Abstract
Repeated blood sampling is required in certain clinical and research settings, which is currently performed by drawing blood from venous catheters requiring manual handling of each sample at the time of collection. A novel body-worn device for repeated serial samples, Fluispotter®, with automated extraction, collection, and storage of up to 20 venous dried blood spot samples over the course of 20 h may overcome problems with current methods for serial sampling. The purpose of this study was to assess the performance and safety of Fluispotter for the first time in healthy subjects. Fluispotter consists of a cartridge with tubing, a reservoir for flushing solution, pumps and filterpaper, and a multi-lumen catheter placed in the brachial vein. We recruited healthy subjects for testing in an in-hospital setting. Fluispotter was attached by an anesthesiologist to 22 healthy subjects of which 9/22 (40.9%) participants had all 20 samples taken, which was lower than the goal of complete sampling in 80% of the subjects (P = 0.02). The main reason for sample failure was clogging of blood flow which was observed in 11/22 (50%) of the participants. No serious adverse events occurred, and the participants rated the pain from the insertion and the removal of catheter as very low. A cortisol profile showed nadir values at midnight and highest values at 05:00 h. Although full sampling was not successful in all participants, the Fluispotter technology proved safe and highly acceptable to the participants producing the expected cortisol profile without the requirement of staff during sample collection.
Introduction
In an everyday endocrine setting, the dynamic response of hormones is assessed for the diagnosis and evaluation of several conditions such as adrenal insufficiency (1), acromegaly (2), and growth hormone deficiency (3), which require repeated venous sampling. The pathology of several other conditions such as major depression, bipolar disease, stress related to night shifts, nocturia, hypertension, and sleep disorders have all been claimed to include disrupted circadian rhythm (4, 5). Repeated sampling is required in both clinical and research settings for diagnosis, evaluation of treatment, and to advance our understanding of disease pathology. Currently, this is obtained by drawing blood from inserted venous catheters. Common problems include occlusion, the need for reinsertion of catheters (6), and disturbance of the subjects during sampling. Collecting samples in a stressful environment, for example, during the night or admission as an inpatient to a hospital potentially influences the hormone concentrations to be studied. Further, the collection of blood samples requires manual handling of each sample at time of the collection, centrifugation, and freezer facilities.
A novel wearable system for automated collection of repeated serial samples, Fluispotter®, may overcome some of these problems associated with repeated sampling or 20-h collection of blood samples (7). Thus, Fluispotter is intended for automated extraction, collection and storage of up to 20 venous dried blood spot (DBS) samples of 3–10 μL over the course of up to 20 h. The system is attached to the patient through a multilumen microcatheter inserted into a peripheral vein in the arm. Fluispotter is a fully automated, programmable, body-worn device for obtaining serial blood samples from humans. This wearable device also allows sampling during situations where wet sampling is problematic, for example, during sleep, work, play, or exercise. Further, it reduces the number of man-hours needed for serial sampling and minimizes the risks of sample loss, wrong timing, misidentification, and contamination. The volumetric DBS technology used in the Fluispotter has previously been tested ex vivo for cortisol assessment by comparing cortisol levels from DBSs with cortisol plasma samples. The interassay accuracy and precision were less than 10% across a range of different hematocrit values (7). DBS technology is increasingly being recognized as a valuable alternative to plasma sampling (8) and is potentially used in different scenarios such as testing for hepatitis C infection (9) or monitoring of anticancer drugs (10), screening at-risk populations for various diseases (11), and general screening (12). The analytical part has similarly advanced from manual handling of DBS samples to a combination of liquid chromotography-tandem mass spectrometry (13) and robotic DBS extraction systems allowing a fully automated analytical process of DBS (14). In the context of classical endocrinology, cortisol is of particular interest for timewise multiple assessments due to the diurnal variation and clinical use of dynamic testing, for example, diagnostic workup for Cushing’s syndrome or adrenal insufficiency. In a small feasibility study in dogs (n = 2), Fluispotter was used to create diurnal profiles of cortisol concentrations in unstressed dogs, showing cortisol levels at the lower end of the reference interval (15).
