Measurement of platelet aggregation functions using whole blood migration ratio in a microfluidic chip

Abstract

Platelets play a major role in maintaining endothelial integrity and hemostasis. Of the various soluble agonists, ADP is an important in vivo stimulus for inducing platelet aggregation. In this study, a simple, rapid, and affordable method was designed for testing bleeding time (BT) and platelet aggregation with a two-channel microfluidic chip. Whole blood migration ratio (MR) from a microchip system was evaluated in comparison to the closure time (CT) from PFA-100 assays (Siemens, Germany) and CD62P expression on platelets. To induce platelet aggregation, a combination of collagen (1.84 mg/ml) and ADP (37.5 mg/ml) were used as agonists. After adding the agonists to samples, whole blood MR from the microchip system was measured. The outcome of the assessment depended on reaction time and agonist concentration. MR of whole blood from the microchip system was significantly correlated with CT from PFA-100 (r = 0.61, p < 0.05, n = 60). In addition, MR was negatively correlated with CD62P expression (r =−0.95, p < 0.05, n = 60). These results suggest that the measurement of MR using agonists is an easy, simple and efficient method for monitoring platelet aggregation in normal and ADP-receptors defective samples, along with the BT test. Thus, usage of the current microfluidic method could expand to diverse applications, including efficacy assessments in platelet therapy.

Keywords

Platelet ADP Collagen PFA-100

1 Introduction

Platelets play a vital role in primary hemostasis, and the evaluation of platelet function is a crucial component of assessing hemostasis status in the preoperative setting, especially when patients are receiving anti-platelet medication such as cyclooxygenase-1 inhibitors, adenosine diphosphate antagonists or glycoprotein IIb/IIIa inhibitors [20 , 27]. Maintenance of optimal platelet function is also critical during surgery [13, 20]. Measuring spontaneous platelet aggregation from platelet-rich plasma (PRP) principally measures changes of optical density with PRP [4 –7]. However, until recently, a bleeding time (BT) assay was widely used for assessing the adequacy of primary hemostasis. Unfortunately, many patients find the BT assay time-consuming and invasive. Furthermore, it has low sensitivity and specificity, and thus is not commonly used in the clinical setting [20, 26].

Platelet function is affected by several preanalytical conditions such as time of sampling, high shear stress induced by syringe cannula, tube constituent characteristics, anticoagulant type, storage conditions, platelet concentration and preparation of blood or plasma before platelet function testing. Platelet function must be measured under near ideal conditions [16]. Even under favorable conditions, high inter-individual variability still exists in the platelet function test. Hence, for many years it has been challenging to design new methods to measure platelet aggregation in vitro, and few successful methods have been developed with whole blood or platelet-rich plasma in the presence and absence of agonists [13, 18]. First, light-transmission aggregometry (LTA) was commercialized and widely used to identify and diagnose platelet functional defects [13, 21], although it required tedious preparation of a large number of platelet-rich plasma samples and exhibited low reproducibility, among other drawbacks [5]. To overcome these limitations, a new platelet function analyser (PFA-100, Siemens, Germany) was introduced [11 , 20]. The PFA-100 was designed to let anticoagulated whole blood flow through a narrow hole (d = 150μm), punched out of coated membranes with collagen and epinephrine, or ADP. Prior to passing the membrane, the blood sample would pass through a capillary (d = 200μm) at a high shear rate (γ> 5000 s-1) to activate platelets and von Willebrand factors (vWFs). These flow conditions simulated the in vivo hemodynamics in the small arteries. Thus, activated platelets tended to adhere to the surface of the aperture and aggregate. Over time, the aperture would gradually become occluded.

Clopidogrel effectively inhibits adenosine diphosphate (ADP)-induced platelet activation and aggregation by selectively and irreversibly blocking the P2Y12 receptor, and plays a role in the antithrombotic regimen [9]. However, the inhibitory effects of clopidogrel on platelet function vary considerably by individual [15]. The PFA-100 System (Siemens, Marburg, Germany) provides a test method to detect antiplatelet drug resistance, the Col/Epi, Col/ADP, INNOVANCE PFA P2Y [17].

In the PFA-100 system, Closure Time (CT) in seconds refers to the time it takes for blood to completely occlude the aperture. Two membranes were used to differentiate between drug-induced platelet defects and other platelet defects. The collagen-epinephrine (C-EPI) membrane was very sensitive to all platelet functional defects, including aspirin-induced platelet dysfunction. In contrast, the collagen-ADP (C-ADP) membrane was relatively insensitive to the short-term effects of aspirin. A prolonged CT from C-EPI and a normal CT from C-ADP would suggest that aspirin is the most plausible cause of platelet dysfunction, whereas prolonged CTs from both C-EPI and C-ADP would suggest abnormal platelet function. However, the PFA-100 system was strongly dependent on vWF function and hematocrits (Hct) [13]. In addition, it is very time-consuming to test a large number of samples through PFA-100. Thus, there is a need for a quick and simple method of evaluating platelet function with whole blood.

