validated clinical reference method that extracted TaC and CsA directly from the same volume of venous whole blood (30,). In the end, a condensed validation was done to show the feasibility and preliminary workflow using MSD+CB sampling with rapid solid phase extraction tandem mass spectrometry (SPE-MS/MS) quantitative analysis to analyze TaC and CsA in a 96-well plate format in < 30 min (31, 32,). This approach has the potential of using less blood volume with a self-collection option at home for transplant patients (31, 32). A survey was also used to assess patient satisfaction with the MSD finger stick collection process compared to a venous collection. If successful, the new protocol would allow less blood to be collected (20 µL vs. ∼4 mL) and home MSD sampling could allow clinicians to monitor patients on TaC or CsA using the same laboratory, independent of patient’s geographic location. This approach could reduce health risks and shipping costs associated with whole blood specimens while lowering the overall TDM burden on transplant patients.

Materials and Methods

Analytical Method and Specimen Extraction

The performance characteristics of the reference method were previously determined by Mayo Clinic in a manner consistent with CLIA requirements. The lower limit of quantitation (LLOQ) was 1 ng/mL (TaC) and 25 ng/mL (CsA). The upper limit of quantitation (ULOQ) was 40 ng/mL (TaC) and 1000 ng/mL (CsA) with <10 % coefficient of variation (%CV) across the analytical measuring range (AMR). The reference method required 200 μL of ethylenediaminetetraacetic acid (EDTA) VB for CsA and TaC quantitation. Briefly, the VB specimen is mixed with 200 μL of clear water (CLRW) and vortexed in order to lyse cells. Lysate is mixed with 300 μL of ascomycin (AsC) which is the internal standard (I.S.) for TaC (final concentration 2.5 ng/mL), cyclosporine A-¹³C₂, D4 which is used as the I.S. for CsA (final target concentration of 50 ng/mL), and 0.17 M ZnSO₄ for protein precipitation in 90% methanol. Both I.S. compounds were purchased from Toronto Research Chemicals. The sample was then vortexed and the centrifuged (3500 rpm; 10 min) to separate insoluble contents and the supernatant was recovered for analysis by SPE-MS/MS.

SPE-MS/MS Method

SPE was performed online with MS/MS quantitation on an Agilent 6495 with the Rapid Fire 365 module (C18 SPE cartridge). The SPE method was as follows: specimen aspiration (0.6 s), approximately 10 µL of specimen was aspirated into the injection loop. The specimen was then loaded/washed into the column with wash 1 (3.0 s with 10 mM ammonium acetate, 0.1% formic acid, and 0.01% trifluoroacetic acid, TFA), followed by wash 2 (2 s with 50% methanol in CLRW), then eluted (5 s with 10 mM ammonium acetate 1%, formic acid, and 0.01% TFA in methanol), and the column was re-equilibrated (0.5 s with 50% methanol in CLRW). The MS/MS method uses electrospray ionization in positive ion mode with source settings: gas temp = 220°C, gas flow = 12 L/min, nebulizer pressure = 40 psi, sheath gas temp = 300°C, sheath gas flow = 10 L/min, nozzle voltage 300 V and capillary voltage positive = 4500 V, chamber current = 0.52 A. The analyzer was set to multiple reaction monitoring with target m/z values for I.S. and target analytes: TaC: m/z = 821.8 > 786.5 (qualifying ion), 821.8 > 768.5 (reporting ion), CsA: m/z = 1219.8 > 1184.8 (qualifying ion), m/z = 1219.8 > 1202.9 (reporting ion), AsC: m/z = 809.6 > 756.5 (I.S.), and CsA ¹³C₂D₄: m/z = 1225.9 > 1208.9 (I.S.). Quantitation used linear least square fitting of a five point standard curve with 1/x² weighting. The same calibrators were used for VB and MSD experiments. Calibrators were prepared in bovine whole blood (Lampire Biological) for both CsA (25, 150, 400, 700, and 1000 ng/mL) and TaC (1, 5, 10, 20, and 40 ng/mL) and verified using purchased calibrators (Chromsystems). Human VB control materials were purchased from UTAK laboratories.

