Novel approaches to quality control and external quality assessment for platelet function testing with a focus on the platelet function analyser (PFA-100 and PFA-200)

Introduction

Platelets represent a key component of primary hemostasis, and deficiency and/or defects in platelets, be they congenital or acquired, lead to bleeding diatheses in affected individuals (1-3). In turn, platelet function tests (PFTs), aiming to identify if platelets are functionally active or instead defective or impaired in some way, represent key diagnostic test processes within hematology laboratories associated with advanced hemostasis diagnostic facilities (4-7).

There are various levels of platelet function tests, from simple screening tests, to complex functional assays and molecular analysis (3-7). Platelet function testing has evolved to now incorporate a wide variety of test processes. These processes include (I) whole blood aggregometry (WBA), which in turn may be driven by a variety of instrumentation (3-8), (II) light transmission aggregometry (LTA) (3-7,9), representing a kind of ‘gold-standard’ in testing by nature of long historical use and wide user experience, (III) testing by automated platelet function analyser (PFA) -100 (or -200) (3-7,10), (IV) assessments by flow cytometry (3-7,11-13), and ‘(V)’ to ‘(XXVI)’ representing a large number of additional methodologies (3,6,7). The reader is referred to Table 1 for a summary of the main types of platelet function tests currently available in diagnostics. However, many other procedures may also be used to monitor anti-platelet therapies and/or in research settings. Therefore, should readers be interested in more detailed listings, they are referred to other excellent reviews (3,6,7,14).

Despite many of these tests being in diagnostic use now for decades, and/or continuously evolving, internal quality control (IQC) and external quality assessment (EQA) for them is limited and made specifically difficult for diagnostic PFTs by the nature of the tests themselves, as well the test material used, with this typically representing functional cellular material (i.e., platelets). The current review therefore aims to overview platelet function testing from the perspective of diagnostic screening and highlights current limitations in IQC and EQA for PFTs, as well as highlighting potential solutions that will enable more effective and accurate testing in the future. However, the primary focus of the review is on IQC and EQA for the platelet function analyser (PFA-100 and PFA-200).

What are PFTs and what are the problems associated with these tests?

Simplistically, PFTs aim to investigate the function of platelets. Although this seems a straightforward statement of fact, how do laboratories actually achieve this, and how can they ensure the quality of PFTs?

To some extent, the answer to such questions depends on the type of tests that are performed. Platelets are complex cellular components of hemostasis (1). There are over 100 million platelets in each mL of our blood, and each platelet expresses a large number of cell surface receptors, and also houses several internal granule types that contain a myriad of (primarily pro-) haemostatic components. Simplistically, then, PFTs aim to investigate whether the platelets are working by investigation of the activity of either the cell surface receptors, or its internal components. In brief, platelets are chiefly involved in so-called primary hemostasis—meaning platelet aided formation of platelet ‘plugs’ to seal sites of vascular injury, and thereby stop bleeding (1,3,15,16). They also assist in secondary hemostasis by providing a scaffold for assembly of coagulation, and by delivery of various hemostasis proteins (as contained in their storage granules) to the site of injury (1,15,16). These activities are achieved by a sequence of steps that involves platelet adhesion [via various surface receptors and von Willebrand factor (VWF; present within plasma and also stored inside the platelets)], platelet activation (which then causes release of the internal storage granule components) and finally platelet aggregation (in which the platelets aggregate (clump together) to help form the ‘brickwork’ that seals the site of injury. Various plasma proteins, some of which are also included within the platelets, but primarily fibrinogen, help to act as the ‘mortar’ for this brickwork.

Irrespective, the important piece of information to remember in terms of IQC and EQA is that platelets represent small cellular components derived from blood. Although collection of platelets (or of whole blood) is very strait forward, platelets are very easily activated, and once activated can no longer be assessed for their activity or their function (since the activity has already essentially taken place). Platelets can easily become activated by (poor) blood collection (procedures), including use of too small-bore needles, and usage of extended stasis or tourniquets (17). Platelets can also become activated by (poor) blood transport, including excessive agitation (‘shaking’) or pressure (e.g., tube transport systems), delays in transport, and extremes of temperature during transport (both high and low temperatures can adversely affect platelet activity).

