Limited Impact of Drone Transport of Blood on Platelet Activation

Abstract

The rapid transportation of blood samples and blood products using drones has high potential in the medical sector. However, before this can be implemented, sufficient evidence that drone transportation is not affecting clinical outcomes is needed. Currently, limited data on the stability of blood, and specifically on platelet activation, after transport using drones are available. Therefore, the impact of drone transportation on platelet activation parameters was analyzed. Blood was drawn from 20 healthy volunteers and lactate dehydrogenase (LDH), potassium, free hemoglobin, PFA-100 closure time, platelet factor 4 (PF4) plasma levels and platelet activation membrane markers were determined in blood that was transported by drone and compared to non-transported samples. In addition, a control group was included where blood samples were transported by car. Transport by both drone and car increased LDH and potassium levels, but the values mostly remained within the total allowable error. Both drone and car transportation impacted platelet activation, as indicated by a small increase in the baseline P-selectin expression and increased PF4 plasma levels. To our knowledge, this is the first study assessing the impact of drone transport on platelet activation. Transportation of blood tubes from healthy individuals using drones has only minimal impacts on blood stability and platelet activation parameters, and is comparable to blood transportation by car. Therefore, the effects observed as a result of drone transportation will likely not impact clinical decision making.

1. Introduction

The logistics sector is encountering several significant challenges. These include increasingly shorter delivery times, a shortage of drivers and staff, traffic congestion, climate issues, intense competition, low profit margins, and rising consumer demands, all of which are straining the industry. Utilizing unmanned aerial vehicles (UAVs), commonly known as drones, presents a promising solution to many of these issues. The medical sector, in particular, is an ideal candidate for this technology due to its high demand for reliable, flexible, and time-sensitive deliveries [1]. Transportation of blood samples and blood products using drones is currently not implemented on a large scale, but is used in several countries such as China [2], Uganda [3], Rwanda [4], and Switzerland [5], where drones are implemented to access remote or hard-to-reach locations. In rural, mountainous, or geographically isolated regions, transporting medical supplies and blood samples can be challenging and time-consuming. Drones overcome these challenges by flying directly to their destinations, even in areas lacking infrastructure. This ensures timely diagnostics and improved healthcare outcomes in underserved areas. Recent changes in legislation make it easier to implement drone transport in current logistics, although it remains challenging to obtain approval from the Civil Aviation Authorities to fly when transporting pathological blood. Nevertheless, blood transportation using drones certainly has high potential in the medical sector. The use of drones can be scaled easily, making them suitable for various healthcare settings, from individual clinics to large hospital networks. The initial investment costs can be offset by long-term savings in fuel, labor, and infrastructure maintenance. As drone technology advances and becomes more affordable, its cost-effectiveness will continue to improve, making it an increasingly viable option for blood sample transportation. Blood and blood products, which are frequently transported between hospitals, patient homes, laboratories, and blood banks, must however adhere to stringent standards for quality. Therefore, maintaining the quality of blood samples during aerial transportation is essential for the widespread utilization of drones for blood transportation. Different studies have demonstrated that while vibrations and turbulence have minimal impacts on blood quality [6,7], temperature fluctuations significantly affect blood quality, particularly glucose, lactate dehydrogenase (LDH), and potassium levels [8,9]. Therefore, to ensure the quality of blood samples, well-insulated and, if necessary, temperature-controlled and even monitored transport containers are required.

However, the cumulative impacts of vibration and temperature during drone transport on platelets, which are highly sensitive to vibration and temperature changes and can easily become activated, has not yet been studied in detail. Platelets are anucleate fragments derived from bone marrow-residing megakaryocytes. In primary hemostasis, platelets adhere to the damaged vessel wall via collagen and von Willebrand factor through various surface receptors. Local prothrombotic factors activate platelets, causing them to change shape and express different surface proteins. They release their granule contents, including serotonin and adenosine diphosphate (ADP), which further promote platelet activation. Activated platelets extend filopodia, aggregate, and form a platelet plug [10]. In secondary hemostasis, activated platelets support fibrin formation to form a stable platelet plug [10]. In the current study, platelet activation parameters were analyzed after transportation by drone. A control group was included where blood samples were transported by a traditionally used car. Parameters were compared to non-transported samples to gain valuable insights into how drone transportation affects platelet-dependent clinical laboratory assays.