Aim
The primary purpose of this study was to assess the performance and safety of Fluispotter in healthy subjects and secondly to explore the diurnal profile of cortisol concentrations using Fluispotter in healthy subjects.
Materials and methods
Design
This study was designed as a prospective single-center, non-randomized, open label, and hospital-based study with non-blinded outcome assessment of the diurnal variation of cortisol concentrations in healthy adults using Fluispotter technology. The study has been approved by the local ethical committee, the Research Ethics Committee in the Capital Region of Denmark (VEK journal n. H-20068538), and has been registered at www.clinicaltrials.gov (NCT04594577). Fluispotter has been approved by the Danish Medicines Agency (LMST j. nu.: 2020100544) and registered with the European database of medical devices (EUDAMED CIV-ID number: CIV-20-10-034866), and consent was obtained from each subject after full explanation of the purpose and nature of all procedures used.
Description of Fluispotter technology
Details of the Fluispotter technology have previously been described (7). In short, the Fluispotter is based on patented technology (16, 17, 18) and consists of a rechargeable control unit with motors and battery, a sterile single-use cartridge with tubing, a reservoir for flushing solution, pumps and filter paper, and a sterile single-use multi-lumen catheter. This allows up to 20 h of unattended serial microsampling of maximum of 20 DBS samples of 3–10 μL venous blood. The blood spots are collected on a strip of PerkinElmer 226 filter paper at user-defined time points. In the tip of the sampling catheter, which is placed in the brachial vein in the upper arm, the blood is mixed 9:1 (v/v) with a 4% sodium citrate flushing solution to prevent coagulation of blood inside the cartridge. Fluispotter is not yet commercially available for use in humans and is intended to be used only by healthcare professionals after appropriate training. A cartoon (Fig. 1) depicts the detailed principle.
Cartoon of the Fluispotter principle. Left side: (A) Reservoir for sodium citrate flushing solution, (B) pump for flushing solution, (C) blood pump, (D) valve for uni-directional flow, and (E) PerkinElmer 226 paper strip. (A)–(E) are all enclosed in the sterile single-use Fluispotter Cartridge. (F) Multi-lumen catheter with (G) tip of catheter placed inside a peripheral vein. The catheter tip is designed to mix sodium citrate flushing solution to the blood flowing into the cartridge and to ensure that return blood is not mixed with the fresh blood sample. Right side: Sketch of Fluispotter placed on the left lower arm, with the sampling catheter placed in the brachial vein in the upper arm (unchanged, with permission: Adhikari K et al. Fluispotter, a novel automated and wearable device for accurate volume serial dried blood spot sampling, Bioanalysis 2020).
Citation: Endocrine Connections 12, 7; 10.1530/EC-23-0087
Assessment of cortisol in dried blood spots
After the collection of blood spots, the filter paper was removed from the disposable cartridge and dried overnight at room temperature before storing it in a zip-lock plastic bag at −20°C until analysis. DBS samples were punched into a U-bottom 96-well plate (Thermo Fisher Scientific, AB-0564) using a Wallac DBS Puncher (PerkinElme). Cortisol was extracted from the DBS with 200 μL extraction solvent (acetonitrile/water: 80:20) containing the stable isotopically labeled cortisol-(13C3) internal standard (30 nmol/L) by vigorously mixing on an Eppendorf MixMate (1200 rpm @ 3mm mixing stroke) for 1 h at room temperature. After centrifugation for 30 min at 7102 g (Rotanta 460RF, Hettich, Hillerød, Denmark), the supernatant was transferred to a 96-well plate and 5 μL was then injected for LC–MS/MS analysis as described elsewhere in detail (7).
where PCplasma is the predicted cortisol concentrations in plasma, CDBS is the observed concentration of cortisol from the DBSs, and HCT is the hematocrit value.
Subjects
Healthy adults for this study were recruited through advertisements on websites (forsøgsperson.dk) and other medias.
