The PFA‐100® does not predict delta‐granule platelet storage pool deficiencies

Summary.  There are no published reports investigating the ability of the platelet function analyzer (PFA‐100^®) to detect the presence of delta‐granule platelet storage pool deficiencies (δ‐PSPD), a common mild bleeding disorder. Prior studies of the PFA‐100^® and congenital platelet disorders have been limited by small numbers of patients with a variety of disorders. We examined PFA‐100^® results in a large paediatric patient population diagnosed specifically with δ‐PSPD, and determined the relationship between PFA‐100^® and platelet electron microscopy (the gold standard for diagnosis). This study is a retrospective review of patients <19 years of age diagnosed with δ‐PSPD at Nationwide Children’s Hospital from 2008 to 2010. To examine the correlation between PFA‐100^® and average number of granules per platelet we used Spearman’s Rho as a non‐parametric measure of dependence. A total of 105 patients diagnosed with δ‐PSPD were included, of which 99 patients underwent PFA‐100^® testing. Of those tested 46% had at least one abnormal closure time, whereas 16% had abnormal results for both cartridges. We found no statistical correlation between C‐EPI closure time and average number of granules per platelet (ρ= ?0.0095, P‐value = 0.9328), nor between C‐ADP closure time and the average number of granules (ρ = 0.0315, P‐value = 0.7798). The PFA‐100^®, a widely used screening test for suspected bleeding disorders, did not correlate with presence or severity of δ‐PSPD as determined by platelet electron microscopy. When evaluating patients with suspected bleeding disorders, PFA‐100^® alone cannot be used to rule out the presence of a δ‐PSPD.     

Platelet aggregation measured by single-platelet counting and using PFA-100 devices

Platelets play a crucial role in haemostasis and thrombosis and evaluation of platelet function in vitro, in particular platelet aggregation responses, has been one of the most common and useful ways of evaluating the risk of bleeding and thrombotic events and assessing the effects of various compounds and conditions on platelets. Traditional approaches to assessing platelet aggregation require specialised equipment and trained laboratory personnel and have other limitations. Studying platelet aggregation in whole blood offers a more physiologically relevant measurement. Additionally, certain approaches could be more widely available than in specialised laboratories. Here we summarise the application of the platelet function analyser (PFA-100), an accessible first point-of-care test for platelet function in whole blood, and the less established, but promising approach of assessing platelet aggregation by single-platelet counting that can also be performed in whole blood. The possibilities of a wider and more accessible application of the latter methodology are also discussed.     

The Platelet Function Analyzer (PFA-100®): a novel in-vitro system for evaluation of primary haemostasis in children

The PFA-100^® system provides an in-vitro method of assessing primary platelet-related haemostasis by measuring the time (the closure time, or CT) taken for a platelet plug to occlude a microscopic aperture cut into a membrane coated with collagen and either epinephrine or ADP. We used the system to establish normal ranges for CTs in healthy children, adults and neonates. Mean CTs of healthy children were independent of the needle gauge used (21G or 23G) for blood sampling; they were very similar to the mean CTs of healthy adults, but longer than mean CTs of healthy neonates. Although children with haemophilia had normal CTs, the PFA-100 system was found to be potentially useful in screening for von Willebrand disease in children.     

Variables influencing Platelet Function Analyzer-100 closure times in healthy individuals

We investigated the relationship between platelet function analyzer (PFA-100) closure times (CT) and bleeding time (BT), platelet aggregation (PA) induced by ADP, arachidonic acid, and collagen, blood cell counts, and von Willebrand factor (VWF) in 120 well-characterised healthy individuals. Pre-analytical and analytical conditions were standardised comprehensively. In a substantial number of cases the differences between duplicate measurements exceeded 15%. The reference range (5th and 95th percentiles) for CT with the collagen/epinephrine (CEPI) and the collagen/ADP (CADP) cartridge was 93-223 s and 64-117 s respectively. Re-examination of 11 individuals with CEPI-CT above the 95th percentile revealed considerable batch-to-batch variation of CEPI-CT. Males had significantly longer CADP than females (P = 0.002). CEPI and CADP-CT measured pm were significantly longer than corresponding values determined am (P = 0.003 and P < 0.0001 respectively). Blood group O was associated with greater CEPI and CADP-CT and lower VWF levels compared with non-O blood groups (P = 0.008, P = 0.0003 and P < 0.0001 respectively). Linear regression analysis revealed association between CEPI-CT, CADP-CT and VWF (P < 0.0001), but no relationship was found between CT and BT or between CT and PA. We conclude that VWF plasma levels modulate PFA-100 CT to a greater extent than platelet function. Establishment of reliable reference ranges and careful standardisation of pre-analytical and analytical conditions is a prerequisite for obtaining reliable PFA-100 results. Duplicate measurements are necessary.     