2. Materials and Methods

2.1. Study Design

The study was approved by the Medical Ethical Committee of AZ Groeninge Hospital (Kortrijk, Belgium; AZGS2023060). Healthy volunteers provided written informed consent in accordance with the Declaration of Helsinki and did not use platelet-inhibiting medication or anti-coagulants in the 10 days prior to blood withdrawal. Venous blood was collected on sodium citrate and lithium heparin (BD Vacutainer; total volume of 21 mL) by a phlebotomist in the clinical laboratory at the AZ Groeninge Hospital in Kortrijk, Belgium. In total, 20 healthy volunteers were included in this study, 14 females and 6 males. Successive samples were divided among three different groups: no transport, transport by car, and drone transport. Over the course of three different days, transport was carried out five times, each time including four blood samples per transport method. Control ‘no transport’ samples remained in the clinical laboratory at room temperature, while the other blood tubes were transported for 30 min either by car or drone. During transportation by car or drone, each blood tube was packed in a closable plastic bag with absorbent paper and placed together with the other samples in an isolated ECO box acclimatized to room temperature (Exam Packaging provided by Inter Healthcare Transport (IHcT)). Temperatures inside and outside the transport box were continuously monitored using KeyTag Kt1Mu (inside) and iMini MX-IN-S-8-L (outside) temperature loggers (Askey). Drone transport was performed using an Alta X drone (Freefly) (Figure 1), a large, heavy-lifting quadcopter with a diameter of 2.27 m (including propellers), an empty weight of 11 kg, and a cargo capacity of 14 kg. The drone was manually flown by the pilot while utilizing QGroundControl software version 4.3 to monitor telemetry and position within the airspace. All these tests took place just south of the AZ Groeninge site, where operating within EASA Open A3 category airspace was allowed. This category permits visual line-of-sight flights with drones weighing up to 25 kg, provided they remain at least 150 m away from residential, commercial, or industrial buildings. The multirotor drone features a carbon fiber cargo frame installed underneath. The transport box was securely fixed to this mounting frame using large elastic bands to keep the box completely locked in place.

2.2. Clinical Laboratory Analysis

Blood samples were analyzed directly after transport, together with the control ‘no transport’ samples, according to standard laboratory protocols (Figure 2). Lithium heparin-anticoagulated blood was used for the analysis of the total cell count on a Sysmex XN3000 cell counter and lactate dehydrogenase (LDH), potassium, and free hemoglobin levels with a Roche Cobas 6000 chemistry analyzer. Sodium citrate-anticoagulated blood was used to analyze the closure time of a collagen/epinephrine cartridge in the PFA-100 system (Siemens Healthcare, Erlangen, Germany) and platelet activation. Plasma was collected after citrated blood was centrifuged at 1000× g for 10 min, was stored at −80 °C, and was later used for the PF4 determination.

2.3. Platelet Activation Assay

Sodium citrate-anticoagulated blood was used to analyze platelet activation via flow cytometry as described [11] using a Cytoflex S flow cytometer (Beckman Coulter, Brea, CA, USA). Whole blood was diluted 4× with 1× Hepes-Buffered Saline (HBS; 10 mM HEPES, 150 mM NaCl, 1 mM MgSO4, and 5 mM KCl, pH 7.3). Diluted whole blood was added to a mixture of agonists and antibodies. The mixtures included either no agonist, 10 µM TRAP−6 (H-Ser-Phe-Leu-Leu-Arg-Asn-OH; #4017752, Bachem, Bubendorf, Switzerland), 0.5 µM ADP (adenosine-diphosphate disodium salt; #01897, Sigma-Aldrich Merck, St. Louis, MO, USA) or 0.1 µg/mL CRP-XL (Triple Helical Peptides). In all mixtures, anti-CD42b-APC (#551061), anti-CD62P-PE (P-selectin; #555524) and anti-activated integrin αIIbβ3-FITC (PAC-1 clone; #340507, BD Biosciences, Franklin Lakes, NJ, USA) antibodies were added. Blood was incubated with the mixtures of agonists and antibodies for 20 min at 37 °C, followed by the addition of 250 µL of fixative solution (137 mM NaCl, 2.7 mM KCl, 1.12 mM NaH2PO4, 1.15 mM KH2PO4, 10.2 mM Na2HPO4, 4 mM EDTA, and 0.5% formalin; pH 6.8). Flow cytometry was performed to analyze the percentage of P-selectin⁺ and activated integrin αIIbβ3⁺ CD42b⁺ platelets.