Inclusion criteria
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Male or female
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Age ≥ 18
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Able to understand verbal and written instructions in Danish
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Able and willing to sign and date the informed written consent form and letter of authority
Exclusion criteria
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Currently participating in a clinical trial evaluating drugs or medical devices
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Known history of coagulation disorders
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Currently taking regular medication (contraceptives, hormonal replacement therapy, and antihistamines exempted)
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Regular smoking or use of nicotine products
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Pregnancy
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Hematocrit < 38% (male) or < 33% (female)
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Hematocrit > 52% (male) or > 48% (female)
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CRP > 10 mg/dL,
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BMI > 30
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Known allergies or hypersensitivity to flushing solution constituents
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Other factors, which in the opinion of the investigator would interfere with the ability to provide informed consent, comply with study procedures/instructions, confound the interpretation of the study results, or put the subject at undue risk.
Procedure
Patients enrolled in the study had safety blood samples taken including hematocrit (Supplementary Table 5, see section on supplementary materials given at the end of this article) and a general physical examination including assessment of pulmonary and cardiac function at the screening visit, and the initial safety samples were therefore always taken at a maximum of 7 days prior to application of the Fluispotter. Within a week from enrollment the patients were admitted to the hospital for application of Fluispotter by a single anesthesiologist. After using standard hospital aseptic procedures, local anesthetics were applied prior to the insertion of a peripheral venous catheter (Venflon 16G). Using the peripheral venous catheter as a guide, the Fluispotter Catheter 45™ was introduced and placed in the brachial vein 1–2 cm distal to the flexure of the basilic vein where it joins the brachial vein near the axilla (confirmed with ultrasound) and fixated. Fluispotter was pre-programmed for 20 samplings of 10 µL during a 20-h period, at 1-h intervals; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 h after sampling has been initiated. Sampling time was initiated at the convenience of trial staff and study participants. After sampling was completed, the Fluispotter catheter was removed using normal aseptic procedures and disconnected from the Fluispotter Cartridge™. The cartridge containing the paper strip was placed in a zip-locked bag, labeled with subject ID until analysis as described earlier. Safety blood samples were repeated immediately after the withdrawal of the catheter. Eight days after the study participants returned for a safety visit.
Outcomes
Primary outcome
The primary outcome of this study, on which the power calculation was based, was the number of successful samplings (DBS) over a 20-h period made by the Fluispotter. A failure was defined as a device which failed to collect 20 samples in a started sampling schedule for 20 samples.
Secondary outcomes
These covered the incidence and severity of adverse effects (AEs), adverse device effects, and device deficiencies. Furthermore, the usability assessment was done by staff and participants, as well as safety biomarkers.
Exploratory outcomes
Finally, an assessment of the diurnal cortisol concentrations profile was performed by measuring the cortisol concentrations in each successful DBS by the LC–MS/MS analysis as mentioned earlier.
Statistical considerations
Data will be presented as proportions, means with standard deviations (s.d.), and medians with minimum and maximum values, as appropriate. The required sample size for the primary endpoint, that is, the number of successful samples, was calculated using a non-parametric binomial reliability demonstration test. Based on the assumption of 0 failures, 22 participants were required to provide a 90% reliability of 0 failures with a confidence level of 90%. Expecting catherization failures, for example, inability to create venous access or incorrect placement of the Fluispotter sampling catheter, up to 25 subjects were planned enrolled. The primary endpoint was assessed by performing an intention-to-treat analysis (ITT) including all subjects who got a Fluispotter attached, while the full analysis set (FAS) included all participants in the ITT analysis who completed the 20-h sampling period without major technical issues from the device. Statistical analysis was conducted by an independent statistical consultant using IBM SPSS version 23.0. P-values ≤ 0.05 were considered significant.
Results
Baseline characteristics
The ITT population included 16 male and 6 female participants with a mean age of 27.0 years (s.d. 9.0), a mean weight of 75.6 kg (s.d. 12.1), and a mean hematocrit of 0.44% (s.d. 0.03). In 4/22 (18.2%) the data collection was not completed and thus 18 participants were included in the FAS analysis. Terminations of data collection in these four cases were categorized as (i) blood pump error, (ii) wrongly placed liner, (iii) clogging of blood/paper jam/power supply error, and (iv) clogging of blood flow/paper jam. Baseline characteristics of the ITT and FAS population are shown in Supplementary Table 1.