This study developed the concept of migration ratio (MR) for assessing platelet function through two channels in a microfluidic device. A whole blood sample was pre-treated with or without collagen and ADP as agonists. This sample was then applied into a two-channel microchip. The present study demonstrated a simple and convenient method to examine platelet aggregation by measuring the difference in migration of whole blood in this two-channel microchip. The microfluidic system performance was compared with various closure times from the PFA-100 test and with CD62P expression levels on platelets.

2 Materials and methods

2.1 Study populations

The study included 60 adults (aged 18–64 yrs, 26 males, 14 females) who were screened preoperatively for coagulation abnormalities according to PFA-100, complete blood count, and platelet count. All patients had normal coagulation function and platelet counts. Preoperative PFA-100 measurements were within normal limits in all patients with the collagen-ADP cartridge (normal cutoff <107 seconds). This study was approved by the Human Use Ethical Committee of Korea University GuroHospital.

2.2 Reagents and blood sample preparation

Human whole blood (n = 60) was collected in sodium citrate and K2EDTA tubes (BD Vacutainer Systems, Franklin Lakes, NJ, USA) by venipuncture. Complete blood counts (CBCs) were measured using an automated blood analyzer (Beckman Coulter, Miami, FL, USA) within 2 hours of collection.

We measured the function of platelets with clinical samples by blood migration ratio on a microfluidic chip, and compared our results with two other commonly used diagnostic techniques, blood closure time from platelet function analyzer (PFA-100; Dade, Miami, FL) and CD62P expression of platelet by flow cytometry after addition of platelet aggregation agonists (ADP/COL). All tests were performed simultaneously and no more than 2 hours after blood collection [16].

Within two hours of blood collection, 20μl of collected blood were pipetted into 1.5-ml micro-centrifuge tubes. For the platelet stimulation, collagen and ADP were added at a final concentration of 0.92–1.84 mg/ml and 30.0–37.5 mg/ml, respectively. After the mixture was vortexed, blood samples were incubated at 4°C or 37°C between 2 and 30 minutes at 50 rpm, in a controlled rotator (Hwashin Technology Co. Seoul. Republic of Korea). At the end of the platelet stimulation process, the samples were used for the platelet functional and flow cytometry analyses.

In order to prepare samples for various lengths of CT, anticoagulated whole blood was pretreated with MRS2179 (at a final concentration of 20, 100 or 200μM) or MRS2395 (at a final concentration of 20, 100 or 200μM) for 20 minutes at room temperature. After pretreatment, the PFA-100 assay was performed, and samples with various lengths of CT were used as controls for comparison in the platelet aggregation tests.

Full blood cell counts revealed normal blood counts including hematocrits (from 42–49%) and platelet counts (from 131–304×10⁹/l).

2.3 PFA-100 assays

PFA-100 assays were performed according to the manufacturer’s instructions. The same batch of each test cartridge was used throughout the entire study. Cartridges were allowed to warm up at room temperature before usage. Following gentle inversion, 0.8 ml of blood was pipetted into the sample reservoir of each cartridge on the carousel holder before being loaded into the device. Real-time data were captured using Comport 13 software. Normal CT established for C-ADP in sodium citrate buffer ranged between 60 and 110 seconds. Maximal CT was at 300 seconds, and values above 300 seconds were recorded as >300 s.

2.4 Assessment of platelet aggregation by light microscopy

Microscopic analyses of samples were performed with the suspension of whole blood to confirm the formation of platelet aggregates in response to agonists. After platelet stimulation, 10μl samples were placed on glass slides and covered with coverslips. The Optical microscope Olympus BX51TF (Olympus, Tokyo, Japan) was used to observe the platelet aggregates. Images were captured using a digital camera (Olympus DP 72; Tokyo, Japan).

2.5 Flow cytometric analysis for CD62P expression of platelet

Immunological staining of whole blood after addition of the agonist and incubation was performed by immediately adding whole blood (5μl) to a polypropylene tube containing 50μl of staining buffer (PBS with 1% BSA) and saturated antibody (5μl of 6.25μg/ml CD62P-PE). After a 20-minute incubation in the presence of the antibody at room temperature, 2 mL of 0.5% paraformaldehyde was added to fix the samples for an additional 20 min. Within 4 hours of blood collection, the samples were analyzed using a flow cytometer (Coulter EPICS XL-MCL flow cytometer equipped with a 488-nm laser, Beckman Coulter, USA), and 20, 000 events were acquired without prior washing and centrifugation to minimize artifacts in platelet activation [27]. Platelets were identified in a sideward scatter/forward scatter dot plot, and the gate for indicating positivity of CD62P was arbitrarily set to include 1% of platelets treated with PE-conjugated control antibody. To compensate for nonspecific immunofluorescence, the percentage of CD62P positive platelets was obtained after subtracting the percentage of positive platelets, when the antibody was replaced by the isotype-matched PE-conjugated immunoglobulin controls for CD62P (isotype IgG1).

2.6 Measurement of whole migration ratio after platelet stimulation

Measurement of whole blood MR was performed using a microchannel chip (AccuChip 2× channel, NanoEnTeck Inc. Seoul, Korea). The microchannel chip was made of polymethylmetacrylate (PMMA). Two microchannels were located in the center of the microfluidic chips; each channel was 50 mm in length, 4 mm in width and 0.1 mm in height (Fig. 1). Fifteen μl of stimulated whole blood with various lengths of CT were added to the microfluidic chip to measure whole blood migration after stimulation with the platelet agonists (Collagen + ADP). Then, sample migrations were determined with and without agonists. Subsequently, MR was calculated as follows:

$MR (%) = [y/Y] \times 100$

In the above equation, “y” and “Y” represent the migration distance of whole blood with and without an agonist, respectively (Fig. 2a).

2.7 Statistical analysis

All data were entered into Microsoft Office Excel, and statistics were presented as means±standard deviations (SDs). Control and test samples from the assay were compared using Pearson‘s correlation test. P-values <0.05 were considered statistically significant.

3 Results

3.1 Effect of collagen and ADP concentrations on migration ratio

Various concentrations of collagen and ADP were prepared and examined to determine the optimal combination of agonist concentrations. ADP concentrations less than 37.5 mg/ml had a relatively small influence on MR, while ADP concentrations of 37.5 mg/ml or greater led to decreases in MR (Fig. 2c). The sample with collagen (1.84 mg/ml) and ADP (37.5 mg/ml) displayed the lowest MR (17.2%) in comparison to other groups with different concentrations of agonists (50.8–97.6%) (n = 6).

3.2 Effect of incubation temperature and time on migration ratio

Stimulating platelets with agonists is dependent on incubation temperature. MRs of blood samples were examined at different incubating temperatures with fixed collagen (1.84 mg/ml) and ADP (37.5 mg/ml) concentrations. In addition, incubation time was adjusted from 2 to 10 min with fixed incubation temperatures at 4°C and 37°C (Fig. 3). At 4°C, MRs did not decrease significantly, even though incubation time was sufficient and there were optimal agonist concentrations (n = 6). These results suggest that platelet stimulation and aggregation seem to be strongly affected by incubating temperature.

Incubation time after the addition of agonists into blood was also found to influence MR. For short incubation times of 2 and 5 min, MRs decreased slightly (by 83% and 75% , respectively), even in the presence of agonists at optimal concentrations (Fig. 3). However, significant decreases in MR were observed at incubation periods of 8 min and 10 min (24.6 % and 22.5% , respectively) (n = 6). Interestingly, MRs after 8 minutes were not significantly different than MRs at 10 minutes in the 30-min incubation set. This suggests that a maximum of 10 min of incubation time would be sufficient to trigger platelet stimulation in aggregating platelets.

These results were further confirmed with microscopic observations of blood samples with different incubation times or varying concentrations of agonists. In fact, the control blood sample without any agonist did not show any platelet aggregation, whereas mixed blood samples with agonists showed aggregations of RBCs and platelets in an incubation time-dependent manner (Fig. 4). This suggests that the performance MR test after 10 min of incubation with collagen/ADP (1.84/37.5 mg/ml) induces platelet aggregation, resulting in different MRs.

3.3 Effect of ADP antagonists on migration ratio and closure time of PFA-100

ADP-induced platelet activation is initiated by the P2Y1 receptor and amplified by the P2Y12 receptor [10, 11], suggesting that ADP-antagonists could prevent platelet activation by blocking the binding of ADP with P2Y1 and P2Y12 receptors. Prior to incubation with agonists for 20 min at 37°C, P2Y1 inhibitor (MRS2179) or P2Y12 inhibitor (MRS2395) was added into whole blood as a pretreatment.

The effects of ADP antagonists on platelets were tested to occlude the aperture in a membrane coated with C-ADP for the PFA-100 device. Figure 5 shows the influence of ADP-antagonists on C-ADP CTs with whole blood. In vitro pre-incubation of whole blood with 200μM MRS2179 resulted in a decreased CT of PFA-100 from 85±12 s in vehicle controls compared to 209±94 s in samples that were pre-incubated with MRS2179 (p < 0.001) (n = 10) (Fig. 5a). Similarly, pre-incubation of whole blood with MRS2395 (200μM) increased PFA-100 CT values from 88±16 s in vehicle controls to 270±39 s in samples that were pre-incubated with MRS2395 (p < 0.0005) (n = 10) (Fig. 5b). The results revealed that ADP-antagonists affected the CT in both experimental conditions by modifying platelet function and causing significant retardation of platelet aggregation.

MRS2179 pretreatment inhibited the agonist-induced decrease in whole blood MR in the microfluidic chip, and thus platelet aggregation was reduced in concentration-dependent manners with antagonists (Fig. 5(c) and (d)). Indeed, a concentration of MRS2179 (200μM) markedly slowed the decrease in agonist-induced whole blood MR (n = 10) from 23±9% to 48±20% . When whole blood was pre-incubated with the P2Y12 receptor antagonist MRS2395, similar results of increased MRs were observed (n = 10). These findings indicate that an agonist-induced decrease in whole blood MR on a microfluidic chip can be inhibited by ADP-antagonists. These antagonists could be useful for preparing quality control materials from platelet function analyzers for future evaluations.

3.4 Effect of ADP antagonist on CD62P expression by flow cytometry

Flow cytometry was used to measure collagen/ADP-induced CD62P expression in whole blood samples. Whole blood alone did not increase expression of surface CD62P; however, induction by collagen/ADP increased CD62P expression (Fig. 6). Flow cytometry showed that pretreatment with MRS2179 and MRS2395 inhibited agonist-induced CD62P when compared to the untreated whole blood samples.

3.5 Relationship between agonist-induced migration ratio, closure time and expression of CD62P

Significant negative correlations (r =−0.95, p < 0.05) were observed between agonist-induced MR on a microfluidic chip and expression of CD62P on platelets using control materials prepared with MRS2179 and MRS2395 (n = 60) (Fig. 7). There were also negative correlations between CD62P expression in whole blood and CT by PFA-100 C-ADP (r =−0.67, p < 0.05) (n = 60). Even though the limited data of CT with the upper limit (<300 sec) were available, significant positive correlations (r = 0.61, p < 0.05) were observed between agonist-induced MR on a microfluidic chip and CT from the PFA-100 CADP using control materials with MRS2179 and MRS2395 (n = 60) (Fig. 7).

4 Discussion

Platelet aggregation with activator is essential for the study of initial platelet plug formation and is used in various platelet function assays. Light transmittance aggregometry (LTA) using an agonist is the most widely studied assay [3]. The VerifyNow^® P2Y12 assay and the vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay (which measures the inhibition of VASP phosphorylation by ADP [14]) are both rapid platelet function assays designed to directly measure the effects of clopidogrel on P2Y12 receptors [32]. A multiplate analyzer was used to implement the principle of impedance platelet aggregometry (IPA) in whole blood (Multiplate analyzer, Dynabyte, Munich, Germany) [23].

A two-channel microchip for in vitro testing was developed to study the effects of a platelet activator on aggregation, by characterizing the MR of a sample in the presence of platelet activator compared to a control. This microchip can be read and analyzed by an optical device, in order to accurately quantify whole blood MR through two channels. This method is a faster and simpler procedure than previously reported commercial techniques [13 , 21], and has the capacity for high throughput without preparation of platelet rich plasma from whole blood samples. MR changes were monitored by varying three experimental conditions: platelet activator concentration, temperature, and reaction time between the platelet activator and the sample.

The greatest decreases in MR were detected with concentrations of collagen and ADP at 1.84 mg/ml and 37.5 mg/ml, respectively (Fig. 2c). If there were insufficient amounts of ADP or collagen in the blood sample, MR did not decrease. These results suggest that agonist-induced platelet aggregation could impact blood flow when there are sufficient concentrations of ADP and collagen. In addition, compared to platelets stored at 22°C, platelets exposed to temperatures less than 20°C quickly change shape and show an increase in glycoprotein (GpIb, GpIIb/IIIa) and platelet activation markers (CD62p and CD63). [29, 34]. Therefore, this study compared changes in MR with platelet activation at 4°C or 37°C.

When platelet activators were added, the MR at 37°C was lower than the MR at 4°C. This is expected, because increasing the temperature to 37°C should restore the discoid shape of the platelets and could induce platelet activation. For this reason, the majority of platelet aggregation tests were performed at 37°C in previous studies [1 , 30]. We also observed the effect of the incubation time on samples with collagen/ADP (Fig. 3). In a previous study, maximum aggregation was observed approximately 10 minutes after adding ADP to citrated whole blood [1]. Hence, in the present study, after adding ADP to citrated whole blood, the basal reaction time for the MR measurement was set for 10 minutes.

The effects of ADP antagonists MRS2179 and MRS2395 on platelet aggregation were monitored based on MR changes. Increased MR was observed in whole blood samples citrated and premixed with higher concentrations of MRS2179 or MRS2395 (Fig. 5). In addition, changes in CD62P expression on platelets were observed with the addition of collagen/ADP in the presence or absence of ADP antagonists. Regardless of the type of ADP antagonist, activated platelets decreased in the presence of ADP antagonists. These results corroborate previous studies demonstrating that MRS2179 and MRS2395 have similar inhibitory effects on platelet activation and platelet aggregation [8 , 33]. These antagonists would be particularly valuable when preparing quality controls for evaluation of platelet function.

The incubation time and anticoagulant in that study were similar to those in the present study [30]. Discrepancies in the findings regarding the inhibitory effects of MRS2179 and MRS2395 were most certainly tied to experimental conditions. First, using both ADP and collagen as enhancing agents for platelet aggregation may have affected the results. During our study, since ADP alone did not cause platelet aggregation in citrated whole blood, collagen was added to provoke platelet aggregation. It has been reported that collagen, unlike ADP, can interact with platelets through the GPIa/IIa and GPVI receptors and influence platelet aggregation [22, 35]. Second, the differences in sample types may have been a factor as well. In the previous study, Spath et al. detected aggregations by using rich platelet plasma, whereas whole blood samples were used to detect aggregations in the current study [30].

Shear stress also had a profound impact on all aspects of platelet function, including platelet activation and aggregation [31]. PFA-100 typically involves a shear-induced platelet activation (SIPA) mechanism with a corresponding shear rate of greater than 5000 s^–1, which simulates in vivo arterial hemodynamic environments. However, this SIPA-based system has critical drawbacks, including a strong dependence on vWF function and hematocrits. In fact, platelet adhesion at high shear rates is possible due to vWF, which functions as an anchor between collagen and platelets. The extended closure time in a PFA-100 measurement could be interpreted as dysfunction of either vWF or platelets. Similar results might occur when low hematocrit blood is tested. However, the present study adopted a low shear stress environment (500-s), which is less dependent on vWF.

Under conditions of high shear, the platelets bind to vWF, and this interaction results in platelet thrombus formation. Similar processes may occur in response to lower shear stress when platelets are exposed to thrombogenic surfaces and agonists generated at sites of vascular injury during thrombus formation [12]. However, in the present study, the rolling machine performed a similar task at 37°C for 10 min, at 30 rpm. Accordingly, the present method may depict the mechanism of platelet activation and aggregation in vein shear stress accurately, and consequently the results may provide more insight into this specific biological process. We measured whole blood migration ratio MR after addition of ADP, with decreased MR indicating increased platelet aggregations in our system. Lastly, our methods did not require any preparation of platelet rich plasma. Even though the low shear system was used for aggregometry, the MR test with a two-channel microfluidic chip was a quick and easy system. Furthermore, while shear test conditions in plasma cannot reflect the exact physiological conditions of primary hemostasis in whole blood, the MR test with agonists using whole blood samples is a reasonable approximation of physiological stress conditions [28]. This method has great potential as a screening test for detecting platelet aggregation at low venous shear stress.

The risk of thrombosis is elevated when platelets are hyper-reactive and spontaneous platelet aggregation is elevated [7]. Like the PFA-100 system, the MR test could be used to diagnose platelet function defects including Bernhard Soulier syndrome, von Willebrand syndrome, and grey platelet syndrome or thrombosis.

In conclusion, an MR test with a two-channel microfluidic chip was developed for measuring platelet function in vitro for both control and test samples with activators. The microfluidic chip system was very sensitive to the presence of inhibitors of ADP-receptors, and the results were comparable to other currently available platelet functional assays. Consequently, this test proved to be a quick and easy method using whole blood samples to observe platelet aggregation in low shear stress, and to detect the effects of ADP antagonists on platelet aggregation. Unlike labor-intensive aggregometry, our method could be used in large-scale screening tests for detecting platelet aggregation. Therefore, this device would be useful as a diagnostic tool for individual blood samples and when studying anti-platelet therapies.

Footnotes

Acknowledgments

This study was supported by a grant from the Korea Health technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI14C0670).

This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI14C0670).

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