Usefulness of PFA-100 Ò testing in the diagnostic screening of patients with suspected abnormalities of hemostasis: comparison with the bleeding time

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1 Journal of Thrombosis and Haemostasis, 5: ORIGINAL ARTICLE Usefulness of PFA-100 Ò testing in the diagnostic screening of patients with suspected abnormalities of hemostasis: comparison with the bleeding time G. M. PODDA,* P. BUCCIARELLI,* F. LUSSANA, A. LECCHI* and M. CATTANEO *Dipartimento di Medicina e Specialità Mediche, IRCCS Fondazione Ospedale Maggiore, Mangiagalli e Regina Elena; and Unità di Ematologia e Trombosi, Ospedale San Paolo, Dipartimento di Medicina, Chirurgia e Odontoiatria, Università di Milano, Milan, Italy To cite this article: Podda GM, Bucciarelli P, Lussana F, Lecchi A, Cattaneo M. Usefulness of PFA-100 â testing in the diagnostic screening of patients with suspected abnormalities of hemostasis: comparison with the bleeding time. J Thromb Haemost 2007; 5: Summary. Background: Global tests of hemostasis that are used to screen patients with clinical suspicion of bleeding disorders should help the physician to identify the phase of the hemostatic system that is abnormal and guide further diagnostic workup. Patients and methods: We compared the performance of Platelet Function Analyzer-100 (PFA-100 Ò ) closure time (CT) with bleeding time (BT), both of which are screening tests for primary hemostasis, in the diagnostic workup of 128 consecutive patients who were screened for bleeding disorders. The sensitivities of BT and PFA-100 CT for known defects of hemostasis were evaluated; in addition, we calculated their correlation with the levels of severity of the bleeding symptoms, which were recorded using a standardized questionnaire. Results: The sensitivity of PFA-100 testing was 71% for von Willebrand disease (VWD) [with both collagen adenosine diphosphate (C-ADP) and collagen epinephrine (C-EPI) cartridges]; 58% (C-EPI) and 8% (C- ADP) for platelet function disorders (PFDs); and the sensitivity of BT was 29% (VWD) and 33% (PFD). C- EPI CT was also prolonged in about 20% of patients with abnormalities of coagulation or fibrinolysis. Only the C-EPI CT was significantly associated with the levels of severity of the patientsõ bleeding scores. Conclusions: BT and C-EPI are insufficiently sensitive to be recommended as hemostasis screening tests. The C-ADP cartridge, which is sensitive to VWD only, might prove useful in further diagnostic workup of defects of primary hemostasis. The association of C-EPI CT with the severity of bleeding symptoms as a useful predictor of risk of bleeding in clinical practise should be tested in properly designed studies. Correspondence: Marco Cattaneo, Unità di Ematologia e Trombosi, Ospedale San Paolo, Università di Milano, Via di Rudinì 8, Milan, Italy. Tel./fax: ; marco.cattaneo@unimi.it Received 17 April 2007, accepted 29 August 2007 Keywords: bleeding time, PFA-100, platelet function disorders, von Willebrand disease. Introduction The screening of patients with bleeding disorders is based on a two-step diagnostic strategy. In step 1, after recording information from the bleeding history and physical examination, tests of global hemostasis are performed. These tests help the physician to identify the phase of the hemostatic system that is abnormal. In step 2, specific tests are performed that study the phase of hemostasis that is suspected to be abnormal in more detail, based on the results of step 1. This strategy aims to limit the number of specific tests that are employed in the diagnostic workup of the patient, because they are usually complex and expensive. Step-1 screening tests should be simple, relatively inexpensive and sensitive to most known disorders of hemostasis. Traditionally, they include two tests that explore the coagulation system, the prothrombin time (PT) and the activated partial thromboplastin time (APTT), and one test that explores primary hemostasis: the bleeding time (BT). PT and APTT are indeed very helpful for diagnosing patients with abnormalities of the coagulation