Diagnosis Of Platelet Function Disorders Research Articles

PB1381 Value of Next-Generation Sequencing in the Diagnosis of Platelet Function Disorders

PB1381 Value of Next-Generation Sequencing in the Diagnosis of Platelet Function Disorders

Stability and utility of flow cytometric platelet activation tests: A modality to bridge the gap between diagnostic demand and supply

Light transmission aggregometry (LTA) is the gold standard for the diagnosis of platelet function disorders (PFDs). The requirement of customized aggregometer, large blood volume, normal platelet count and processing within 4 hours of venipuncture for LTA makes platelet function testing inaccessible to wider population. Flow cytometric platelet activation test (PACT) may overcome these limitations. This study compares the performance of PACT with LTA, characterizes diagnostic patterns of PFDs on PACT and assesses the stability of PACT beyond 4 hours of venipuncture in controls (n = 5) at different temperature conditions. LTA and PACT were performed in 121 healthy controls and 66 patients with suspected PFD. PACT had excellent agreement (kappa = 0.93) with LTA and 94.1% sensitivity, 98.5% specificity. PACT had distinct patterns in Bernard Soulier Syndrome (n = 10), Glanzmann Thrombasthenia (n = 24), δ-granule disorder (n = 7), and other PFDs (n = 12). PACT could assess platelet function in patients (14%) with thrombocytopenia/lipemia wherein LTA was inconclusive. PACT was stable up to 24 hours in samples stored/transported at 2–8◦C. The results of utility and stability are only valid for the specific markers, agonist concentrations, and conditions investigated in this paper. PACT is a useful modality for the diagnosis of PFD, especially in children, thrombocytopenia cases or in the setup where an aggregometer is not readily available.

Diagnosis of Platelet Function Disorders: A Challenge for Laboratories.

In patients with normal plasmatic coagulation and bleeding tendency, platelet function defect can be assumed. Congenital platelet function defects are rare. Much more commonly they are acquired. The clinical bleeding tendency of platelet function defects is heterogeneous, which makes diagnostic approaches difficult. During the years, a large variety of tests for morphological phenotyping and functional analysis have been developed. The diagnosis of platelet function defects is based on standardized bleeding assessment tools followed by a profound morphological evaluation of the platelets. Platelet function assays like light transmission aggregation, luminoaggregometry, and impedance aggregometry followed by flow cytometry are commonly used to establish the diagnosis in these patients. Nevertheless, despite great efforts, standardization of these tests is poor and in most cases, quality control is lacking. In addition, these tests are still limited to specialized laboratories. This review summarizes the approaches to morphologic phenotyping and platelet testing in patients with suspected platelet dysfunction, beginning with a standardized bleeding score and ending with flow cytometry testing. The diagnosis of a functional defect requires a good collaboration between the laboratory and the clinician.

Flow Cytometric Assessment of AKT Signaling in Platelet Activation: An Alternative Diagnostic Tool for Small Volumes of Blood.

The diagnosis of platelet function disorder in children is challenging. Light transmission aggregometry is the gold standard for platelet function disorders. However, large blood volumes are required. Currently, there are no existing tools for the diagnosis of platelet function disorders that use small blood volumes. AKT signaling plays a central role in platelet activation during hemostasis and might be visualized by flow cytometry. Platelet-rich plasma obtained by centrifugation of citrated blood from healthy volunteers was activated with arachidonic acid, thrombin receptor activating peptide-6 (TRAP-6), collagen, adenosine diphosphate ADP, collagen-related peptide (CRP), and epinephrine. After platelet activation, the phosphorylation of AKT was assessed by flow cytometer using a Navios cytometer. Healthy volunteers showed a reproducible phosphorylation of AKT upon activation. In comparison to nonactivated platelets, we documented an increase in pAKT expression with all agonists. Especially TRAP-6 and CRP caused considerable increase in percentage of pAKT expression throughout all the tested healthy volunteers. An activation of the AKT-signal pathway by different agonists can clearly be detected on the flow cytometer, indicating that the visualization of signaling in platelets by flow cytometry might be an efficient alternative for light transmission aggregometry to test platelet function in children.

Platelet preparation for function testing in the laboratory and clinic: Historical and practical aspects

Laboratory tests of platelet function are instrumental in studying platelet physiology and inherited or acquired platelet abnormalities. Light transmission aggregometry, developed in the early 1960s, is still considered the gold standard for the identification and diagnosis of platelet function disorders. Since then, novel techniques have been developed, including flow‐based assays and flow cytometry. In this tutorial, we describe the basic methodologies for the preparation of citrated platelet‐rich plasma and washed platelet suspensions and discuss their respective advantages and limitations as well as important factors to consider to perform high‐quality tests of platelet function. In addition, the methodologies of the main platelet function tests (light transmission aggregation, flow‐based assays, and flow cytometric assays) are described, and their respective strengths and limitations are discussed to assess various aspects of platelet biology.