In general, PFTs need to be completed within a few hours of (a well-managed) blood collection, so that tests usually need to be performed within the same general location as the blood collection, and after collection of blood by experienced phlebotomists. This is unlike most assays of hemostasis, which can either be performed soon after collection, or else, the plasma can be separated from the centrifuged blood and then frozen for later (even geographically distant) testing. Platelets cannot be frozen for later testing, as freezing platelets destroys them. Platelets cannot be transported over large distances.

IQC and EQA for most tests of hemostasis can typically be achieved using lyophilised or frozen plasma control material. Again, this is simply not possible for PFTs. For most tests of hemostasis, before patient samples can be tested, the process needs to be ‘controlled’ by use of IQC material that can identify whether or not the test processes are working appropriately. The IQC samples must yield test results that are within an acceptable pre-defined range of expected values. Typically, IQC is performed using commercially available (plasma) materials representing several ‘levels’ of the analyte to be controlled, usually meaning a normal sample to control test results around the normal range (or reference interval) and also a ‘pathological’ sample, to control test results above or below the normal range. Sometimes, laboratories utilise a sample that mimics an ‘anticoagulated sample’ to control testing performed around the ‘therapeutic range’ (as an alternative to a ‘pathological’ sample). Such processes can be exemplified, for example, for routine tests of hemostasis, such as prothrombin time (PT), international normalized ratio (INR) and activated partial thromboplastin time (APTT) (18,19), with 2–3 levels of IQC normally applied (20). Indeed, the same set of commercial materials can typically be used for all routine tests, as well as additional tests of hemostasis (e.g., same control set can be used for PT, APTT, thrombin time and fibrinogen).

For PFTs, there are no such commercial controls, simply because as stated, lyophilisation or freezing of platelets, or whole blood, effectively destroys the platelets (21), and there are no commercial sources of stabilised native platelets. Thus, the only way to control PFTs is to collect fresh whole blood or platelets from human donors. To control testing that provides ‘normal’ results, an ostensibly normal individual would need to be collected. This needs to be done each and every time that PFTs are performed—meaning that either a normal individual is punctured every day, or else that the laboratory needs an armamentarium of normal individuals from which to regularly source fresh whole blood/platelets. Neither of these options is ethical or sustainable. Even should such options be available, the arising IQC does not provide control cover for ‘pathological’, ‘therapeutic’ or ‘abnormal’ test results, which for PFTs may involve a multitude of distinct activities depending on the test performed. As a comparative example, for the INR, a ‘therapeutic’ IQC could be a warfarin-like plasma to cover vitamin K antagonist (VKA) therapy, and for the APTT, a ‘therapeutic’ IQC could be a (unfractionated) heparin-like sample (18,19). For PFTs involving LTA and WBA, for example, this might need to include ‘aspirin-treated’ platelets to assess arachidonic acid/cyclooxygenase pathway defects, ‘clopidogrel-treated’ platelets to assess P2Y12 defects and ADP responsiveness, ‘GPIb-denuded’ platelets to assess platelet glycoprotein 1b pathway defects, and so on and so forth. Thus, even if ‘normal’ platelet function testing could be controlled by collection of normal individuals, the results of ‘abnormal’ PFTs cannot be easily controlled, since this would require collection of a wide range of ‘abnormal’ test samples, representing an armamentarium of ‘abnormal’ individuals, or else, the ‘construction’ of abnormal test samples covering a wide variety of potential defects. This is simply not feasible.

EQA for PFTs poses additional challenges. EQA would theoretically require collection of a huge amount of normal blood (to assess/control for normal PFT results) as well as much larger quantities of blood from various abnormal individuals, and/or samples be purpose constructed to reflect a wide variety of potential platelet function abnormalities. This material would then need to be transported to a multitude of EQA participating laboratories, within a few hours (to maintain platelet integrity) and to mitigate adverse effects on platelet function. This is simply a current logistic impossibility.