system, not only because they are accurate and highly reproducible but also because they help to identify the coagulation pathway in which the abnormality is likely to be found. In contrast, the usefulness of BT for the diagnostic workup of patients with defects of primary hemostasis is questionable. It is an invasive and poorly reproducible technique (and should therefore be carried out by experienced personnel), its sensitivity seems unacceptably low and it is of no help in differentiating among the various types of defects of primary hemostasis [1,2]. As a matter of fact, because BT can be equally prolonged in von Willebrand disease (VWD) and platelet function defects, it is of no help in differentiating between these two common disorders of primary hemostasis. These last drawbacks of BT created the need for new, in vitro tests that could substitute for BT in the diagnosis of defects of primary hemostasis. The Platelet Function Analyzer-100 (PFA-100 Ò, Dade Behring, Marburg, Germany)

2 2394 G. M. Podda et al was proposed as a useful screening test for defects of primary hemostasis, because it is simple, non-invasive, reproducible and does not require specialized personnel [3,4]. In several published studies, PFA-100 proved to be more sensitive than BT to type-1 VWD [5 8], which usually causes mild abnormalities of primary hemostasis and is associated with mild bleeding diathesis. In contrast to its sensitivity to VWD, PFA-100 is poorly sensitive to platelet secretion defects [9,10], which represent the most common abnormalities of platelet function [11]. Many published studies comparing PFA-100 with BT were performed in selected patients whose diagnosis had previously been established. None of the studies evaluated the association of these tests with the severity of the bleeding history. We compared the sensitivity of BT with the sensitivity of PFA-100 in 128 consecutive patients who had been referred to our outpatient clinic to be screened for bleeding disorders, and we evaluated the correlation of the two tests with the severity of the bleeding history. Patients and methods Patients and controls One-hundred and twenty-eight patients (57 men, 71 women, median age 32 years, range 4 87 years) were referred to the A. Bianchi Bonomi Hemophilia and Thrombosis Centre from June 2002 through June 2003 in order to be screened for hemostasis disorders due either to previous bleeding episodes (n = 90) or to the occasional finding of abnormal hemostasis tests (n = 38). This study was approved by the institutional review board and all participants provided informed consent. Evaluation of the severity of the bleeding history The severity of the bleeding history was evaluated in all patients using a standardized questionnaire, which was based on the one reported by Sramek et al. [12], with slight modifications. The questionnaire was filled out by a physician from our outpatient clinic, during the medical interview of the patients. The score was calculated and recorded by the same physician, who was not involved in the subsequent diagnostic workup of the patient. Table 1 lists the types of bleeding episodes that were included in the questionnaire. Patients were assigned 1 point per every type of bleeding manifestation and additional points (up to a maximum of 5 points per each type, as indicated in Table 1), depending on its nature (spontaneous vs. posttraumatic or following surgical procedures), severity, duration, localization and frequency. The sum of all the points assigned to each patient represented his/her global bleeding severity score. Seventy-one individuals (24 men, 47 women, median age 31.5 years, range years) with a negative history for abnormal bleedings were enrolled as healthy controls in order Table 1 Types of bleeding episodes and additional items that were considered in the standardized questionnaire used to evaluate the severity of the bleeding history in patients enrolled in the study (slightly modified from Srameck et al. [12]) Type of bleeding episode (items considered to assign additional points) Bleeding after tooth extraction 5 (frequency, duration, recurrence, therapy) Bruising (frequency, size, location, causes) 5 Nosebleeds (frequency, location, causes, therapy) 5 Bleeding after surgery (duration, hematoma, therapy) 4 Hemarthrosis (frequency, causes, therapy) 4 Hematoma (frequency, causes, therapy) 4 Bleeding after tonsillectomy (duration, therapy) 3 Gumbleeds (causes, severity) 3 Profuse menstruation (duration, therapy) 3 Profuse bleeding after delivery (frequency, therapy) 3 Intracranial bleeding (causes) 2 Bleeding after trivial wounds, blood 1 in feces or urine, familiarity to establish the normal range of PFA-100. They were enrolled from amongst laboratory personnel and friends or partners of the index patients. None of the patients and healthy controls had been taking drugs known to affect hemostasis during the preceding 10 days. Hemostasis tests Maximum points Patients were assigned 1 point per every type of bleeding episode and additional points (up to a maximum of 5 points per each type), depending on its nature (spontaneous vs. post-traumatic or following surgical procedures), severity, duration, localization and frequency, which were predefined for each bleeding episode. The sum of all the points assigned to each patient represented his/her global bleeding severity score. Blood collection Blood collected in buffered 129 mm trisodium citrate (9:1 v/v) was used as such in the PFA-100 system. Blood samples for all other hemostasis measurements were collected in 129 mm trisodium citrate (9:1 v/v) and centrifuged at 1200 g at 20 C for15mintoobtainplateletpoor plasma or at 130 g at 20 C for 15 min to obtain plateletrich plasma. The PFA-100 system The PFA-100 system creates an artificial vessel consisting of a sample reservoir, a capillary and a biologically active membrane with a central aperture, coated with collagen plus adenosine diphosphate (C-ADP) or collagen plus epinephrine (C-EPI). The application of a constant negative pressure aspirates the anticoagulated blood of the sample from the reservoir through the capillary (mimicking the resistance of a small artery) and the aperture (mimicking the injured part of the vessel wall). A platelet plug forms that gradually occludes the aperture; as a consequence, the blood flow through the aperture gradually decreases and eventually stops. The time required to interrupt blood flow [Ôclosure timeõ (CT)] is recorded.

3 PFA-100 Ò in the diagnostic screening of bleeding disorders 2395 Bleeding time BT was measured with modified Ivy technology and performed on the volar forearm using Simplate II (General Diagnostics, Milan, Italy). For ethical reasons, BT was not performed in healthy volunteers. Other hemostasis tests First-step screening tests of hemostasis included PT, APTT and platelet count. Further tests exploring hemostasis were performed after the screening tests, when indicated. Platelet agglutination induced by ristocetin (Mascia Brunelli, Milan, Italy) and platelet aggregation and secretion induced by ADP, collagen, U46619, and the thrombin receptor (proteaseactivated receptor-1) activating peptide (Sigma, St Louis, MO, USA) in the presence of luciferine/luciferase reagent were measured in a lumiaggregometer (Lumi-aggrometer, Chrono-log Corp., Havertown, PA, USA). Further tests exploring platelet function disorders (PFDs) included measurements of serum thromboxane B 2 levels and platelet adenine nucleotides, serotonin and fibrinogen [13]. Plasma VWF was measured using a home-made ELISA for VWF antigen and an aggregometric assay with formalin-fixed platelets for VWF ristocetin cofactor [14]. The plasma levels of factor (F) VIII, FIX, FXI, FV, FVII, FX and FXII were measured by one-stage clotting assays, using congenitally deficient plasma. The concentration of FXIII-A in plasma was determined by an ELISA assay [15]. Fibrinolytic parameters, including the activities of plasminogen activator inhibitor type-1 (PAI-1), tissue plasminogen activator and antiplasmin, were determined as previously described [16]. The presence of lupus-like anticoagulant was assessed according to the guidelines issued by the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis [17]. Diagnoses of VWD types and subtypes and of PFDs were carried out according to established criteria [9,11,18]. Prothrombin fragment (F 1+2 ) was measured using a commercially available kit (Dade Behring). Statistical analysis Data from laboratory tests and bleeding scores were analyzed by two authors (G. M. P. and F. L.), who were not involved in the clinical and laboratory evaluation of the patients. Values were expressed as medians and ranges. Differences among groups were assessed using a non-parametric test for independent samples (Kruskal WallisÕ test), while the Wilcoxon test was performed to compare means of paired samples. The non-parametric v 2 -test was used to determine the significance for bivariate tabular analysis. The upper limits of the normal ranges of PFA-100 CTs were set at the 95th percentiles of distribution of values obtained in healthy controls: s for C-EPI and s for C-ADP. The upper limit of normal range for the BT in our institution is set at 8:00 min. SPSS for Windows version 12.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Results As shown in Table 2, VWD was diagnosed in seven patients (6%; five type 1 and two type 2A); PFDs in 12 (9%; eight primary secretion defect, three d-storage pool deficiency and one Glanzmann thrombasthenia); defects of clotting factors in 22 (17%; FVII, n =6;FVIII,n = 4; multiple factors, n =7; FV, FIX, FX, FXI and FXIII, n = 1 each); deficiency of PAI- 1 in six (5%); and abnormalities that affect PT or APTT, but do not usually cause bleeding diathesis, in 18 (14%; defect of FXII, defect of high-molecular-weight kininogen, presence of lupus anticoagulant). With the exception of patients with lupus anticoagulant, all abnormalities of hemostasis that were diagnosed in the studied patients were most likely to be congenital, as the presence of inhibitory antibodies was ruled out and no patient was affected by diseases known to be associated with acquired VWD or PFD. No known abnormalities of the hemostatic system were diagnosed in 63 patients (49%). Of these, seven had been referred to our centre for occasional findings of mild prolongation of PT or APTT: because the presence of these abnormalities of hemostasis tests was not confirmed and their history of abnormal bleedings was silent, these patients were considered normal and additional screening was not performed. Of the remaining 56 patients with a wide range of bleeding severity scores (from 0 to 17), 20 were screened for both VWD and PFDs and 15 were screened for VWD only. The remaining 21 refused further testing. Sensitivity of PFA-100 and BT for abnormalities of hemostasis Table 3 summarizes the sensitivity of PFA-100 and BT for the bleeding disorders that were diagnosed in the cohort of patients enrolled in the study. In general, PFA-100 CT was more sensitive than BT for VWD and PFDs. C-ADP and C-EPI cartridges were both sensitive to VWD (sensitivity 71% for both cartridges), while only the C-EPI cartridge was sensitive to PFDs (sensitivity 58%), with C-ADP giving essentially normal CTs in most of these patients. In particular, both C-ADP and C-EPI CTs were prolonged in the only patient with the severe PFD Glanzmann thrombasthenia, while among the 11 patients with milder defects (primary secretion defect and storage pool deficiency), six had prolonged C-EPI CT (sensitivity 55%) and none had prolonged C-ADP CT (sensitivity 0%). Table 2 Abnormalities of hemostasis that were diagnosed in the patients enrolled in the study Diagnosis No. of patients (%) von Willebrand disease 7 (6) Platelet function disorders 12 (9) Defects of clotting factors or fibrinolytic factors 28 (22) Abnormalities of laboratory tests 18 (14) not associated with bleeding risk* No known abnormalities 63 (49) Total 128 (100) *Lupus anticoagulant and defects of the contact phase of coagulation