Analytical Validation for MSD Collection Method

Since this was only a pilot study with limited supplies, an abbreviated analytical MSD method validation was performed to determine stability, carry-over, precision, linearity, and accuracy using VB on the MSD. The overall analytical scope of this study was to compare accuracy of results with direct comparisons using VB vs. VB spotted on the MSD, and VB vs. CB spotted on the MSD. The ultimate goal was to assess the accuracy of TaC and CsA quantitation in capillary blood using a MSD. Matrix effects between capillary and venous whole blood were not explored at this time. Samples were prepared by spotting specimens on a 20 μL Mitra^® MSD. Spotted samples were dried for at least 24 h at room temperature, extracted, and analyzed using the SPE-MS/MS analytical protocol. Briefly, dried MSD tips containing standards, controls, and patient specimens were mixed with 150 μL CLRW and I.S (2.5 ng/mL AsC and 50 ng/mL CsA-d4) in a 96-well plate and shaken for 20 min at 2500 rpm. 50 µL of CLRW with 0.2 M ZnSO₄ was added and samples mixed for 5 min at 2500 rpm. Finally, 100 µL of methanol was added and the plate was shaken for 5 min at 2500 rpm. Samples were centrifuged at 3500 rpm for 10 min and the supernatant transferred to a clean 96-well plate for SPE-MS/MS analysis.

Clinical Validation for MSD

The clinical validation study was approved by the Mayo Clinic Institutional Review Board (IRB). Fifty solid organ transplant patients prescribed TaC and 50 patients prescribed CsA were enrolled. During routine visits, two MSDs were collected from each patient using 20 μL of CB per MSD from one finger-stick. Specimens were collected between June, 2016 and June, 2017. The MSD+CBs were collected immediately prior to a VB draw for routine TaC or CsA TDM. Patient demographics are shown on Supplemental Table 1 (Supplementary Information, SI). Prior to each collection, patients were instructed on how to perform the capillary collections and presented with two options; 1) self-collect a CB sample (patient performs finger-stick and the MSD sample collection) or 2) a member of the study team assists by performing the finger-stick, but the patient collects the MSD sample (assisted collection). Following each paired collection (VB & MSD+CB), patients completed a survey on the MSD+CB collection procedure. All samples were sent to the laboratory for analysis.

Results and Discussion

Analytical Validation of Blood Spot Method

MSD stability

MSD blood spot analyte stability (MSDs+VB) was assessed using freshly prepared residual patient pools of whole blood which contained 56, 263, or 945 ng/mL CsA or 1.2, 10, or 21 ng/mL TaC along with purchased human whole blood controls spiked with immunosuppressant with concentrations spanning the analytical measuring range (115, 312, and 521 ng/mL CsA; 5.2, 14.8, 29 ng/mL TaC). The MSD+VB were dried in foil pouches (room temperature) and tested in duplicate on day 1, 2, 3, 7, 14, and 28. Acceptable criteria for stability on the MSD was ±20% variation from the initial concentration at day zero (8). Our data showed CsA MSD+VB were stable at room temperature with an average difference of <10% (actual −8.2%) for the first 7 days, and <20% for up to 14 days. TaC MSD+VB showed more variability with <20% change up to 7 days.

Carry-over

Carry-over for the MSD method was determined using EDTA whole blood pools with TaC or CsA spiked-in at a concentration 3x the ULOQ. These samples were extracted and analyzed by SPE-MS/MS followed by 5 consecutive blank samples to assess carry-over. TaC showed some carry-over at 43% of the LLOQ ( LLOQ = 1 ng/mL) and CsA displayed minimal carry-over at 10% of the LLOQ (LLOQ = 25 ng/mL). Given the therapeutic target ranges for TaC and CsA discussed below, this amount of carry-over was deemed not significant and acceptable.

MSD precision

The MSD precision was determined using 3 levels of human VB control material for TaC and CsA purchased from UTAK. Intra-assay precision was calculated using 10 data points from each control material in a single run. Inter-assay precision was determined with 10 data points collected from each control material over a 3-day period. MSD intra/inter-assay precision was <10% CV for all level I and II concentrations for each immunosuppressant (Supplemental Table 2). The target concentrations for TaC in level I and II control material was 5 and 15 ng/mL and covers the trough (C₀ = 5–15 ng/mL) reference range for TaC monitoring (8, 33,). For CsA, level I and II controls also spanned the trough (C₀ = 100–400 ng/mL) target concentration at 118 and 330 ng/mL, respectively (8). MSD intra-assay precisions on level III were 11.5% CV for TaC and 10.1% CV for CsA (Supplemental Table 2).

Expert recommendations suggest a target intra/inter-assay precision of <±10% CV for TDM TaC and CsA assays, whereas the FDA has set a goal of <±15% CV for approved TDM assays (8). Our assay met the <±10% CV goal for all levels that cover the target trough levels (Supplemental Table 2). However, level III controls for CsA and TaC just exceeded the (±10% CV limit, but was within the ±15% CV limit (Supplemental Table 2). Since both level III controls have concentrations targeted well above the trough targets (<15 ng/mL for TaC and <350 ng/mL for CsA), the observed precision was deemed acceptable for the pilot study.