Primary outcome – number of successful samples
As illustrated in Fig. 2, 9/22 (40.9%) participants in the ITT population had all 20 samples taken, which was lower than the expected 80% (P = 0.02). As reported in Table 1, the main reason for sample failure was clogging of blood flow which was observed in 11/22 (50%) of the participants. Similarly, the results in the FAS-population obtaining 9/18 successful samples were less than the expected 80% (P = 0.004).
Number of successful venous blood samples obtained in the ITT population (n = 22) using Fluispotter, an automated device for serial blood sampling.
Citation: Endocrine Connections 12, 7; 10.1530/EC-23-0087
Distribution of successful samples and adverse device events.
| Population | Intention-to-treat | Full analysis set |
|---|---|---|
| N | 22 | 18 |
| Complete sampling, n (%) | 9 (41) | 9 (50) |
| Samples, n of 20 | ||
| Mean (s.d.) | 11.6 (7.9) | 13.7 (7.2) |
| Median (min–max) | 11.0 (0–20) | 17 (2–20) |
| Percentile, 25th–75th | 2–20 | 2–20 |
| Adverse device events | ||
| Clogging of blood flow, n (%) | 11 (50.0) | 9 (50.0) |
| Paper jam | 2 (9.1) | 0 (0.0) |
| Use error | 1 (4.5) | 1 (5.6) |
| Blood pump error | 1 (4.5) | 0.0 (0.0) |
| Wrongly placed liner | 1 (4.5) | 0 (0.0) |
| Power supply error | 1 (4.5) | 0 (0.0) |
Exploratory analysis – cortisol profile
Based on all included samples (Supplementary Table 6), a diurnal cortisol profile was created as shown in Fig. 3, with nadir at midnight, while maximum values were observed at 05:00 and 06:00 h in the morning. A similar profile can be observed from the measurements from the first participant as shown in Supplementary Fig. 1. Based on analysis from the 10 participants with complete data, that is, 20 successful samples, the mean cortisol in the first sample was 203.8 nmol/L compared to 218.0 nmol/L in the second sample (P = 0.64).
Cortisol profile based on samples in healthy subjects obtained using Fluispotter, an automated device for serial venous blood sampling.
Citation: Endocrine Connections 12, 7; 10.1530/EC-23-0087
Secondary endpoints and exploratory analysis
No serious adverse events occurred. As shown in Table 2, the observed number of participants experiencing adverse event was 7/22 (31.8%) in the ITT population and 5/18 (27.8%) in the FAS population.
Adverse events.
| Adverse event | ITT population n = 22 | FAS population n = 18 |
|---|---|---|
| Erythema, n (%) | 4 (18.2) | 3 (16.7) |
| Bleeding | 1 (4.5) | 0 (0.0) |
| Headache | 1 (4.5) | 1 (5.6) |
| Vasovagal syncope | 1 (4.5) | 1 (5.6) |
The mean number of catheter attempts in the ITT population was 1.36 (s.d. 0.58) with a median of 1.0 (min–max: 1–3) while the corresponding mean was 1.33 (s.d. 0.59) in the FAS population with a median of 1.0 (min–max: 1–3).
One anesthesiologist performed all Fluispotter insertions and reported a usability statement after the first and eleventh (halfway) insertion as shown in Supplementary Table 2. Overall, the anesthesiologist was neutral to mildly satisfied with the device and procedures.
As shown in Table 3, the ITT population had low pain scores during the insertion and removal of the Fluispotter. The item which scored the highest with a mean of 13.6 (s.d. 17) on a scale from 0 to 100 (0 = no disturbance to 100 = most disturbance) was a disturbance from the Fluispotter during sleep. Similar data for the FAS population is shown in Supplementary Table 3. On the same scale, user assessment of pain or bleeding at follow-up was estimated to be 1.2 and 0.6 in the ITT population and 0.7 and 1.4 in the FAS population (Supplementary Table 4).