4 2396 G. M. Podda et al Table 3 Sensitivity of collagen epinephrine (C-EPI) closure time (CT), collagen adenosine diphosphate (C-ADP) CT and bleeding time (BT) for abnormalities of hemostasis Sensitivity(%) von Willebrand disease C-EPI (71) C-ADP (71) BT (29) Platelet function disorders C-EPI (58) C-ADP (8) BT (33) Defects of clotting factors or fibrinolytic factors Abnormalities of laboratory tests not associated with bleeding risk* C-EPI (21) C-ADP (4) BT (4) C-EPI (22) C-ADP (6) BT (17) Unknown abnormalities C-EPI (11) C-ADP (10) BT (6) The upper limits of the normal ranges of the PFA-100 Ò CTs were set at the 95th percentiles of distribution of values obtained in healthy controls: s for C-EPI and s for C-ADP. The upper limit of normal range for the BT in our institution is set at 8.00 min. Unexpectedly, C-EPI CT was also prolonged in some patients with clotting factor deficiency (Table 3). This finding raised the hypothesis that the passage of normal blood through the pore in the PFA-100 C-EPI cartridge could generate trace amounts of thrombin (despite the presence of citrate anticoagulant), which would contribute to the formation of the occluding platelet thrombus, and that a defect of this component in patients with clotting factor deficiency may account for their prolonged CTs. However, this hypothesis was not supported by the findings that, in 35 individuals with normal levels of clotting factors, the plasma levels of F 1+2 in the effluent blood from C-EPI and C-ADP were not significantly different (C-EPI 0.97 nmol L 1 ± 0.30, C-ADP 0.94 nmol L 1 ±0.29, P = 0.194) and were lower than those in baseline blood (1.24 nmol L 1 ±0.31,P <0.001). Association of the bleeding severity score with PFA-100 CT and BT In our patient population the bleeding scores ranged between 0 and 19 (median 5; Fig. 1). We divided the patients into four groups, according to the severity of their bleeding scores, which were arbitrarily termed Ôvery mildõ (score 0 3, 41 patients), No. of patients ÔmildÕ (score 4 7, 53 patients), ÔmoderateÕ (score 8 11, 22 patients) and ÔsevereÕ (score 12, 12 patients). Most of the patients with VWD or PFDs were in the ÔmildÕ and ÔmoderateÕ groups, while some patients with other abnormalities of hemostasis also had Ôvery mildõ bleeding scores. We then tested whether or not there was an association between the severity of the bleeding score and the extent of prolongation of the BT or PFA-100 CT. The median C-EPI CT progressively increased from the Ôvery mildõ score group (130 s) to the ÔsevereÕ score group (153 s, P = 0.004, Kruskal WallisÕ test), while the median C-ADP CT and BT did not significantly differ among the four groups (Table 4). Prolonged C-EPI CT, C-ADP CT and BT were found in eight (20%), six (15%) and two (5%) patients, respectively, in the Ôvery mildõ score group; in 11 (21%), 12 (23%) and five (9%) patients in the ÔmildÕ score group; in six (27%), five (23%) and five (23%) patients in the ÔmoderateÕ score group; and in four (33%), three (25%) and two (17%) patients in the ÔsevereÕ score group (v , P = for C-EPI; v , P =0.25 for C-ADP; v , P = 0.19 for BT). Discussion Bleeding severity score Fig. 1. Distribution of the bleeding severity scores in the 128 patients enrolled in the study. In this study, we compared the performances of BT and PFA- 100 as screening tests in 128 consecutive patients who were referred to our outpatient clinic in order to undergo diagnostic screening for hemostasis abnormalities, due either to previous bleeding episodes or to the occasional finding of abnormal hemostasis tests. In addition, we evaluated the correlation of Table 4 Median (ranges) of collagen epinephrine (C-EPI) closure time (CT), collagen adenosine diphosphate (C-ADP) CT and bleeding time (BT) as function of the bleeding severity score Quartile of bleeding severity score Very mild (0 3) Mild (4 7) Moderate (8 11) Severe (12 19) P for trend Patients (n) C-EPI CT (s) 130 (85 244) 142 (88 300) 152 (74 300) 153 (95 300) C-ADP CT (s) 85 (67 179) 89 (57 182) 89 (59 100) 93 (73 207) BT (min) 5.3 (3.0 10) 5.0 ( ) 5.0 ( ) 4.8 ( ) 0.870