The PFA‐100®: a potential rapid screening tool for the assessment of platelet dysfunction

The PFA‐100^® is a device that simulates high shear dependent platelet function in vitro and thus is particularly useful for screening for von Willebrand's disease (VWD). The aim of this study was to assess the overall potential of the PFA‐100^® as a primary clinical screening tool using the wide spectrum of clinical samples assessed for platelet function within our institution. The PFA‐100^® test was performed using both collagen/ADP (CADP) and collagen/epinephrine (CEPI) cartridges on samples from 337 patients with a wide variety of haemostatic defects. One hundred and eighty‐two patients were defined as having normal platelet function based on classical laboratory tests and von Willebrand factor levels. The overall clinical sensitivity of the PFA‐100^® for platelet abnormalities (including VWD) was 81% for CADP and 86% for CEPI. The overall specificity was found to be 82% for CADP and 80% for CEPI. When utilizing both cartridges in combination (with both results either higher or lower than the upper cutoff of the normal ranges), the overall false positive and false negative rates were 12% and 6%, respectively. The PFA‐100^® proved to be sensitive in detecting classical defects by giving prolonged closure times in samples from patients with major platelet function defects (e.g. von Willebrand's disease, Glanzmann's thrombasthenia and Bernard Soulier syndrome). However, there were a small number of false negative results (6%) obtained with various milder platelet defects (e.g. Hermansky Pudlak syndrome, storage pool and release defects, type I VWD and macrothrombocytopenia). The PFA‐100^® test provides a useful rapid screening tool and should increase the efficiency and reduce the cost of the routine diagnosis of platelet dysfunction.     

The Platelet Function Analyzer (PFA-100) may not be suitable for monitoring the therapeutic efficiency of von Willebrand concentrate in type III von Willebrand disease

 We describe a type-III von Willebrand patient who was admitted to the hospital with severe deformity and functional deficit of the left knee joint due to recurrent hemarthrosis. Orthopedic intervention was necessary. To prevent bleeding episodes, von Willebrand factor (vWF) replacement therapy was given during and after surgery. APTT, plasma FVIII activity (FVIIIc), vWF antigen (vWF Ag), and vWF ristocetin cofactor (vWF Rco) were measured. Primary hemostasis was monitored using the PFA-100. This "Platelet Function Analyzer" is designed to measure platelet adhesion and aggregation capacities. Whole blood is aspirated through a capillary and is forced to flow through the central hole of a membrane coated with collagen and epinephrine (COL/EPI) or ADP (COL/ADP) as platelet activators. Irreversible platelet aggregation results in the formation of a stable platelet plug, closing the central hole. The result is expressed as "closure time" (CT), i.e., time necessary to stop the blood flow, and is a measure of platelet hemostasis capacity. Laboratory investigations during substitution therapy revealed no shortening of closure times with both COL/EPI and COL/ADP cartridges despite normalization of plasma vWF Ag, vWF Rco, and FVIIIc levels. These observations suggest that intraplatelet vWF, which is totally absent in type-III von Willebrand disease, plays an important function in the adhesion of platelets to the collagen-coated membrane of the PFA-100 system, simulating an injured vessel wall. Consequently, we conclude that the PFA-100 may not be suitable for monitoring the therapeutic efficacy of von Willebrand concentrate in type-III von Willebrand patients during substitution therapy. Received: February 1, 1999 / Accepted: April 30, 1999     

Systemic inflammation increases shear stress-induced platelet plug formation measured by the PFA-100

The PFA-100 measures platelet plug formation under shear stress and is strongly dependent on von Willebrand Factor (VWF) levels in plasma. We therefore hypothesized that elevated VWF levels, possibly as a result of acute inflammation, adversely influence PFA-100 results. Healthy volunteers received either 2 ng/kg endotoxin or placebo in a randomized controlled trial. Four hours after endotoxin (but not placebo) infusion VWF levels increased by 85%, collagen epinephrine-induced closure time (CT) decreased by 47% and collagen ADP-CT decreased by 38% (P < 0.0001) respectively. In conclusion, systemic inflammation has a major impact on the results obtained by PFA-100 and may confound interpretation of platelet function.     