Validation of flow cytometric analysis of platelet function in patients with a suspected platelet function defect

Background Light transmission aggregometry (LTA) is the most commonly used test for the diagnosis of platelet function disorders (PFDs), but has moderate sensitivity for mild PFDs. Flow cytometry has been recommended for additional diagnostics of PFDs but is not yet standardized as a diagnostic test. We developed a standardized protocol for flow cytometric analysis of platelet function that measures fibrinogen binding and P-selectin expression as platelet activation markers in response to agonist stimulation. Objectives To determine the additional value of flow cytometric platelet function testing to standard LTA screening in a cross-sectional cohort of patients with a suspected PFD. Methods Platelet function was assessed with flow cytometry and LTA in 107 patients suspected of a PFD in whom von Willebrand disease and coagulation factor deficiencies were excluded. Both tests were compared in terms of agreement and discriminative ability for diagnosing patients with PFDs. Results Out of 107 patients, 51 patients had an elevated bleeding score; 62.7% of the patients had abnormal platelet function measured with flow cytometry and 54.2% of the patients were abnormal based on LTA. There was fair agreement between LTA and flow cytometry (κ = 0.32). The discriminative ability of flow cytometric analysis in patients with an elevated bleeding score was good (AUC 0.82, 0.74-0.90), but moderate for LTA (AUC 0.70, 0.60-0.80). Both tests combined had a better discriminative ability (AUC 0.87, 0.80-0.94). Conclusion Flow cytometric analysis of platelet function has added value in diagnostics of PFDs in patients with unexplained bleeding tendency.

Diagnosis of platelet function disorders: A standardized, rational, and modular flow cytometric approach

A high proportion of patients with mucocutaneous bleeding diathesis and suspected inherited or acquired platelet disorder remain without diagnosis even after comprehensive laboratory testing. Since flow cytometry allows investigation of resting and activated platelets on the single cell level by requiring only minimal amounts of blood, this method has become an important assay within the diagnostic algorithm, especially in pediatrics. We therefore developed a standardized and modular flow cytometric approach that contributes to clarify impaired platelet function in a rational step-by-step manner.Due to simultaneous analysis of four fluorophores in a basic panel design, we are able to readily detect the most common and clinically significant platelet disorders: Glanzmann thrombasthenia or Glanzmann-like diseases (fibrinogen receptor GPIIb-IIIa), Bernard–Soulier syndrome (von Willebrand-factor receptor complex GPIb-IX-V) and less well characterized β1-integrins that serve as the collagen, laminin or fibronectin receptor (CD29-CD49b, e and f, respectively). Platelet reactivity was investigated in response to the agonists adenosine diphosphate (ADP) and thrombin receptor activator peptide 6 (TRAP6) in suboptimal and optimal concentrations by quantifying surface expression of activation markers CD62P and CD63 as well as binding of PAC-1 antibody to the high affinity conformation of the fibrinogen receptor. For advanced diagnostic questions, several further modules were implemented: (i) calcium mobilization for evaluation of early signal transduction, (ii) a kinetically resolved mepacrine assay for estimation of delta-granule content and release, and (iii) a module to determine platelet reactivity upon additional agonists like the thromboxane A2-analogue U46619 or collagen. Blood withdrawn from a healthy control cohort allowed generating preliminary standard values for all parameters. The modules were validated by analysis of patients with known or suspected platelet defects (leukocyte-adhesion deficiency type III, Wiskott–Aldrich syndrome, acute myeloid leukemia, sickle cell disease and chronic immune thrombocytopenia).

Screening for platelet function disorders with Multiplate and platelet function analyzer