2.4. Platelet Factor 4 (PF4) ELISA

Citrated plasma was used to determine PF4 levels using a human PF4 ELISA kit, according to the manufacturer’s instructions (#EHPF4, Invitrogen ThermoFisher, Waltham, MA, USA), using a 1/1000 sample dilution. The absorbance was read at 450 nm and unknown concentrations were calculated from the standard curve.

2.5. Statistics

Graphpad Prism version 10.1.1 (GraphPad Software, LLC, La Jolla, CA, USA) was used for the statistical analysis. To analyze clinically relevant changes due to transportation, the minimum and desirable total allowable errors (TAEs) for clinical parameters were calculated using the EFLM database [12]. To study the correlation between platelet activation parameters, values are expressed as the percentages of control measurements (no transport). The effect of sample transport was analyzed by performing a Friedman test (non-parametric data with repeated measures) with Dunn’s post hoc test (to perform pairwise comparisons between each independent group), comparing non-transported samples to drone-transported samples and non-transported samples to car-transported samples. Simple linear regression was performed to analyze correlations. A p-value of <0.05 was considered significant.

3. Results

3.1. Drone Flights and Car Transport

Five drone flights were performed on three different days with different outdoor weather conditions. Two flights were performed in December, one flight in March, and two flights in June. No precipitation occurred during these flights. Wind conditions varied across the multiple testing days, ranging from calm winds to gusts exceeding 40 m/s on one test day. Ambient temperature ranged from 8.1 °C to 20.5 °C. During the 30 min flight, the drone covered an area of 0.17 km² at a maximum horizontal speed of 80 km/h and a maximum takeoff speed of 5 m/s. During the drone flights, car transport occurred for the same duration, covering a distance of approximately 25 km at a maximum speed of 70 km/h. The temperature measured on the outside of the drone transport box ranged between 7.9 °C and 19.8 °C, and the temperature measured inside the car ranged between 11.7 °C and 25.8 °C. The average decrease in temperature compared to room temperature in the lab (21 °C) was 6.3 ± 4.5 °C inside the drone transport box and 4.1 ± 4.8 °C inside the car transport box, despite the use of isolated transport boxes. Continuous temperature registration demonstrated the largest drop in temperature upon opening the transport boxes outside to place the samples inside the box.

3.2. Clinical Laboratory Parameters

Upon arrival in the clinical laboratory, blood samples that were transported by either car or drone were directly analyzed, together with the control samples that remained in the lab during the duration of transport. LDH, potassium, and free hemoglobin were measured as markers of cellular damage. For LDH and potassium, the minimum and desirable TAEs could be calculated using the EFLM database [12]. A significant increase in the mean LDH level was observed for samples transported by car compared to control samples that were not transported, and four samples (20%) exceeded the minimum level of TAE (Figure 3A). No significant increase in the mean LDH level was observed for samples transported by drone, but three samples (15%) exceeded the minimum level of TAE (Figure 3A). Also, mean potassium levels increased significantly as a result of transport by both drone and car compared to control samples, with one sample (5%) exceeding the minimum level of TAE in the drone transport group (Figure 3B). Surprisingly, no significant increase in free hemoglobin levels was observed in samples transported by either drone or car (Figure 3C), suggesting that the cellular damage markers LDH and potassium are most likely not derived from the lysis of red blood cells but from other blood cells such as platelets. Indeed, a significant decrease in the platelet count was observed as a result of transportation by either drone or car (Figure 3D). Possible clumping of activated platelets caused this decrease, which could fit with increased LDH and potassium levels measured in the same blood tubes. Of note, since platelet counts were determined in lithium heparin-anticoagulated blood rather than with the gold standard EDTA anticoagulated blood for complete blood counts, no TAE values were available for this parameter.

3.3. Platelet Activation During Drone Transportation

To further study the effects of drone and car transportation on platelet activation, baseline platelet activation (in the absence of agonists) was measured using flow cytometry. Blood transportation by car or drone significantly increased baseline percentage of P-selectin⁺ platelets (Figure 4A), while no increase in the percentage of activated integrin αIIbβ3⁺ platelets was observed compared to control samples (Figure 4B). In addition, platelet factor 4 (PF4) levels, indicative of platelet activation, were significantly elevated in plasma samples from car- and drone-transported blood (Figure 4C). The increase in baseline percentage of P-selectin⁺ platelets correlated with the decrease in the platelet count (Figure 4D) and increase in PF4 plasma levels (Figure 4E), indicating that the decreased platelet count as a result of blood transportation by both car and drone could be a result of platelet activation and aggregation.