MSD linearity

MSD linearity across the desired measuring range was assessed using verified TaC and CsA whole blood standards (Fig. 1). The calibrators were extracted and analyzed 5 times on 3 separate days with 3 different technicians performing extractions. MSD calibrator results were plotted against calibrator values obtained from the reference method and linearity was assessed using the coefficient of determination (r²). Figure 1 shows the linear regression analysis. For CsA, MSD results (x-axis) gave a linear correlation with y = 1.07x – 6.63 (r² = 0.98) for a range spanning 25 −1000 ng/mL (Fig. 1, A). For TaC, MSD results (x-axis) reveal a linear relationship with y = 1.31x −1.21, r² = 0.99 over 1–40 ng/mL (Fig. 1, B). It is unclear why the recovery was higher for the TaC standards (slope = 1.31) compared to CsA (slope = 1.07) when plotted against the target values. One possible explanation is that the verified standards were prepared using bovine whole blood. However, this same effect was not seen when patient samples were compared during the accuracy studies which used the same verified standards and MSD+VB vs. VB had slopes of 0.97 and 1.08 for TaC and CsA, respectively (Fig. 2).

Fig. 1

Assessment of MSD linearity using CsA (A) and TaC (B) calibrators. The MSD data were plotted against the target value and analyzed using linear regression. The data represent 5 experiments conducted over 3 different days between 9/15 and 10/15. The best fit line from linear regression is shown in solid red. The 95% CI for the fit is shown in shaded red. Fit parameters for (A) slope = 1.07 (95% CI = 0.82 to 1.32), y-int = −6.64 (95% CI = −153 to 139), r² = 0.98, and (B) slope = 1.31 (95% CI = 1.14 to 1.48), y-int = −1.21 (95% CI = −4.50 to 2.09), r² = 0.99.

Fig. 2

Accuracy comparison comparing MSD+VB vs. VB results obtained from residual VB specimens. (A) Linear regression for CsA, MSD+VB vs. VB, slope = 0.97 (95% CI = 0.93 to 1.02), y-int = 1.48 (95% CI = −6.56 to 9.56), r² = 0.97. (B) Bland-Altman plot for CsA, MSD+VB vs. VB, Mean bias = −1.50% (solid red line with the 95% CI in red dotted lines). (C) Linear regression for TaC, MSD+VB vs. VB, slope = 1.08 (95% CI = 1.00 to 1.16), y-int = −0.81 (95% CI = −1.55 to 0.07), r² = 0.95. (D) Bland-Altman plot for TaC, MSD+VB vs VB, Mean bias = −2.19% (solid red line with the 95% CI in red dotted lines). The best fit line from linear regression is shown in red. The 95% CI for the fit is shown in shaded red.

MSD accuracy

Initial MSD accuracy was determined with paired VB and MSD+VB results from 45 de-identified residual whole blood (EDTA) specimens received for TaC and CsA TDM. Linear regression and Bland-Altman plots were generated for method comparison. Regression analysis for CsA MSD+VB results vs. VB results (n = 45) gave a best fit line with slope = 0.97 (95% CI = 0.93 to 1.02), y-intercept = 1.48 (95% CI = −6.56 to 9.56), r² = 0.97 (Fig. 2, A). The data gives a standard error of the estimate (Syx) = 11.1% of the average result from the comparison. A Bland-Altman plot revealed a nonsignificant mean bias of −2.44 ng/mL (95% CI = 30 to –35 ng/mL) for CsA in MSD+VB compared to VB (Fig. 2, B). For TaC (n = 45), regression gave a fit with slope = 1.08 (95% CI = 1.00 to 1.16), y-intercept = −0.81 (95% CI = −1.55 to 0.07), r² = 0.95 and Syx = 14.2% (Fig. 2, C). Bland-Altman plot also reveals a nonsignificant mean bias of −0.13 ng/mL (95% CI = 2.33 to −2.58 ng/mL) for TaC in MSD+VB compared to VB (Fig. 2, D). Most importantly, the MSD+VB and VB methods show clinical concordance of 98% and 89% for CsA and TaC, respectively (Fig. 3, A). All of the discordant samples were ≤20% from the target value (Fig. 3, B).