Light transmission aggregation (LTA) is the gold standard for the diagnosis of platelet function disorders (PFDs), but it is time-consuming and limited to specialized laboratories. Whole-blood impedance aggregometry (Multiplate) and platelet function analyzer (PFA) may be used as rapid screening tools to exclude PFDs. The aim of this study is to assess the diagnostic performance of Multiplate and PFA for PFDs, as detected by LTA.Data from preoperative patients, patients referred to the hematologist for bleeding evaluation, and patients with a diagnosed bleeding disorder were used. PFDs were defined as ≥2 abnormal LTA curves. Diagnostic performance of Multiplate and PFA for detecting PFDs was expressed as sensitivity and specificity. The ability of Multiplate agonists and PFA kits to detect corresponding LTA curve abnormalities was expressed as area under the receiver operating characteristic curve.Prevalence of PFDs was 16/335 (4.8%) in preoperative patients, 10/54 (18.5%) in referred patients, and 3/25 (12%) in patients with a diagnosed bleeding disorder. In preoperative and referred patients, the sensitivity of Multiplate and PFA for detecting mild PFDs varied between 0% and 40% and AUCs for detecting corresponding LTA curve abnormalities were close to 0.50. In patients with a diagnosed bleeding disorder, both assays could detect Glanzmann thrombasthenia (GT) with sensitivity of 100% and AUCs of 0.70–1.00.Multiplate and PFA cannot discriminate between preoperative and referred patients with and without mild PFDs, meaning that they cannot be used as screening tests to rule out mild PFDs in these populations. Both Multiplate and PFA can detect GT in previously diagnosed patients.

The coming of age of the megakaryocyte‐platelet

The coming of age of the megakaryocyte‐platelet

Testing for inherited platelet defects in clinical laboratories in Germany, Austria and Switzerland. Results of a survey carried out by the Permanent Paediatric Group of the German Thrombosis and Haemostasis Research Society (GTH)

Inherited platelet defects are a rare and heterogeneous group of disorders. The majority of affected patients present with mild to moderate bleeding tendencies. However, in trauma and surgery, bleeding may be difficult to control. Laboratory tests for diagnosis are necessary for the prevention and treatment of critical bleeding. The aim of the THROMKID study was to obtain information on the means of investigating platelet function employed by clinical centres in German-speaking countries. For this purpose a patterns-of-practice survey was conducted from 2005 to 2007, the results of which are reported here. A total of 37 out of 41 identified clinical centers serving 98 million people completed the survey questionnaire. The number of tests offered for assessment of platelet function varied between 1 and 11, median 4.Aggregometry continued to be the most popular and helpful technique for evaluation of suspected platelet function disorders (100%). The PFA-100® CT (76%) and in vivo bleeding time (54%) were used to screen patients with suspected platelet function disorders. Selection of tests was based on a case-by-case decision at most centres (82%). The majority of centres performed specific platelet function tests less than 50 times per month. This survey illustrates the preferences of clinical centres in the selection, performance and interpretation of platelet function tests. These practices may considerably influence the detection and diagnosis of platelet function disorders. There is an urgent need for existing tests to be improved and new, fast and reliable tests of platelet function to be developed.

Screening Tests of Platelet Function: Update on Their Appropriate Uses for Diagnostic Testing

Global tests of platelet function are often used as screening tests during the laboratory investigation of individuals with suspected hemostatic defects. Because global tests of platelet function do not enable specific diagnosis of platelet disorders, they are normally performed as the first part of a two-step strategy that requires further testing with more specialized assays of platelet function to confirm or refute the diagnosis. The most commonly proposed rationale for testing global platelet function as a first-line investigation is that normal test results may exclude a diagnosis of platelet function disorder so that further specialized testing can be avoided. For this reason, global platelet function tests are usually initially performed at the same time as global assays of coagulation pathway function (prothrombin time and activated partial thromboplastin time, von Willebrand factor screening tests [VWF:Ag, VWF:RCo, and FVIII:C]) and measurement of platelet number. The most widely performed tests for screening platelet function disorders are currently the template bleeding time and the closure time within the Platelet Function Analyzer. These tests will therefore be considered in detail in this review, and we will discuss the limitations of these methods in screening individuals for platelet disorders.