Assessment of the response to acetylsalicylic acid in patients with myeloproliferative neoplasms by whole blood assays: A comparison of the PFA-100 with multiple electrode aggregometry

Abstract

Since thrombotic and haemorrhagic complications are the most important causes of morbidity and mortality in myeloproliferative neoplasms (MPN), establishing valid techniques for the monitoring of antiaggregatory treatment would be beneficial. The aim of this study was to assess the aspirin responsiveness in patients with MPN by multiple electrode aggregometry (MEA) and the PFA-100, to determine the concordance rate between the two techniques and to examine a potential clinical impact. Twenty-two consecutive outpatients with polycythaemia vera and essential thrombocythaemia receiving long-time treatment with 100?mg of aspirin were included and clinically re-evaluated within six months after study entry. All subjects were identified as aspirin responders using the PFA-100, whereas only nine (41%) study participants were detected as responders by MEA. The difference in the response rates was statistically highly significant (p?<?0.0001). The median aggregation result was 55.5?U?(8–123) in the ASPI test, and the median PFA-100 closure time (CT) was 300?sec (221 to 300) in the COL-EPI test. Within the clinical observation period no thrombotic or haemorrhagic events occurred in the study population. In this study we concluded that MEA and the PFA-100 are suitable devices for the detection of a response to aspirin treatment in patients with MPN, but differ significantly in the response rates and thus show a low concordance rate.     

Platelet function analyser (PFA‐100) results and von Willebrand factor deficiency: a 16‐year ‘real‐world’ experience

The platelet function analyser (PFA‐100) is a biological tool designed to explore primary haemostasis. This system has thus been widely demonstrated as reliable in detecting von Willebrand factor (VWF) deficiency. However, most studies were based on patients benefitting from regular medical care and accurate diagnosis, and it would seem probable that the results were somewhat optimistic, and do not reflect its performances in ‘real‐world’ situations. We have chosen to study the reliability of PFA‐100 for screening VWF ristocetin cofactor (VWF:RCo) deficiency. We retrospectively analysed the results (n = 6431) of 4027 patients referred to our centre between October 1997 and June 2013 and in whom PFA‐Epi, PFA‐ADP, and VWF:RCo activity had been evaluated. We studied the influence of blood group on the results and the performances of each method in a subgroup of 213 patients with genetically confirmed von Willebrand disease. We have shown that the PFA‐100 system, in our experience, constitutes an excellent screening test for detecting VWF:RCo deficiency, whatever the clinical situation, in ‘real‐world’ conditions. The negative predictive value (NPV), the positive predictive value, the sensitivity and the specificity were respectively: 0.98, 0.51, 0.98 and 0.40. When values adjusted for blood group are used, NPV and sensitivity are inferior to those using normal values which have not been adjusted for blood group. We have shown the PFA‐100 method to be more efficient in screening for VWF deficiency than the VWF:RCo technique.     

The role of PFA-100 testing in the investigation and management of haemostatic defects in children and adults

The PFA-100 provides a simple global measure of high shear-dependent platelet function, and as such is not diagnostic or specific to any disorder. Prolonged closure times must be interpreted in conjunction with a full blood count, von Willebrand factor (VWF) screen and other platelet tests. The PFA-100 may also give false negative results with relatively common platelet defects. If clinical suspicion is high, further detailed platelet function testing and VWF screening are required to exclude abnormal platelet function, even if the PFA-100 is normal. In more recent studies the PFA-100 has been used for preoperative identification and management of surgical patients with haemostatic defects and for assessing the clinical effectiveness of platelet transfusion therapy. This review highlights the up to date, evidence-based, advantages and disadvantages of the PFA-100 test in the investigation and management of haemostatic disorders in both children and adults.     

血小板功能评价预测冠脉介入——臆度还是希望?

抗血小板治疗与经皮冠脉介入治疗患者临床结果密切相关,药物剂量和作用时间直接影响患者预后,而患者对血小板治疗的反应多变,能否制定出个体化的最佳治疗方案,或许血小板功能的研究会给我们带来希望。     

Utility of the PFA-100® for assessing bleeding disorders and monitoring therapy: a review of analytical variables, benefits and limitations

The PFA-100 (platelet function analyser) is a relatively new tool for the investigation of primary haemostasis. Recent studies have shown its utility in monitoring antiplatelet therapy (including aspirin) and as a screening tool for investigating possible von Willebrand disease (vWD) and various platelet disorders. More recently, the PFA-100 has been shown to be of value in monitoring DDAVP therapy in both vWD and platelet disorders. This paper reviews current findings, details the utility of the PFA-100 for some of these purposes, as well as reviewing analytical variables that may complicate the interpretation of results. The author highlights the benefits, as well as noting the limitations, of its use. Ultimately, the greatest strengths of the PFA-100 are its simplicity in use and excellent sensitivity to particular haemostatic disturbances such as vWD, platelet disorders and platelet-affecting medication. However, because it is thus a 'global' test system, this also creates a significant limitation, as the PFA-100 is not specific for, nor predictive of, any particular disorder. However, utilized appropriately, the PFA-100 can be considered a worthwhile addition to any haemostasis laboratory involved in the diagnosis or therapeutic-monitoring of bleeding disorders including vWD and platelet-dysfunctions. This review should be of value to both haemostasis scientists and clinical specialists.     