The (potential) solutions to IQC and EQA for PFTs—focus on the PFA-100/-200

Given the difficulty of IQC and EQA for PFTs (summarised in Table 2) as otherwise ‘classically defined’ for hemostasis tests, there is a need to think ‘outside the box’ and devise alternate strategies to control laboratory performed PFTs and thereby ensure the quality of such testing.

The feasibility of alternate approaches to IQC and EQA for PFA-100/-200 testing has been explored by two EQA groups. The College of American Pathologists (CAP) reported on one potential strategy in 2007 (22). This approach utilised an inhibitor of platelet function to generate ‘pathological’ PFT results in the PFA-100 after addition of whole blood from a normal individual, as collected fresh on site by the participating laboratory. Thus, the EQA (CAP in this case) sent the laboratory a ‘wet-challenge’ tube, with this containing a platelet function antagonist, and the EQA participating laboratory then tested normal whole blood collected fresh on site on their PFA-100, either without any manipulation (to generate a normal PFA-100 EQA test sample result) and also repeating the test(s) after addition of this normal whole blood to the ‘wet-challenge’ tube (to generate a ‘pathological’ PFA-100 EQA test sample result). Test results obtained by participant laboratories were then compared with other laboratories in a peer-assessment process. CAP reported this produced a successful EQA for the PFA-100 (22), but inter-laboratory co-efficient of variation (CVs) produced for the wet-challenges were as high as 50%, as compared to about 20% for the normal (non-manipulated test sample), thereby potentially limiting the overall usefulness of the EQA. CAP recently published an update (23) to the original report (22), and the PFA-100 based program remains available more than a decade later in 2019 (https://www.cap.org/). Indeed, this EQA program now also has wet-challenges for ‘Platelet aggregation’ and ‘Helena Plateletworks’, according to the latest CAP catalogue (available at https://www.cap.org/). Moreover, the specific nature of these ‘wet-challenges’, although not identified in the catalogue, is elaborated on in the recent publication (23). It seems that essentially the same approach is used for all the platelet function options—PFA-100, Platelet aggregation, Helena Plateletworks and also PlateletMapping (23). Two specimen challenge tubes are sent with each platelet function survey. One tube contains saline, while the other contains tirofiban, a platelet GPIIb/IIIa inhibitor. If a normal donor is collected, the saline challenge should provide normal results. In contrast, tirofiban would simulate a severe platelet aggregation defect similar to homozygous GPIIb/IIIa deficiency (Glanzmann thrombasthenia). Thus, tirofiban would be expected to produce an abnormal result for all types of platelet function testing. Participating laboratories are instructed to pipette citrated whole blood from a normal donor into each of the challenge tubes and mix gently by inversion 8 to 10 times.

Platelet function testing is then to be performed on these samples according to the standard procedure for each laboratory.

As a summary of their most recent report (23), and using proficiency testing data from 2012–2016, a total of 1,200 laboratories participated for PFA-100, with the coefficient variation (CV) of cartridge closure times for saline being was 22%. The CV for the tirofiban challenge was not reported. Nevertheless, 44,952 of 45,616 survey responses (99%) provided an interpretation, and 42,934 of 44,952 (96%) were correct. This indicated that the wet-challenge process for the PFA-100 worked as expected for the vast majority of participants. For optical platelet aggregation, 190 laboratories participated, and the CV for saline was 17%. Again, the CV for the tirofiban challenge was not reported. Nevertheless, 7,444 of 7,813 survey responses (95%) provided an interpretation, and 7,015 of 7,444 (94%) were correct. This again indicates that this wet-challenge process, this time for optical platelet aggregation, also worked as expected for the vast majority of participants. For PlateletWorks, 60 laboratories participated, and the CV was 3% to 11% (for saline challenge). Of 2,454 survey responses, 2,412 (98%) provided an interpretation, and 1,207 of 1,276 (95%) were correct for adenosine diphosphate (ADP) and 936 of 1,136 (82%) for collagen. For PlateletMapping, 200 laboratories participated; for ADP, 1,128 of 2,697 survey responses (42%) provided an interpretation, but only 927 of 1,128 (82%) were correct. For arachidonic acid, 1,139 of 2,604 survey responses (44%) provided an interpretation and 964 of 1,139 (85%) were correct. Thus, PlateletWorks using collagen and PlateletMapping showed worse interpretive accuracy than the other methods.