Overall, specimens with elevated TaC (>15 ng/mL) or CsA (>400 ng/mL) were not prevalent in our patient population. Only one specimen beyond the therapeutic range was available for each analyte (Fig. 1: TaC = 28 (33) ng/mL and CsA = 546 (538) ng/mL, values from the MSD method are in parentheses). Recommendations for acceptable criteria for immunosuppressant method comparison include a slope of 1.0 (±)0.1, y-intercept spanning zero, and Syx ≤10% of the average target concentration (8). The comparison for MSD+VB and VB methods meet the slope and y-int criteria, but not the Syx ≤10% criteria for both analytes (Syx = 14.2% for TaC and 11.1% for CsA) which was deemed acceptable for the pilot study since clinical interpretation was not affected.

Pilot Clinical Study

Patient demographics

Following IRB approval, paired MSD+CB and VB specimens were collected from 100 adult allogeneic organ transplant patients who consented to the study. MSD+CB were collected by applying the MSD tips directly to capillary blood following finger-prick. Since the tips do not overfill, they are kept in contact with capillary blood for 3 s after the entire tip turns red. Paired specimens from 50 patients were collected for each analyte. Our cohort included 61 (61%) male patients and 39 (39%) female patients. The final data for MSD+CB vs. VB comparisons included 96 specimens from 58 male (60.5%) and 38 female (39.5%) patients from the original cohort. Four CsA results were excluded; one due to ion suppression, one because the CsA concentration was below our LLOQ, and two because a valid venous VB result was not obtained for comparison. Detailed demographics for the final 96 specimens are shown on Table 2. The average patient age is 57.8 years (Std.Dev = 12.1, Min = 25, Max = 85, Median = 61).

Table 2

Cohort patient demographics and data overview.

	All recruits	Age avg (St.Dev) years	Tac avg (St.Dev) ng/mL	CsA avg (St.Dev) ng/mL
Total	100	57.4 (12.6)	6.6 (3.1)	82.6 (50)
Male	61	61.51 (12.6)	6.1 (2.8)	95.8 (43)
Female	39	52.7 (13.1)	7.2 (3.3)	88.9 (43)
Transplant Type
Liver	48	59.3 (10.7)	5.7 (2.4)	85.5 (41)
Kidney	25	53.5 (15.5)	6.7 (3.5)	110 (49)
Heart	22	61.5 (10.7)	10.4 (4.0)	93 (58)
Lung	3	53.3 (12.2)	10.0 (1.6)	N/A
Pancreas	2	42.5 (2.1)	8.7 (2.4)	N/A

MSD accuracy

Since capillary blood samples may behave differently from whole blood samples, MSD+CB from finger-prick results were compared to VB results collected from the same patient. Data were analyzed using linear regression and Bland-Altman plots. A summary of the data along with comparisons from other clinical studies comparing MSD+CB vs. VB are listed on Table 1. Linear regression for CsA data (n = 47) reveals a fit with slope = 0.95 (95% CI = 0.84 to 1.05), y-intercept = 13.95 (95% CI = −3.95 to 23.94), r² = 0.88, and Syx = 17.5%) (Fig. 4, A). The Bland-Altman plot showed a mean bias = −9.85 ng/mL (95% CI = 23.7 to −43.42 ng/mL) for CsA on MSD+CB compared to VB (Fig. 4, B). For TaC (n = 49), linear regression gave a best line fit with slope = 0.98 (95% CI = 0.84 to 1.11), y-int = 0.99 (95% CI = −0.15 to 1.83), r² = 0.82, and Syx = 18.1%) (Fig. 4, C). The Bland-Altman plot has a mean bias of −0.88 ng/mL (95% CI = 1.64 to −3.40 ng/mL) for TaC in MSD+CB compared to VB (Fig. 4, D). Compared to the MSD+VB data, MSD+CB revealed higher bias and a lower correlation to VB compared to MSD+VB (Figs. 2–4). Overall, both CsA and TaC performed well, but given approximately the same precision for the TaC and CsA in our MSD assay (Supplemental Table 2), CsA performed better than TaC. Clinical concordance for MSD+CB and VB was also higher for CsA (94%) compared to TaC (79%) (Fig. 3, C). The concordance table for MSD+CB revealed a total of 12/91 discordant results, all occurring at the lower end of the TaC or CsA reference ranges with MSD+CB running lower compared to VB (Fig. 3, D). 9/12 of these results have % differences of −28 to −48% below the target VB concentration (Fig. 3, D). Overall, the MSD+CB vs VB comparison showed linear correlation with slopes and mean bias values which were similar to other published reports (Table 1) (34).

Fig. 3

Summary of concordant and discrepant results from MSD+CB vs. VB immunosuppressant measurements. (A) CsA and TaC concordance table for MSD+VB vs. VB results. (B) CsA and TaC discrepant results from MSD+VB vs. VB comparisons. (C) CsA and TaC concordance table from the clinical study comparing MSD+CB vs. VB results. (D) CsA and TaC discrepant results from the clinical study comparing MSD+CB and VB. Discordant results are highlighted in red while concordant results are highlighted in green.