Standardization of the PFA-100® platelet function test in 105 mmol/l buffered citrate: effect of gender, smoking, and oral contraceptives

The PFA-100(R) (PFA) diagnostic system for the detection of platelet dysfunction was evaluated to determine reference ranges in a normal population. The PFA determines the primary haemostasis capacity (PHC) of anticoagulated whole blood, expressed by the system's closure time (CT). In this study the CT reference ranges were determined for blood samples collected in 105 mmol/l (3.2%) buffered citrate and the effect of gender, smoking, and use of oral contraceptives on reference ranges was assessed. Each of the 309 healthy blood donors from five blood centres was confirmed to have normal platelet function before inclusion in the study. Blood samples were tested in duplicate with both the collagen/epinephrine (Col/Epi) and collagen/ADP (Col/ADP) test cartridges. PFA reference ranges (90% central intervals of measured closure times) for both cartridge types were similar for all groups. Subgroup analysis showed that neither gender nor oral contraceptive usage had any effect on PHC. The 95% cut-off value for the Col/Epi CT was slightly higher for smokers than for non-smokers, an effect more pronounced in female than in male donors. However, the small difference did not justify establishment of specific reference ranges for smokers. Data from all included subjects were pooled to calculate the CT reference ranges for blood samples collected in 105 mmol/l buffered citrate (Col/Epi 82-150 s; Col/ADP 62-100 s). Normal levels of fibrinogen, as well as normal platelet counts and normal haematocrit levels, appeared not to influence the PHC. Because slight but significant differences of the reference ranges were observed between some of the participating sites, in-house confirmation of these reference range guidelines is recommended.     

Long term follow-up after splenectomy performed for immune thrombocytopenic purpura (ITP)

Splenectomy is the only treatment of ITP known to have "curative" effects in a substantial fraction of patients. However, the true long-term outcome is uncertain and controversial because published series have not adjusted for the duration of follow-up. This IRB-approved retrospective study included all patients with ITP who underwent splenectomy between 1988-1993 at three major medical centers and required a minimum postoperative 5-year follow-up. Complete response (CR) was defined as all postsplenectomy platelet counts >150 x 10(9)/L without treatment; partial response (PR) as platelet counts > or =50 x 10(9)/L without treatment; and failure as platelet counts <50 x 10(9)/L or receiving therapy after splenectomy. Seventy-five patients identified with ITP underwent splenectomy from 1988 to 1993. Three patients died prior to 5-year follow-up, and 56 of the 72 patients (78%) were evaluable with follow-up for five years or longer, median 7.5 years. The immediate postoperative complete remission rate was 77%; 57% of patients have remained in prolonged CR. Thirty-seven patients (66%) have not required any therapy after splenectomy. Eight patients had platelet counts >150 x 10(9)/L for 4-8.5 years before relapsing; no clear plateau was attained in the remission curve. There was no operative mortality. Ten patients (18%) reported minor postoperative bleeding episodes. No life-threatening infections, significant heart disease, or pulmonary hypertension developed after splenectomy in the 434 patient-years of follow-up. This study helps to define the long-term results of splenectomy for ITP.     

Abnormal Platelet Function in a Case of Megakaryoblastic Leukaemia

A platelet function study in a patient with megakaryoblastic leukaemia is reported. The abnormalities of the platelet function suggest a probable platelet membrane injury and a platelet release defect. The reduced platelet half-life and the non changing splenohepatic ratio confirm the clinical and histological features of the systemic disease.     

The Assessment of the Platelet Function During the Acute Phase of ST-segment Elevation Myocardial Infarction in Essential Thrombocythemia

We encountered a case of ST-segment elevation myocardial infarction (STEMI) as the first clinical manifestation of essential thrombocythemia (ET). Platelet function tests revealed high thrombogenicity during primary percutaneous coronary intervention compared with general cardiovascular patients, whereas the platelet function two weeks after admission was effectively suppressed by dual antiplatelet therapy. The patient, who lacked cytoreduction, suffered from recurrent STEMI because of poor compliance with antiplatelet drugs. The risk of acute coronary occlusion may be high during the acute phase of STEMI in ET patients because of high thrombogenicity. Insufficient antiplatelet therapy and no cytoreduction are also risk factors for recurrent coronary events.