As a hemostasis advisor to the Royal College of Pathologists of Australasia (RCPA) hematology quality assurance program (QAP), the author developed an Australasian program for PFA-100 EQA in 2008, and the results of this EQA have since been reported in several publications (24-28). The premise of this EQA is similar to that of CAP, but alternate use of various (propriety) formulations to tirofiban (as used by CAP) has seemingly achieved a much tighter CV than that reported by CAP (22,23). A summary of the anticipated test patterns obtained for the PFA-100/-200 from the perspective of clinical scenarios is provided in Table 3, whereas the corresponding perspective of EQA is shown in Table 4.

A summary of the EQA material sent out by the RCPA QAP over the past 10 years is identified in Table 5, and the resultant outcomes summarised in Table 6. This EQA provides both positive (‘abnormal’ PFA) and ‘negative’ (‘normal’ PFA) wet challenges, and thus like CAP also provides an EQA wet-challenge for both normal and ‘pathological’ test results. Figure 1 shows data from Table 1 summarised according to challenge type, for main categories of ‘baseline’, ‘no additive challenge’, ‘mild/moderate defect’ challenges (various samples) and ‘severe defect’ challenges (various samples). Inter-laboratory CVs tend to be <20% for baseline test results and no additive ‘wet-challenges’ and are often <15% for ‘positive’ (severe defect’) challenges yielding grossly prolonged PFA test times (Table 6; Figure 1). CVs for ‘mild/moderate defect’ challenges tend to be higher, but are generally <30%, albeit representing quite a heterogeneous group of challenge samples. The EQA has been shown to be effective for both the PFA-100, and the newer PFA-200 model (not yet available in the USA). To date, a total of 47 separate EQA challenges have been distributed and undertaken by participants (Table 5), with results published for most challenges (24-28). A summary of data for baseline PFA test times, the negative ‘wet-challenges’, and some positive ‘wet-challenges’ is shown in Figure 2.

Figure 1 Summary of data findings from Table 6. Data summarised according to challenge type, for main categories of ‘baseline’ (B), ‘no additive challenge’ (NA), ‘mild/moderate defect’ challenges (M; various samples) and ‘severe defect’ challenges (S; various samples). (A) Median values for PFA closure times (CTs) in seconds (s) for collagen/ADP (C/ADP) and collagen/epinephrine (C/Epi) cartridges. The dotted horizontal lines represent the upper limit of normal using manufacturer reported ranges. (B) Inter-laboratory coefficient of variation (CVs). The dotted horizontal lines represent 10%, 20% and 30%.

Figure 2 Summary of external quality assessment (EQA) data for PFA-100/-200 from Royal College of Pathologists of Australasia (RCPA) hematology Quality Assurance Program (QAP), for years 2008–2017 inclusive. Data shown as box plots, with the bars representing the 10^th–90^th percentiles, and the box representing the 25^th–75^th percentiles. Left y-axis collagen/ADP (C/ADP) cartridge closure time (CT) in seconds. Right y-axis collagen/epinephrine (C/Epi) cartridge closure time (CT) in seconds. X-axis identifies the EQA challenge. The dotted horizontal lines represent the upper limit of normal using manufacturer reported ranges. (A) Baseline (pre-wet-challenge) data; (B) ‘negative’ (no additive) wet challenges; (C) ‘positive’ (additive) wet challenges.