Fig. 4

Accuracy comparison comparing MSD+CB collected by finger-stick vs VB collected from venipuncture. (A) Linear regression for CsA, MSD+CB vs. VB, slope = 0.95 (95% CI = 0.84 to 1.05), y-int = 13.95 (95% CI = −3.95 to 23.94), r² = 0.88. (B) Bland-Altman plot showing % difference for CsA, MSD+CB vs. VB, Mean bias = −10.72% (solid red line with the 95% CI in red dotted lines). (C) Linear regression for TaC, MSD+CB vs. VB, slope = 0.98 (95% CI = 0.84 to 1.11), y-int = 0.99 (95% CI = −0.15 to 1.83), r² = 0.82. (D) Bland-Altman plot showing % difference for TaC, MSD+CB vs VB, Mean bias = −10.69% (solid red line with the 95% CI in red dotted lines). Regression parameters are discussed in the main text. The shaded red area represents the 95% CI for the linear fit.

Patient satisfaction with MSD

The pilot clinical study also included a survey designed to gain insight into the patient’s experience using the Mitra^® MSD (Fig. 5). Out of the 100 patients who participated in our study, 53% chose the assisted collection option (Fig. 5, A). Some of these patients cited general fear of blood collection or sharp objects (22%) as the main reason for choosing assisted collection. A majority of the patients (78%) reported other reasons for their choice, including being tired (Fig. 5, B). Up to 90% of patients who chose assisted collection indicated they would be comfortable performing a self-collection after observing the entire process (Fig. 5, C). In the end, 81% of all patients would prefer the capillary blood collection if the option were to become available for immunosuppressant monitoring (Fig. 5, D).

Fig. 5

Survey results for patient experience and satisfaction using the Mitra^® MSD.

A total of 48% of all patients chose to collect their own MSD+CB specimens following initial training with the Mitra^® MSD (Fig. 5, A). Out of these, 81% thought the capillary collection was better than traditional venous blood draw (Fig. 5, E). Many of these patients (85%) also found the capillary collection easy to perform (Fig. 5F–G), and reported the finger prick as less painful compared to a traditional venous draw (Fig. 5, H). To date, only one other study has captured the patient’s preference for the blood collection method used for immunosuppressant monitoring (34), but our study explored this using a volumetric MSD. In the end, the patient experience survey clearly revealed that MSD sampling was preferred by the majority of transplant patients who participated in this study.

Conclusion

Transplant patients favored having the ability to collect capillary samples at home for TaC/CsA monitoring. The study also demonstrated good correlation between MSD+CB and VB samples for TaC and CsA using the MSD with 94% (CsA) and 79% (TaC) clinical concordance. However, further studies for optimizing recovery along with a larger cohort study looking at the variability and stability of collecting and shipping samples back to the laboratory are ultimately needed to prove feasibility.

Nonstandard Abbreviations

%CV, percent coefficient of variation; AMR, analytical measuring range; AsC, ascomycin; BC, blood cards, usually made from cellulose; CB, capillary blood; CDB, capillary dried blood; CI, confidence interval; CLIA, Clinical Laboratory Improvement Act; CLRW, clinical laboratory reagent grade water; CsA, cyclosporine; DBS, dried blood spots, typically used with finger-stick/capillary blood; EDTA, ethylenediaminetetraacetic acid; LC-MS/MS, liquid chromatography tandem mass spectrometry; LLOQ, lower limit of quantitation; MpA, mycophenolic acid; MSD, Mitra® Microsampling Device; SI, Supplemental Information; SPE-MS/MS, solid phase extraction tandem mass spectrometry; TaC, tacrolimus; TAT, turn-around-time; TDM, therapeutic drug monitoring; ULOQ, upper limit of quantitation; VB, venous blood.

Author Contributions

All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved.

M.M. Mbughuni, statistical analysis; M.A. Stevens, administrative support, provision of study material or patients; L.J. Langman, statistical analysis; Y.C. Kudva, provision of study material or patients; W. Sanchez, provision of study material or patients; P.J. Jannetto, statistical analysis.

Employment or Leadership: None declared. Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: None declared. Research Funding: Mayo Clinic Department of Laboratory Medicine and Pathology Discretionary Funds were awarded and used to fund the study. Neoteryx provided the Mitra microsampling devices used in the study at no charge. Expert Testimony: None declared. Patents: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, preparation of manuscript, or final approval of manuscript.