Consideration of platelet function disorders in patients with reduced VWF levels

We read with interest the comprehensive National Heart, Lung, and Blood Institute (NHLBI) guidelines for the diagnosis and management of von Willebrand disease (VWD) [1]. However, we would like to draw attention to a practically important diagnostic issue that we frequently encounter in our clinical practice and that can mislead the unwary. The considerable overlap between von Willebrand factor (VWF) levels in patients with VWD and healthy controls is well established [2]. Although the authors acknowledge the potential difficulties in attributing bleeding symptoms to lowered VWF levels, particularly in cases with modest reductions in VWF levels, no mention is made of the possibility of co-existing bleeding disorders, including defects in platelet function and their consideration in the investigation of patients with possible VWD. The clinical manifestations of platelet function disorders and VWD are interchangeable [1,3], and it is our experience, as a tertiary referral centre, that abnormalities of platelet function are not infrequently found in patients with reduced or borderline levels of VWF. We have 25 patients registered at this centre with dual defects of various kinds, at least half of whom have a diagnosis of both VWD and a defect in platelet function. While the population frequency of platelet function disorders is unknown, as we have 208 patients registered with platelet function disorders and 566 with VWD, it is not surprising that the combination turns up. Failure to recognize this would lead to inappropriate treatment. We are planning to undertake a systematic survey of all our patients to establish the frequency of platelet function disorder in VWD at this tertiary centre. In view of the low specificity of the platelet function analyser (PFA-100) test reported in mild VWD phenotypes, the authors do not support the routine use of such primary haemostatic screening in the investigation of VWD. High false-negative rates on PFA-100 analysis have also been described in patients with platelet function defects [4–5]. Furthermore, measurement of the bleeding time is also known to lack specificity and sensitivity in the diagnosis of these patients. It is our practice to perform platelet aggregometry studies and quantify platelet nucleotides at an early stage in the investigation of patients with a history of mucocutaneous bleeding and patients with a diagnosis of VWD in the absence of significantly lowered VWF levels. While the use of optical platelet aggregometry is limited to specialist laboratories, the development of impedance aggregometers (multiplate analysis) may widen the availability of these studies, and their validation in the diagnosis of platelet function disorders is required. In summary, the early consideration of platelet function disorders when investigating patients for VWD reduces the frequency of incorrectly attributing bleeding symptoms to mild reductions in VWF levels resulting in improved clinical management and outcomes in these patients.

Platelet Adrenergic Receptor Dysfunction Is Associated with Both Aggregation and Adhesion Defects: Application of the Cone and Plate(let) Analyzer for Diagnosis and Monitoring of the Response to DDAVP Therapy.

Diagnosis of platelet function disorders is based on the nature of bleeding symptoms, a positive family history and abnormal laboratory tests. However, currently available methods are time and labour consuming and lack sensitivity and specificity. In the present study we evaluated the performance of the Cone and Plate (let) analyzer (CPA) for screening patients with a suspected platelet function disorder and for monitoring therapy.Consecutive patients presenting with spontaneous or post surgical bleeding were studied. All patients underwent primary and secondary hemostasis evaluation including CPA testing. In this test a volume of 200μL of citrated whole blood is placed onto polystyrene wells and submitted to flow (shear rate of 1800 sec. −1) by a Teflon cone for 2 minutes. The resulting platelet deposition is then read by an image analyzer and expressed as % of the well surface covered ( SC, %) with adhered platelets and as the average size (AS, μm2)of platelet aggregates. Patients with a platelet function disorder were further tested with a modified CPA test in which blood samples are pre-incubated (1 min.) with a sub-optimal concentration of an agonist prior to subjecting the sample to the regular CPA test. Response of platelets to the agonist leads to agglutination and micro aggregates formation in the suspension phase of the test tube with consequent decline in platelet adhesion.165 consecutive bleeding patients were evaluated and compared to 54 healthy controls. We diagnosed 51 patients as suffering from a platelet function disorder, of whom 8 patients (GTA – 7, BSS – 1) were excluded. The remaining 43 patients presented complete unresponsiveness to epinephrine in repeated platelet aggregation tests while all other coagulation, liver and renal function tests were normal and similar to healthy controls. In the CPA test these 43 patients had a significantly lower SC values compared to the other patients or to healthy controls. To further explore the CPA's diagnostic capability 6 patients were compared to 16 randomly chosen controls using the modified CPA test. Whereas SC declined significantly by 73% in healthy controls after pre-incubation with epinephrine a non significant decline of only 15% was noted in patients with platelet adrenergic receptor dysfunction (table). Seventeen patients with platelet adrenergic receptor dysfunction underwent surgery. Following pre-surgical administration of DDAVP SC values followed closely and the VWF:RCo levels whereas the response to epinephrine in routine aggregometry did not change. All patients underwent successful operations without excessive bleeding.Our data suggest that patients with adrenergic receptor dysfunction suffer from a combined aggregation and adhesion platelet defects. The CPA test was found useful for screening both adhesion and aggregation of platelet disorders including adrenergic receptor dysfunction as well as for monitoring DDAVP therapy in these patients.Adrenergic receptor dysfunctionNo disorderHealthy controlsN4311454Bleeding time (min.)5.6±2.55.5±2.36.7±3.1Aggregomatry *23%82%81%SC%6.2±2.411.5±3.412.3±3.0Modifioed CPA-pre6.5±2.29.4±1.8Modified CPA-post**5.4±1.32.5±0.8* max. response to epinephrine in aggregometry ** 1 min. incubation with epinephrine