The PFA EQA has also been shown to be potentially useful in the setting of IQC (26-28). For the PFA-100/-200, the manufacturer only suggests performance of a normal test sample (normal fresh donor) with each change in lot of test cartridges, or after any major instrument maintenance, as IQC. The EQA ‘wet-challenges’ may be therefore potentially utilised to also provide some reassurance of PFA functionality around the ‘abnormal’ test region, and test data can be depicted in Levy-Jennings graphs (25-28) (samples shown in Figure 3).

Figure 3 Levey-Jennings-like plots of an internal quality control (IQC) like process using some external quality assessment (EQA) challenge samples. (A,B) Normal baseline whole blood closure time (CT) values can act as ‘normal IQC’ and sequential test data from EQA samples PF12-08b (A) and PF15-08a (B) can act as ‘pathological IQC’. Data reported as CTs in seconds (s; y-axis in each figure), for a theoretical timeline of 12 months. Here, IQC limits for the ‘normal QC’ sample (= baseline whole blood CTs) would be values below the normal/abnormal cut-off value (manufacturer values used in this example). For PFA-12-08b and PF15-08a, representing ‘pathological’ IQC samples, the IQC limit could be assigned as a value above a predefined cut-off (e.g., 200s as used in this example). (C,D) Data from Figures A and B, but now expressed in terms of x-fold of baseline. In this potential IQC scenario, the IQC limits might be expressed by predefined limits of test data (for example above 2x baseline value).

Other EQA solutions for PFTs

Another EQA provider, ECAT (External Quality Control of Diagnostic Assays and Tests; http://www.ecat.nl/) in partnership with NASCOLA (North American Specialized Coagulation Laboratory Association; https://www.nascola.com/) offer a variety of ‘electronic’ surveys to support laboratories involved in PFTs. These comprise: (I) Post Analytical Platelet Function EQA (electronic survey); (II) Platelet Dense Granule exercise (electronic survey); (III) case studies on bleeding disorders (distribution separately from the regular surveys); (IV) pre- and post-analytical electronic surveys in haemostasis. Some publication around these exercises are available for the interested reader (29-32).

Conclusions

IQC and EQA for PFTs remains challenging, but some support is available from a variety of sources, especially in relation to EQA. Wet-challenges are so far limited to two EQA providers, RCPAQAP and CAP, although ECAT expects to also offer the same challenges as available to RCPAQAP participants in the near future. In terms of EQA for PFTs, the main alternative to ‘wet-challenges’ comes in the form of educational support, for example by electronic surveys, and typically ‘post-analytical’ (meaning that results of PFTs are provided the EQA to participants for their interpretation – and then these interpretations are then relayed to the EQA for review and assessment/peer comparison/reporting). Irrespective, ‘thinking outside the box’ is a critical requirement when contemplating IQC and EQA for PFTs, since standard hemostasis approaches simply remain infeasible (Table 2).

Acknowledgments

This review includes some data derived as a result of an ongoing collaboration with the RCPAQAP. This data is presented in summary form, and/or may derive from prior studies in which the RCPAQAP and its staff contributed, either by provision of raw data, or by prior data analysis, or by other logistical support. This EQA program is therefore acknowledged as facilitating some of the work described in this paper. Several individuals within the RCPA can also be personally acknowledged, in particular Roslyn Bonar, who has recently retired from the workforce to concentrate on life and family.

Footnote

Provenance and Peer Review: This article was commissioned by the editorial office, Annals of Blood for the series “External Quality Assurance”. The article has undergone external peer review.

Conflicts of Interest: The author has completed the ICMJE uniform disclosure form (available at: http://dx.doi.org/10.21037/aob.2019.01.02). The series “External Quality Assurance” was commissioned by the editorial office without any funding or sponsorship. EJF served as an unpaid Guest Editor of the series. The author has no other conflicts of interest to declare.

Ethical Statement: The author is accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Disclaimer: The opinions expressed in this review are those of the author, and not necessarily those of NSW Health Pathology or of the RCPAQAP. This manuscript reflects findings from a single EQA provider, the RCPAQAP, and the opinions of other EQA organizations may differ.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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