Usability assessment by participants, intention-to-treat population.
| ITT population, n = 22 | Mean (S.D.) | Median (min–max) | Percentile, 25th–75th |
|---|---|---|---|
|
5.4 (4.9) | 4.5 (0–15) | 1– 9 |
|
5.0 (4.6) | 4.0 (0–14) | 0–8 |
|
3.1 (5.8) | 0.0 (0–24) | 0–4 |
|
7.5 (7.7) | 6.0 (0–24) | 1–10 |
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0.5 (2.6) | 0.0 (0–12) | 0–0 |
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11.4 (13.6) | 6.5 (0–52) | 1–18 |
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2.4 (3.8) | 0.0 (0–13) | 0–5 |
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13.6 (17.1) | 6.5 (0–63) | 3–14 |
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1.6 (0.9) | 1.0 (1–4) | 1–2 |
Questions 1–8 were assessed on a VAS ranging from 0 (no disturbance) to 100 (maximum disturbance). Question 9 was assessed on a Likert scale from 1 (much less interruption) to 5 (much more interruption).
Additional analysis did not suggest that hemoglobin levels explained (P = 0.15) the number of DBS comparing participants in the ITT with 20 samples to participants with less than 20 samples. Neither did hemoglobin levels (P = 0.16) or sex (P = 1.00) explain exclusion from the FAS population. Sampling prior to insertion and after removal showed a fall in thrombocytes from 247.5 × 109/L (s.d. 55) to 237.6 × 109/L (s.d. 58), and an increase in hematocrit values from 0.44 (s.d. 0.03) to 0.45 (0.04) while none of the other markers, for example, leucocytes, C-reactive protein, or APTT changed (Supplementary Table 5).
Discussion
In this study, we investigated Fluispotter, an automated novel automated body-worn system for a serial sampling of volumetric venous DBS, for the first time in healthy human participants. In 9/22 (41%) participants, all 20 samples were collected successfully, with the main reason for sampling failure being clogging of blood flow. AE was reported in 7/22 (32%) of the participants with erythema at the site of insertion predominant. Usability assessment by the performing anesthesiologist reported moderate to mild satisfaction with the device while the participants reported noise from the device as the main complaint, but no pain from the application.
This study has thus demonstrated good feasibility of Fluispotter in a setting of healthy human subjects. The median number of successful samples was 11 in the ITT population and 17 in the FAS population, with only 41% of the ITT population obtaining all 20 samples. Although this is below our pre-defined criteria for success, it is considered sufficient to continue finetuning the Fluispotter technology. The main reason for sampling failure was technical and related to clogging of blood flow, and it is very likely that this failure could be mended by increasing the flow of anticoagulants in future trials. Importantly, no serious adverse events occurred, and blood sampling before and after insertion suggested a clinically non-significant fall in thrombocytes but otherwise no indications of affected biochemical markers.
The important exploratory analyses of diurnal variation of cortisol concentrations were based on 10 participants with complete data, that is, 20 successful samples each. The mean cortisol concentration in the first sample was 203.8 nmol/L compared to 218.0 nmol/L in the second sample (P = 0.64), suggesting a minimal stress response to the insertion of the Fluispotter catheter, which must be considered one of the clinically important exploratory outcomes of the study even though it was not part of the power calculation. The diurnal curve pattern found in the present study was very similar to a typical curve as previously presented (19). However, a later meta-analysis indicated that there are major ethnic differences in the cortisol concentrations (20), and quantitative comparisons between the cortisol concentrations in the present study and those of others would require using the same laboratory method for the quantification of plasma cortisol (21). Similarly, concerning the assessment of the low concentrations of the diagnostic midnight cortisol in the low measurement range would also require a much higher number of samples for the calculation to overcome the lack of power due to a small sample size in the present study. Furthermore, in the previous comparison of DBS-predicted cortisol concentrations vs plasma cortisol samples, the number of samples below 50 nmol/L was very low, and therefore not representative of the overall excellent correlation of r2 0.982 over the entire concentration range (7).