5 PFA-100 Ò in the diagnostic screening of bleeding disorders 2397 PFA-100 CT and BT with the severity of the bleeding history, which was determined using a standardized questionnaire. Confirming the results of previous studies of historical patients and a similar study of consecutive patients [19 22], we found that PFA-100 was more sensitive than BT to abnormalities of primary hemostasis due either to VWD or to PFDs. Both C-EPI and C-ADP cartridges of PFA-100 gave abnormal results in 71% of VWD patients. In contrast, only the C-EPI cartridge was sensitive to abnormalities of platelet secretion (prolonged in 55% of patients), while the C-ADP cartridge gave normal results. The lack of sensitivity of the C-ADP cartridge to defects of platelet secretion is probably because of the presence of ADP on the cartridge surface. By interacting with its platelet receptors, this can compensate for the lack of secreted ADP in pathologic platelets, which amplifies the aggregation of normal platelets and is essential for the formation of a normal hemostatic plug. Based on the results of our study and previous reports [19 22], we think that the use of PFA-100 for the initial screening of patients with clinical suspicion of bleeding disorders should not be recommended. Although the sensitivity of PFA-100 to VWD Patients with clinical suspicion of defect of primary hemostasis PFA-100 C-ADP closure time Prolonged Screening for VWD No VWD Screening for PFD normal Screening for VWD and PFD Fig. 2. Proposed diagnostic algorithm for further evaluation in the studies of the potential utility of the Platelet Function Analyzer-100 (PFA-100 Ò ) in patients with clinical suspicion of abnormalities of primary hemostasis. The use of the collagen epinephrine (C-EPI) cartridge of PFA-100 is not recommended, because, albeit more sensitive than the bleeding time (BT), it is insufficiently sensitive to both von Willebrand disease (VWD; sensitivity about 70%) and the most common, mild platelet function disorders (PFDs; sensitivity about 50%). As a consequence, patients with clinical suspicion of defects of primary hemostasis should undergo screening for VWD and PFDs even if their C-EPI closure times (CTs) are normal. In the algorithm shown, the use of the collagen adenosine diphosphate (C-ADP) cartridge PFA-100 is proposed as an aid to differentiate between VWD and PFD. This is based on the consideration that this cartridge gives prolonged CTs in most patients with VWD, while it gives normal results in patients with the most common PFDs, which are characterized by abnormalities of platelet secretion, such as primary secretion defects and d-storage pool deficiency. When VWD is ruled out, patients with prolonged C-ADP CT should undergo platelet function tests to diagnose the very rare and severe platelet function disorders, such as Glanzmann thrombasthenia, in which C-ADP CT is usually prolonged. Whether or not this diagnostic approach will indeed cost-effectively reduce the number of laboratory tests and the time to diagnosis in the diagnostic workup of these patients should be tested in ad hoc studies. defects of primary hemostasis, and in particular to VWD, is much higher than that of BT, we think that it is still unsatisfactory. In fact, about 30% of patients with VWD and 50% of patients with PFDs would be classified as normal based on the results of PFA-100. Therefore, patients with clinical suspicion of defects of primary hemostasis should be studied using specific diagnostic tests for VWD and PFDs, independently of the results of PFA-100. Although PFA-100 is of very little help in identifying which patients need further diagnostic testing for defects of primary hemostasis, it could still be useful in guiding the physician in the diagnostic workup of these patients. As a matter of fact, the use of the C-ADP cartridge might help in differentiating patients with VWD from patients with mild PFDs, because, as already mentioned, the C-ADP CTs are normal in platelet secretion defects and prolonged in most patients with VWD. We speculate that patients with clinical suspicion of defects of primary hemostasis should undergo an initial screening with the C-ADP cartridge of PFA-100 (Fig. 2). In the case of prolonged C-ADP CT, patients should be studied with specific diagnostic tests for VWD, without undergoing tests of platelet function; when VWD is ruled out, patients