3.4. Drone Transportation Did Not Affect the Platelet Response Toward Strong Agonist Stimulation

Next, an agonist-induced platelet activation assay using flow cytometry was performed. This assay can be used to determine whether transportation affects the platelets’ sensitivity toward agonist stimulation. Platelets in whole blood were activated with threshold concentrations of either TRAP-6 (Figure 5A,D), ADP (Figure 5B,E) or CRP-XL (Figure 5C,F) to avoid fully activating all platelets, allowing for the detection of subtle changes in activation responses. As shown in Figure 5, drone transportation did not affect the sensitivity to platelet activation, whereas platelets from samples transported by car showed a slightly reduced sensitivity to TRAP-6 and CRP-XL-mediated activation of αIIbβ3. Next, using the PFA-100, the closure time using a collagen/epinephrine cartridge was determined. The closure time was significantly increased in the drone-transported samples, and a trend was observed in the samples transported by car (Figure 5G). The minimal TAE was calculated using the results from Božič-Mijovski et al. [13] and demonstrated that nine samples (45%) transported by car and four samples (20%) transported by drone exceeded the minimum level of TAE. For both car- and drone-transported samples, PFA closure times did not lead to clinically abnormal values, as closure times without and with transportation were not longer than the normal closure time threshold of 180 s, except for the sample from one donor in the car transport group.

4. Discussion

Drone transport could revolutionize the delivery of blood tubes and blood products. It significantly enhances the efficiency of transporting blood samples due to their speed, direct flight paths, and ability to bypass ground traffic congestion. Traditional transportation methods often involve delays caused by road conditions, traffic, or reliance on human couriers. Drones can cover distances more quickly and predictably, reducing the time between sample collection and laboratory analysis. Additionally, operational costs can be lower, especially for repeated or urgent deliveries, as drones require minimal fuel and maintenance compared to conventional vehicles. However, limited data are available on blood stability during drone transportation. To our knowledge, this is the first study assessing the impact of drone transport on platelet activation. A comprehensive analysis demonstrated that transport by drone can cause changes in platelet activation, though the extent of these changes does not appear to be clinically significant, as car transportation gives similar results. Overall, our study demonstrates that the transportation of blood tubes using drones does not impact platelet stability differently than the currently used transportation by car.

Amukele et al. were the first to study the effects of drone transportation on blood sample stability. They compared samples transported by car or drone at ambient temperatures, finding that drone transport did not affect the accuracy of routine tests but slightly reduced the precision for certain analytes [8]. This was recently confirmed by Weekx et al., who also demonstrated a limited increase in CVs for both LDH and potassium levels, indicating a potential clinically relevant effect due to drone transport [14]. In a subsequent study by Amukele et al., blood specimens were transported by drone, and higher glucose and potassium levels were observed when compared to car-transported samples, possibly due to a 2.5 °C temperature difference rather than drone-induced vibration or turbulence [6]. These findings were supported by the absence of changes in laboratory parameters after shaking blood for 3 h at 3000 rpm [6]. Perlee et al. compared drone-transported blood samples with car-transported and stationary samples, finding a significant increase in LDH levels, and attributed this to hemolysis due to the movement of samples during transport [9]. However, Johannessen et al. supported the hypothesis that temperature changes induce changes in blood values rather than the drone vibrations, as their in vitro model simulating vibration and turbulence showed that patient blood samples tolerated substantial vibration and turbulence over 2 h [7]. Therefore, various studies have shown that while vibrations and turbulence have minimal impacts, temperature fluctuations significantly affect blood stability, particularly glucose, LDH, and potassium levels. In our study, the LDH level was significantly increased due to transportation by car. Drone transportation did not significantly increase LDH levels; however, 15% of samples exceeded the minimum level of TAE. Potassium levels were significantly increased due to transport by both car and drone. During transportation, temperature decreases of 6.3 °C and 4.1 °C were observed inside, respectively, the drone and car transport boxes, mostly due to (accidentally) opening the transport boxes outside to place the samples inside the box during the first drone flight. These results suggest that the drone vibrations have not caused the impacts on LDH and potassium levels, but the decrease in temperature inside the transport box did.