The same anesthesiologist performed the insertion of the Fluispotter catheter and reported ‘mildly’ agreement with the statement ‘Overall, I was satisfied with the procedure’, corresponding to 4 of a maximum score. Important findings from the participant’s usability assessment were minimal pain at insertion and removal, as well as almost no disturbance from the staff during sampling suggesting that the collection of samples took place in a stress-free environment. If the insertion of Fluispotter had caused pain, we would have expected higher cortisol concentrations at the first sampling compared to the second sampling. The main participant complaint was noise from the device, however, even these ratings were minor.
The perspectives for automated sampling appeared convincing in the current study: insertion of the device was rather uncomplicated and insertion as well as removal of the device caused very little discomfort to the participants, and especially when assessing cortisol the lack of stress otherwise associated with sampling procedures appeared to be minimal. Obtaining a diurnal curve for hormones like cortisol is usually only possible at a high workload in a rather large setup requiring staff for sampling and handling of patients as well as samples throughout the procedure. The technical performance of the system also had limitations, and especially the small number of successful samples makes the current version of limited interest for use in a clinical or research setting. However, it is possible that adjusting the mix ratio of anticoagulants to blood in the catheter would be able to solve this problem. Due to inclusion criteria, the current results are restricted to individuals with hematocrit values within the normal range. Also, the need for an anesthesiologist for insertion could be considered a limitation as could the number of possible sampling hours of 20, just short of a full diurnal rhythm of 24 h, which is an important limitation for understanding diurnal patterns in the blood-borne factors. The 20-h limit is due to design choices prioritizing small size over sampling duration and number of samples for this first version of Fluispotter. Design led to limitations in the dimensions of the battery, volume available for anticoagulant fluid, and room for more blood samples. The observed blood clotting issues were also related to the design and can be solved by re-design. The current study was an explorative study of a novel medical device and future studies should include larger populations in order to further explore safety issues and clinical utility. A similar, portable system has previously been developed by Lightman’s group using microdialysis catheters to collect samples from the subcutaneous tissue for 24-h sampling (22). While studies have shown a good correlation between subcutaneous samples and plasma cortisol during stimulation test, that is, synacthen test and dexamethasone suppression test, the correlation was lower when sampling was carried out without stimulation tests. Also, the pulsatility observed in the blood compartment was not replicated in samples from subcutaneous tissue (22). However, both the microdialysis system and the Fluispotter have obvious advantages in the form of portable stress-free sampling potentially allowing for improved and more accessible evaluation of diurnal rhythm.
In conclusion, Fluispotter has shown to be a potentially important advance for automated samplings in humans with acceptable AEs, and at minimal discomfort to the patients, Fluispotter is the first wearable device to automatically maintain peripheral venous catheter patency for blood withdrawal for up to 20 h and is able to maintain catheter patency in a peripheral vein in 9 subjects for 20 h in a safe, comfortable, and painless way. The cortisol measurements cannot be directly compared to others due to methodological differences even though one might think that the cortisol concentrations obtained using conventional techniques may be over-estimated due to white coat syndrome and stress associated with the sampling process. The perspectives of Fluispotter with its ability of repeated venous sampling are assessment of dynamic responses of hormones for diagnosis and evaluation of a number of important clinical conditions. Repeated sampling is also required in both toxicological analyses and pharmacokinetic evaluations, as well as to advance our understanding of disease pathology. Fluispotter may also be useful in the earlier assessments in children due to the lack of serious complications and positive patient acceptance.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/EC-23-0087.
Declaration of interest
MS and SV are co-owners of Fluisense. The other authors state no conflict of interest.
Funding
This study was funded by a grant from Innovation Fund Denmark (5154-0008).
Author contribution statement
All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
Acknowledgements
The authors are grateful for the excellent technical support provided by Christina Boesgaard Knudsen, Department of Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark. UFR's research salary was sponsored by an unrestricted grant from The Kirsten and Freddy Johansen's Fund.
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