should undergo platelet function tests to diagnose the very rare and severe PFDs, such as Glanzmann thrombasthenia, in which C-ADP CT is usually prolonged (Fig. 2). Whether or not this diagnostic approach will indeed cost-effectively reduce the number of laboratory tests and the time to diagnosis in the diagnostic workup of these patients should be tested in ad hoc studies. Based on the results of our study, we think that the use of the C-EPI cartridge in the diagnosis of defects of primary hemostasis is unjustified, not only because of its insufficient sensitivity and inability to differentiate between VWD and PFDs but also because of its low specificity, as it gave abnormal results in about 20% of patients with defects of clotting factors. The reasons for the unexpected prolongation of the C-EPI CT in patients with defects of clotting factors are presently unknown. The hypothesis that trace amounts of thrombin, formed in citrated blood during its flow through the C-EPI cartridge, could contribute to the formation of the occluding platelet thrombus was not supported by the finding that the concentration of F 1+2 was similar in effluent blood from C- EPI and C-ADP cartridges. However, absorption of F 1+2 on the surface of the cartridges, which is most likely to account for the observed reduction in effluent blood compared with baseline blood, does not allow firm conclusions on this issue. A potential useful application of global tests of primary hemostasis would be in the prediction of the bleeding risk [23 27]. To the best of our knowledge, our study is the first to demonstrate that the C-EPI cartridge CT correlates with the severity of bleeding history. We evaluated the severity of bleeding history using a standardized questionnaire based on the one published by Sramek et al. [12], which has many similarities with that recently validated by Rodeghiero et al. [28]. In contrast to C-EPI CT, C-ADP CT and BT did not correlate with the severity of the bleeding history. Whether or not this property of C-EPI will be useful in clinical practise in

6 2398 G. M. Podda et al order to predict the risk of bleeding in patients at risk should be tested in properly designed, prospective studies. Disclosure of Conflict of Interests The authors state that they have no conflict of interest. References 1 Rodgers RPC, Levin J. A critical reappraisal of the bleeding time. Sem Thromb Haemost 1990; 16: Cattaneo M. Are the bleeding time and PFA-100 useful in the initial screening of patients with mucocutaneous bleedings of hereditary nature? JThrombHaemost2004; 2: Kundu SK, Heilmann EJ, Sio R, Garcia C, Davidson RM, Ostgaard RA. Description of an in vitro platelet function analyzer PFA- 100 TM. Sem Thromb Hemost 1995; 21: Kundu SK, Heilmann E, Sio R, Garcia C, Ostgaart R. Characterization of an in vitro platelet function analyzer, PFA-100 TM. Clin Appl Thromb Hemost 1996; 2: Fressinaud E, Veyradiers A, Truchaud F, Martin I, Boyer-Neumann C, Trossaert M, Meyer D. Screening for von Willebrand disease with a new analyzer using high shear stress: a study of 60 cases. Blood 1998; 91: CattaneoM,FedericiAB,LecchiA,AgatiB,LombardiR,StabileF, Bucciarelli P. Evaluation of the PFA-100 Ò system in the diagnosis and therapeutic monitoring of patients with von Willebrand disease. Thromb Haemost 1999; 82: Favaloro EJ, Facey D, Henniker A. Use of a novel platelet function analyzer (PFA-100 TM ) with high sensitivity to disturbances in von Willebrand Factor to screen for von WillebrandÕs Disease and other disorders. Am J Hematol 1999; 62: Dean J, Blanchette V, Carcao M, Stain A, Sparling C, Siekmann J, Turecek P, Lillicrap D, Rand M. Von Willebrand disease in a pediatric-based population comparison of type1 diagnostic criteria and use of the PFA-100 and a von Willebrand factor/collagen-binding assay. Thromb Haemost 2000; 84: CattaneoM,LecchiA,AgatiB,LombardiR,ZighettiM.Evaluation of platelet function with the PFA-100 system in patients with congenital defects of platelet secretion. Thromb Res 1999; 96: Hayward CP, Harrison P, Cattaneo M, Ortel TL, Rao AK; The Platelet Physiology Subcommittee of the Scientific and Standardization Committee of the International Society on Thrombosis And Haemostasis. Platelet function analyzer (PFA)-100 closure time in the evaluation of platelet disorders and platelet function: reply to a rebuttal. JThrombHaemost2006; 6: Cattaneo M. Inherited platelet-based bleeding disorders. J Thromb Haemost. 