Free hemoglobin levels were measured in plasma to analyze whether the increased LDH and potassium levels were derived from hemolysis, but increased free hemoglobin levels were not observed. A decrease in the platelet count together with increased baseline platelet P-selectin exposure and plasma PF4 levels suggest that platelet activation was responsible for the increased LDH levels. Platelets are very sensitive cells and easily become activated. The drop in the platelet count observed in our study is not clinically meaningful because the platelet count was not measured in EDTA-anticoagulated blood; hence, no clinical decisions can be made on platelet counts performed using lithium heparin-anticoagulated blood. EDTA prevents platelet activation and subsequent granule release by strong Ca²⁺ chelation, and is therefore the gold standard for complete blood counts [12]. Other studies investigating the effect of drone transport on platelet counts using EDTA-anticoagulated blood have shown mixed results regarding platelet counts; while Perlee and colleagues showed no effect on the platelet count after drone transportation [9], Weekx et al. did find a significant decrease in the platelet count [14]. Our results suggest that the increase in LDH and potassium levels, as observed by others, is not necessarily a result of hemolysis but can also be the result of platelet activation. This is supported by a study where RBC concentrates, and hence free of platelets, were transported using a drone, and no effect on LDH or potassium levels was observed [15].

Closure times measured with the PFA-100 were significantly elevated in drone-transported samples, and 45% of car-transported samples exceeded the minimum level of TAE. However, all (except one car-transported sample) remained below the normal closure time threshold of 180 s. The increase in closure time could be a result of the decreased platelet counts, as closure times are prolonged when platelet counts are lower [16]. The PFA-100 provides measures of both platelet adhesion and aggregation, and is sensitive to determine severe platelet function defects (e.g., storage pool disorders, type I von Willebrand disease, and primary secretion defects), but small changes in platelet function will not be noticed using this clinical assay [17]. Light transmission aggregometry (LTA) is an often used assay to test platelet function in the clinical lab. With LTA, different agonists can be added to platelet-rich plasma and light transmission as a result of platelet aggregate formation is measured in time [18]. However, LTA is very time-consuming, relatively insensitive to small changes in platelet function, and the outcome is affected by an abnormal platelet count as well as hemolysis [17,19]. Therefore, we have used a flow cytometry-based platelet activation assay to gain more insights into platelet activation as a result of blood transportation by drones. This assay has the advantage that (i) the results combine both the aggregation potential as well as the granule release capacity of platelets; (ii) platelets are analyzed in their physiological milieu of whole blood and no centrifugation is needed, thereby limiting possible artefactual pre-activation; (iii) the effect on a large number of individual platelets can be studied; and (iv) it is a simple and stable assay with acceptable intra-assay variability [11]. Our results indicated slight baseline platelet activation when transported with either car or drone, and a small decrease in the sensitivity of platelets toward low-concentration agonist stimulation when transported by car.

Future studies should however confirm these findings using different types of UAVs. This is supported by studies analyzing the effect of blood transport via a pneumatic tube transport system (PTS). Blood transported through the PTS is subjected to changes in air pressure, accelerations, decelerations, radial gravity forces, and vibrations, resulting in blood cell damage comparable to blood transported using drones. While some studies showed no effect of blood transport via PTS on platelet function assays [20,21], others showed that PTS induced a decreased platelet aggregation response and increased PFA closure time [22,23,24,25]. The conflicting results between studies could possibly be explained by differences in the local PTS (e.g., number of bends, length, transport time, air pressure, and speed), hence demonstrating the need to evaluate the effect of a local PTS in platelet function assays [26]. This also applies to our study where an Alta X multirotor drone was used to study the effect on platelet function. Our results cannot automatically be extrapolated to the other types of UAVs, such as fixed-wing drones. Therefore, testing platelet activation using different UAVs is needed to support our findings.

5. Conclusions

To our knowledge, this is the first study assessing the impact of drone transport on platelet activation. The transportation of blood tubes from healthy individuals using drones has only minimal impacts on blood stability and platelet activation parameters, which are comparable to blood transportation by car. Therefore, the effects observed as a result of drone transportation will likely not impact clinical decision making. While the results are very promising and drone transportation of blood tubes demonstrated similar stability to car transportation, conclusions should be drawn with caution. In this study, only healthy individuals were included, thereby lacking pathological samples outside the reference intervals. Therefore, future studies are needed to analyze the impact of drone transport on pathological patient samples. In addition, our results are limited to the multirotor drone used in this study and cannot automatically be extrapolated to other types of UAVs. Also, although drones could enhance current blood transport, legal regulations and safety considerations in case of the transport of pathological blood must be thoroughly examined before it can be implemented on a larger scale.