Remote platelet function testing – Significant progress towards widespread testing in clinical practice

• Blood cells fixation
• flow cytometry
• platelet function testing

Platelets play a significant role in the physiological process of haemostasis by ensuring the integrity of the vessel wall and initiating primary haemostatic mechanisms when the vessel wall is damaged. They also contribute to the formation of the blood clots that give rise to heart attack and stroke. It is well known that platelet function can be impaired – either elevated or decreased – due to inherited or acquired factors. Reduced platelet function is most often the result of inherited defects in platelet morphology/function or a consequence of the use of a variety of pharmacological agents that either specifically inhibits platelets to reduce the risk of heart attacks or stroke or those with alternative therapeutic targets that can also affect platelet function. Several dietary components and herbs are also known to affect platelet responsiveness [1].

Accordingly, there are several clinical scenarios when platelet function needs to be assessed. A range of techniques are available to test platelet reactivity, however, one of the major limitations of all available methodologies is that the testing needs to be performed on a fresh blood sample within 2–4 hours following venipuncture [2]. Even if the technique is not too complex and time consuming, it still limits platelet testing to a specialized laboratory or at least a laboratory where the appropriate equipment is installed and experienced personnel are available to perform the tests. Such a limitation is a significant obstacle. For example, in the case of diagnosis of platelet function defects, there may be an underestimation of their prevalence with cases tending to be clustered near specialised diagnostic centres [3].

One of the most commonly used methodologies for measuring platelet function is light transmission aggregometry (LTA), which is still considered the “gold standard” method despite the fact that it was developed long ago in the early 1960s [4]. This method can assess platelet aggregation together with its potentiation via amplification mechanisms, such as TxA₂ synthesis and granule release, and register a two-phase aggregation response. This dynamic information is perhaps the most useful characteristic of the LTA and is not available with any other aggregation technique. Although informative, the whole procedure is time- and labour-intensive, and involves multiple steps from preparation of platelet-rich plasma (PRP) to the actual aggregation measurements. There is also significant variability in performance of LTA between different laboratories [5, 6], and this reduces the diagnostic value of the test as the results become incomparable. Recently, recommendations on standardisation of the LTA procedure were issued [2] with the aim to improve reproducibility and comparability of the LTA; however, the technique is still too cumbersome for routine clinical use. Another limitation of LTA is that the method requires large volumes of blood for PRP preparation, which presents a significant challenge when platelet function needs to be assessed in individuals in whom the volume of blood sample is limited.

To overcome some of these limitations, modern technologies have been developed, such as the VerifyNow system (Accumetrics, San Diego, CA) and Multiple Electrode Aggregometry (MEA, Multiplate®, Roche Diagnostics International Ltd, Rotkreuz, Switzerland). Currently, their application is mostly restricted to assessing the effectiveness of antiplatelet therapy, although MEA has been used in two studies to diagnose one of the most prominent inherited platelet defects – Glanzmann thrombasthenia [7, 8]. These tests are certainly simpler, and hence less time consuming and labour intensive, they are performed on whole blood and are much better standardised than the traditional LTA. However, their downside is that they utilise a dedicated piece of sophisticated equipment and use of specific consumables, which together make these methodologies quite expensive to run.

Another important application of platelet function testing is within the drug discovery area, when novel drugs designed to affect platelet function are being developed or when the effect on platelets needs to be excluded as an unwanted effect for a drug with the therapeutic target other than platelets. In this scenario, complex or expensive methodology is not ideal, as pharmaceutical industry studies often require multiple samples to be processed in a limited time period, in multiple centres, and require that methodology is standardized across all participating centres.

Professor Stan Heptinstall has led the Platelet Research Group at the University of Nottingham for more than 30 years and during this time his group has performed both basic research on platelets and multiple projects with a range of pharmaceutical companies, meeting different requirements of platelet function assessment and developing technologies and assays to meet these requirements. Development of procedures designed to meet the needs of standardized testing of platelet function across several centres changed the approach to platelet testing within the group. Utilization of state of the art flow cytometry on fixed cells became a major research focus for the group and has generated a widespread interest in this approach. The technology provides a unique way of stabilizing platelet activation markers through the use of a solution known as PAMFix (patent reference PCT/GB2008/050169) which stabilises platelet activation markers on activated platelets in whole blood for up to 9 days. The activation markers can then be measured by flow cytometry in a very small quantity of whole blood. Recent studies have shown that, at least for the expression of P-selectin, an extended stability of 28 days can be achieved using PAMFix (unpublished data).

Measurement of platelet activation as the amount of P-selectin expressed on the platelet surface in response to stimulation with specific agonist(s) was shown to provide a robust and sensitive way to assess the level of platelet reactivity [9]. High-platelet reactivity measured using this approach was shown to predict the risk of recurrent thrombotic events in patients who developed acute coronary syndrome (ACS) during treatment with the antiplatelet agent clopidogrel [10]. Overall, the technique was easy to perform, requiring only a small volume of blood and the fixed blood samples were stable and could be batch-analysed at a later stage. However, at that time, this technique was still laboratory based and required experienced personnel to perform blood handling and pipetting.

In order to simplify the step of platelet stimulation and make the technique usable in a non-laboratory setting by clinical personnel, a remote platelet function test (RPFT) kit was developed. This was developed together with Hart Biologicals (Hartlepool, UK) who partly manufacture the kit to provide freeze-dried reagents and tubes containing PAMFix which are then assembled by Platelet Solutions Ltd (Nottingham, UK), a spin out company from the University of Nottingham. This spin out company was founded to undertake the development and validation of this technology and take it into clinical practice to improve management of patients. The RPFT kit contains all that is needed to easily process a blood sample in various settings without the need of any laboratory equipment. The kit is stable and may be stored unused for more than 1 year at 4 °C. The main components of the test kit are the vials with lyophilised platelet agonists and the tubes with PAMFix (Figure 1), which allow processing of the blood in a closed system that can be performed by almost anyone. The kit stabilizes the blood sample for at least 9 days, which is then shipped at ambient temperature to a central laboratory for analysis, performed at a later stage. At present, the fully developed RPFT is being used in the platelet function substudy of TARDIS (Triple Antiplatelets for Reducing Dependency after Ischaemic Stroke, NCT 01661322) to assess the effectiveness of several antiplatelet therapy regimes in patients following ischaemic stroke and transient ischaemic attack.

Figure 1. A typical kit supplied by Platelet Solutions Ltd for remote platelet function testing.

A similar approach to remotely assess platelet function has been tested as a diagnostic tool for inherited platelet function disorders (PFDs) within the GAPP study (Genotyping and Platelet Phenotyping, ISRCTN 77951167) using the laboratory version of the test with liquid reagents. In this study, the RPFT included a wider set of platelet stimulants and two platelet activation markers were measured, P-selectin and CD63. Overall, this simple to use test showed fairly good agreement in detecting platelet abnormalities with the measurement of platelet aggregation and dense granule secretion by lumi-aggregometry [11]. This initial evaluation suggests that a similar test could be developed to provide a screening pre-test for patients with suspected PFDs. At present, a specially manufactured lyophilised version of the RPFT for bleeding disorders is being used in subjects with suspected PFD who continue to be recruited into the GAPP study.

In addition to platelet activation markers, platelet aggregation in whole blood can also be assessed by flow cytometry in fixed whole blood samples. The method has been developed within our group and is based on measuring the decrease in the number of single platelets as they form aggregates in stimulated and stirred whole blood [12]. The platelet number can be measured in small sub-samples removed from the test tube and fixed at different time points to provide kinetic information on the platelet aggregation. Platelets are then labelled with platelet-specific antibody and then counted using flow cytometry with the number of red cells being used as a reference for counting individual platelets. The method is sensitive to microaggregate formation and can also provide information on platelet disaggregation. There is an existing commercially available test for assessing platelet aggregation in whole blood, which is also based on counting the decrease in single platelets, Plateletworks (Helena Laboratories, Beaumont, TX). However, the counting is performed using impedance cell counters, which are far less accurate than flow cytometric-based counting. More importantly, the analysis is performed on fresh unfixed samples, which means that it has to be performed immediately after the blood sample is taken and therefore cannot be used between different centres. Therefore, the assessment of platelet aggregation in fixed samples offers a more robust and easy to perform assay.

The current version of the assay requires significant laboratory skills and thus we have recently modified the method and applied it to a 96 well-plate in order to simplify the test and perform simultaneous assessment of several pathways of platelet activation using only a very small volume of blood [13]. In this version of the test, whole blood is directly activated with agonists and stabilised with fixing solution AggFix (patent reference PCT/GB2009/050482, Platelet Solutions, Nottingham) after which the remaining single platelets and aggregates are stabilised for at least 9 days and thus can be assessed remotely. The method proved reliable and easy to use generating full dose–response curves to ADP, AA, collagen and PAR1 agonist using 1.2 ml of whole blood, which makes this method suitable when the blood sample volume is very limited, such as paediatric patients, and could be particularly useful in drug discovery when high throughput methods are needed to screen multiple compounds. Additionally, the AggFix solution is also designed to stabilise platelet–leucocyte conjugates, although the measurement should be performed within 3 rather than 9 days after fixation. Hence, the use of this fixative can provide reliable simultaneous assessment of platelet aggregation and platelet–leucocyte conjugates formation in the same small volume of whole blood sample [14].

Overall, the invention of designated fixing solutions within the research group led by Stan Heptinstall offers significant improvements and new opportunities in the assessment of platelet function. The most prominent breakthrough developed by Stan’s group in this field is the possibility to assess platelet function remotely using fixed whole blood samples. This novelty opens up new possibilities for testing platelets. Firstly and most importantly, the simplicity of the initial stimulation of the blood sample using remote kits makes platelet function testing easily accessible in any healthcare settings without the need for any equipment or technical expertise. The developed approach provides the testing in a standardised way using a closed system and long-term stability of the blood sample – all these characteristics offer potential for better standardisation of the platelet function testing, which can reduce variability in results and make them more comparable between different testing centres.

So far, several applications of the technology have been validated: assessment of platelet activation by measuring P-selectin expression, assessment of platelet dense granule secretion by measuring CD63 expression and assessment of platelet aggregation and platelet–leucocyte conjugate formation. This list can be expanded, and other techniques to measure platelet function using flow cytometry could be improved and facilitated by the use of the described fixing solutions. Certainly, flow cytometry-based techniques hold promise as they require a small volume of whole blood and can cover multiple parameters of platelet function. Indeed, the technology can be applied to measure different aspects of platelet function and therefore bespoke kits can be modelled to answer specific research questions. The latter can help research groups that work with platelets to improve and simplify their methodologies and facilitate the development and testing of novel compounds within pharmaceutical industry. However, the most important outcome of this development would be the availability of simple and reliable platelet testing kits in clinical practice and potential for improved patient care – the application of research findings in real life – something that Stan Heptinstall has always wanted to achieve.

Declaration of interest

All authors are shareholders and Natalia Dovlatova is a part-time employee at Platelet Solutions Ltd.