2003; 1: Sramek A, Eikenboom JC, Briet E, Vandenbroucke JP, Rosendaal FR. Usefulness of patient interview in bleeding disorders. Arch Intern Med 1995; 155: Balduini CL, Cattaneo M, Fabris F, Gresele P, Iolascon A, Pulcinelli FM, Savoia A; Italian Gruppo di Studio delle Piastrine. Inherited thrombocytopenias: a proposed diagnostic algorithm from the Italian Gruppo di Studio delle Piastrine. Haematologica 2003; 88: Federici AB, Stabile F, Castaman G, Canciani MT, Mannucci PM. Treatment of acquired von Willebrand syndrome in patients with monoclonal gammopathy of uncertain significance: comparison of three different therapeutic approaches. Blood 1998; 92: Katona EE, Ajzner E, Toth K, Karpati L, Muszbek L. Enzyme-linked immunosorbent assay for the determination of blood coagulation factor XIII A-subunit in plasma and in cell lysates. J Immunol Methods 2001; 258: Tripodi A, Moia M, Bottasso B, Tenconi PM, Gianese F, Mannucci PM. Effects of subcutaneously administered dermatan sulfate (MF 701) on the coagulation and fibrinolytic parameters of healthy volunteers. Thromb Res 1991; 62: Brandt JT, Triplett DA, Alving B, Scharrer I. Criteria for the diagnosis of lupus anticoagulants: an update. On behalf of the Subcommittee on Lupus anticoagulant/antiphospholipid Antibody of the Scientific and Standardization Committee of the ISTH. Thromb Haemost 1995; 74: Castaman G, Federici AB, Rodeghiero F, Mannucci PM. Von WillebrandÕs disease in the year 2003: towards the complete identification of gene defects for correct diagnosis and treatment. Haematologica 2003; 88: Favaloro EJ. Utility of the PFA-100 for assessing bleeding disorders and monitoring therapy: a review of analytical variables, benefits and limitations. Haemophilia 2001; 7: Kerenyi A, Schlammadinger A, Ajzner E, Szegedi I, Kiss C, Pap Z, Boda Z, Muszbek L. Comparison of PFA-100 closure time and template bleeding time of patients with inherited disorders causing defective platelet function. Thromb Res 1999; 96: Posan E, McBane RD, Grill DE, Motsko CL, Nichols WL. Comparison of PFA-100 testing and bleeding time for detecting platelet hypofunction and von Willebrand disease in clinical practice. Thromb Haemost 2003; 90: Quiroga T, Goycoolea M, Munoz B, Morales M, Aranda E, Panes O, Pereira J, Mezzano D. Template bleeding time and PFA-100 have low sensitivity to screen patients with hereditary mucocutaneous hemorrhages: comparative study in 148 patients. J Thromb Haemost 2004; 2: Cammerer U, Dietrich W, Rampf T, Braun SL, Richter JA. The predictive value of modified computerized thromboelastography and platelet function analysis for postoperative blood loss in routine cardiac surgery. Anesth Analg 2003; 96: Slaughter TF, Sreeram G, Sharma AD, El-Moalem H, East CJ, Greenberg CS. Reversible shear-mediated platelet dysfunction during cardiac surgery as assessed by the PFA-100 platelet function analyzer. Blood Coagul Fibrinolysis 2001; 12: LasneD,FiemeyerA,ChatellierG,ChammasC,BaronJF,AiachM. A study of platelet functions with a new analyzer using high shear stress (PFA-100 Ò ) in patients undergoing coronary artery bypass graft. Thromb Haemost 2000; 84: Fattorutto M, Pradier O, Schmartz D, Ickx B, Barvais L. Does the platelet function analyser (PFA-100) predict blood loss after cardiopulmonary bypass? Br J Anaesth 2003; 90: Wahba A, Sander S, Birnbaum DE. Are in-vitro platelet function tests useful in predicting blood loss following open heart surgery? Thorac Cardiovasc Surg 1998; 46: Rodeghiero F, Castaman G, Tosetto A,Batlle J,Baudo F, Cappelletti A,CasanaP,DeBoschN,EikenboomJC,FedericiAB,LethagenS, Linari S, Srivastava A. The discriminant power of bleeding history for the diagnosis of type 1 von Willebrand disease: an international, multicenter study. JThrombHaemost2005; 3: