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PubMed1999

A simple assay for platelet-mediated hemostasis in flowing whole blood (PFA-100): reproducibility and effects of sex and age.

BACKGROUND: A simple assay for platelet-mediated hemostasis in flowing blood (PFA-100) has become available. Whole blood is aspirated through the central opening of a membrane coated with platelet agonists; the time required for a platelet thrombus to occlude the opening is defined as closure time; the shorter the closure time the greater the platelet aggregability. There are limited data on the normal range of values for this test, and the effects of sex and age are not known. The aim of this study was to determine the effects of sex and age on closure time in normal volunteers and to assess the reproducibility of the test in our laboratory. METHODS: Closure time using collagen/adenosine-5'-diphosphate was measured in 62 apparently healthy individuals 35 to 75 years of age (11 men and 17 women < 55 years and 22 men and 12 women > 55 years). RESULTS: Closure time was 96.6 +/- 24 s in men and 93.1 +/- 16 s in women (p = 0.20). In the entire group, closure time did not significantly correlate with age (r = -0.17, p = 0.18). However, men < 55 years tended to have a longer closure time than men > 55 years (109.7 +/- 23 vs 90.0 +/- 22 s, p = 0.08), whereas in women closure time was similar in those younger or older than 55 years (93.1 +/- 16 vs 93.3 +/- 18 s, respectively). In 20 samples tested in duplicate, the mean closure time was 80.9 +/- 10 s on the first determination and 81.5 +/- 12 s on the second (r = 0.89, p < 0.001). There was no significant correlation between closure time and hematocrit, platelet number, mean platelet volume, or leukocyte count. CONCLUSIONS: The platelet function analyzer PFA-100 showed a good reproducibility in apparently healthy subjects. No significant difference in closure time was found between men and women, nor between subjects younger or older than 55 years, although a tendency towards shorter values was found in older compared with younger men.

Adult↗
PubMed1990

The bleeding time: its potential utility among patients receiving thrombolytic therapy.

The BT as a test of hemostatic function was first described 80 years ago. It has generally been considered a primitive and tedious test. Improvements in technique and standardization have increased the BT's reliability and led to its consideration as a preoperative screening measure. Current use has not been widespread, however, except for patients undergoing neurosurgery and organ biopsy. Recently, though, there has been a renewed interest in the BT for patients receiving thrombolytic therapy because levels of fibrinogen and fibrin(ogen) degradation products have been only weak predictors of hemorrhagic complications. The rationale for using the BT in this setting is that thrombolysis appears to impair platelet function, either through depletion of platelet granules or through direct proteolytic actions on platelets. Further research will determine whether these platelet effects are manifest as BT prolongation; whether increased BT will correlate with hemorrhagic complications; and, finally, whether patients who fail to achieve clot lysis or those at risk for bleeding can be identified prospectively.

Bleeding Time↗
PubMed2002

Antithrombotic effect of tissue factor inhibition by inactivated factor VIIa: an ex vivo human study.

FFR-rFVIIa is an inactivated recombinant factor VIIa (rFVIIa) that inhibits the binding of factor VIIa to tissue factor (TF). It has been shown to prevent TF-induced thrombosis in animals. The present study is a substudy of the Active Site Inhibited Seven (ASIS) trial and examines the antithrombotic effect of 3 doses of FFR-rFVIIa in 24 patients undergoing percutaneous coronary intervention (PCI). Group 1 (n=9) received 400 microg/kg FFR-rFVIIa and 40 to 50 U/kg heparin, group 2 (n=7) received 200 microg/kg FFR-rFVIIa and 100 U/kg heparin, and group 3 (n=8) received 50 microg/kg FFR-rFVIIa and 100 U/kg heparin. Blood thrombogenicity was assessed as total thrombus area and fibrin deposition on the perfusion chamber at shear rate conditions typical of mild-moderate coronary stenosis. Baseline blood thrombogenicity was evaluated a day before PCI, after heparin administration. A second perfusion chamber study was performed just before PCI, 15 minutes after the administration of heparin and FFR-rFVIIa. Thrombus formation at a high shear rate was markedly reduced in groups 1 and 2 after drug administration, by 79% to 84% and 76% to 87%, respectively (P<0.004 [group 1], P<0.04 [group 2]). In group 3, moderate thrombus reduction of 46% to 48% was achieved (P<0.04). Fibrin deposition in all 3 groups was nearly eliminated after drug administration. Our data demonstrate that FFR-rFVIIa has a potent antithrombotic effect at different shear rates and severe arterial injury conditions.

Angioplasty, Balloon, Coronary↗
PubMed1995

Preliminary data from a field trial of the PFA-100 system.

The PFA-100 system (Dade International Inc., Miami, FL) is a platelet function analyzer, the design of which is based on the technology of the Thrombostat 4000 VDG, Seeon, Germany. It was developed to measure primary, platelet dependent hemostasis in citrated whole blood in vitro. A first pilot study was conducted with the instrument to assess performance characteristics. Healthy subjects (normals) who had not ingested any medications, and patients (abnormals) with primary, platelet-related hemostasis defects, which included users of aspirin, were studied with two test cartridges; collagen/ADP and collagen/epinephrine. Before the study certain variables were tested that ascertained that blood drawn into either 3.8% or 3.2% sodium citrate containing vacutainers (rather than syringes) could be used for testing. Tests must be performed within a five-hour time span from drawing to testing, and blood must be kept at room temperature. Normal reference values were 77-133 seconds closure times for collagen/ADP and 98-185 seconds for collagen/epinephrine. Precision testing revealed a CV of < 10% for within-day and between-day (five days) analyses on collagen/ADP cartridges and a CV of 5-14% for both runs on the collagen/epinephrine cartridges. No clinically important differences were found between measurements in the two positions of the instrument, although one follows the other.(ABSTRACT TRUNCATED AT 250 WORDS)

Bleeding Time↗
PubMed2004

In vitro aspirin resistance detected by PFA-100 closure time: pivotal role of plasma von Willebrand factor.

The in vitro closure time (CT), determined by the Platelet Function Analyzer (PFA-100), is used to monitor patients treated with aspirin. A relatively high percentage of in vitro aspirin resistance was reported despite an adequate inhibition of platelet response to arachidonic acid and we investigated whether high plasma levels of von Willebrand factor ristocetin cofactor activity (vWF:RCo) may contribute to this profile. Platelet aggregation test, CT [collagen adrenaline (CEPI-CT) and collagen adenosine 5'-diphosphate (ADP) (CADP-CT)], and vWF:RCo levels were evaluated in 55 consecutive patients receiving aspirin (75-250 mg/d) versus 32 untreated control subjects. All the aspirin-treated patients showed platelet aggregation responses that reflected the aspirin intake. However, CT data analysis enabled aspirin good-responder (GR) and aspirin bad-responder (BR) patients to be identified. All GR group subjects (n = 27), had a CEPI-CT and a CADP-CT longer than 300 s and 96 s respectively. The BR group (n = 28) had CEPI-CT values below 200 s and all CADP-CT were in the normal range (77 +/- 19 s). Interestingly, the BR plasma vWF:RCo levels were significantly higher (159 +/- 43%) than those of the GR group (121 +/- 34%) (P < 0.01), which were similar to control values (114 +/- 31%). A negative correlation between vWF:RCo and CT values was established. We demonstrate that in vitro aspirin-resistance, revealed by PFA-100 CT prolongation failure, is correlated to increased plasmatic vWF:RCo levels, reinforcing its particular importance in PFA-100 cartridges performance.

Adult↗
PubMed1998

A multi-laboratory evaluation of in vitro platelet assays: the tests for extent of shape change and response to hypotonic shock. Biomedical Excellence for Safer Transfusion Working Party of the International Society of Blood Transfusion.

BACKGROUND: There is no consensus regarding the use of specific in vitro tests for the assessment of the quality of platelet components. A literature review found that the platelet discoid shape as measured photometrically by the extent of shape change (ESC) and hypotonic shock response (HSR) correlated well with in vivo viability. The purpose of this study was to determine whether multiple research laboratories can perform the ESC and HSR assays in an accurate, reproducible manner, with acceptable sensitivity and comparable results. STUDY DESIGN AND METHODS: Eleven laboratories conducted five identical experiments, each with a different unit of platelet-rich plasma (PRP). For each experiment, 2 half-units of PRP were prepared and stored overnight: 1 half-unit at 20 to 24 degrees C in CPD (CPD-PRP) and the other at 1 to 6 degrees C with 2 mg per mL of EDTA (cold EDTA-PRP) added to produce spherical platelets with reduced HSR. Platelet suspensions having different proportions of the two PRPs were prepared and evaluated in duplicate by ESC and HSR assays, and morphologically scored by microscopy. One-way ANOVA and Duncan multiple-range tests were performed to determine significant differences in assay results for suspensions having different proportions of CPD-PRP. RESULTS: Comparable ESC (mean range: 20-28% for CPD-PRP and 1-6% for cold EDTA-PRP) and HSR (mean range: 58-81% for CPD-PRP and 12-31% for cold EDTA-PRP) measurements were obtained by nine laboratories. Duplicate testing showed high reproducibility of ESR and HSR results /in all laboratories. A 25-percent difference in the proportion of CPD-PRP (indicative of a difference of approximately 25% in the proportions of discoid and spherical platelets) was detected with a sample size of five (p<0.05) for both the ESC and HSR assays. A high correlation was found for the ESC assay and morphology score (r = 0.93, n = 345). CONCLUSION: Multiple laboratories were able to obtain comparable results with the ESC and HSR tests. They were able to show that the tests can be performed in an accurate, reproducible manner and with acceptable sensitivity.

Blood Platelets↗
PubMed2003

A prospective, double-blind study on the efficacy of the bioline surface-heparinized extracorporeal perfusion circuit.

BACKGROUND: We evaluated the newly introduced Bioline heparin coating and tested the hypothesis that surface heparinization limited to the oxygenator and the arterial filter will ameliorate systemic inflammation and preserve platelets during cardiopulmonary bypass (CPB). METHODS: In a prospective double-blind study, 159 patients underwent coronary revascularization using closed-system CPB with systemic heparinization, mild hypothermia (33 degrees C), a hollow-fiber oxygenator, and an arterial filter. The patients were randomly divided in three groups. In group A (controls, n = 51), surface heparinization was not used. In group B (n = 52), the extracorporeal circuits were totally surface-heparinized with Bioline coating. In group C (n = 56), surface heparinization was limited to oxygenator and arterial filter. RESULTS: No significant difference was noted in patient characteristics and operative data between groups. Operative (30-day) mortality was zero. Platelet counts dropped by 12.3% of pre-CPB value among controls at 15 minutes of CPB, but were preserved in groups B and C throughout perfusion (p = 0.0127). Platelet factor 4, plasmin-antiplasmin levels, and tumor necrosis factor-alpha increased more in controls during CPB than in groups B or C (p = 0.0443, p = 0.0238 and p = 0.0154 respectively). Beta-thromboglobulin, fibrinopeptide-A, prothrombin fragments 1 + 2, factor XIIa levels, bleeding times, blood loss, and transfusion requirements were similar between groups. Intensive care unit stay was shorter in groups B and C than in controls (p = 0.037). CONCLUSIONS: Surface heparinization with Bioline coating preserves platelets, ameliorates the inflammatory response and is associated with a reduced fibrinolytic activity during CPB. Surface heparinization limited to the oxygenator and the arterial filter had similar results as totally surface-heparinized circuits.

Aged↗
PubMed1980

Persistent abnormalities of fibrinolytic activity and platelet function in patients with reversible oestrogen-associated hypertension.

Normotensive women with a previous history of oestrogen-associated hypertension were found to have raised platelet factor 3 (PF3) availability, raised triglycerides and pre-beta-lipoprotein and impaired fibrinolytic activity and capacity, but were shown to have normal platelet lifespan and renal platelet localization. The significance of these abnormalities in relation to the development of hypertension and to the intrarenal vascular disease found in these patients is discussed.

Adult↗
PubMed2004

Changes in hematological, hemostatic and biochemical parameters induced experimentally in rabbits by Loxosceles gaucho spider venom.

Human accidents caused by Loxosceles spiders may result in local dermal necrosis and, in some cases, severe systemic reactions - such as intravascular hemolysis, disseminated intravascular coagulation (DIC), renal failure and death. Since many aspects of envenomation by Loxosceles spiders remain unclear, we studied the hematological and hemostatic responses induced by the i.d. injection of 10 microg/kg Loxosceles gaucho venom in rabbits. For this purpose, total blood cell count, platelet function, coagulation tests and biochemical parameters were analysed at 3, 24, 48, 72 and 120 hours after venom administration. Thrombocytopenia and leukopenia were noted at 3 and 24 hours. Histopathological analysis of the skin lesion, performed at 24 hours after venom administration, showed a massive presence of leukocytes and platelets, hemorrhage and thrombus formation at the injection site. At 72 and 120 hours, neutrophilic leukocytosis and thrombocytosis were observed. Platelet hyperaggregation was noticeable at 48 and 72 hours. Haptoglobin and fibrinogen levels were elevated early and remained in high levels over time. Significant increases in coagulation factors V, VII, VIII, IX, X and XI were noted at 120 hours. The results showed that neither intravascular hemolysis nor DIC occurred. However, the early onset of thrombocytopenia and leukopenia are important findings that may be related to dermal necrosis formation during loxoscelism.

Animals↗
PubMed2006

Aprotinin shows both hemostatic and antithrombotic effects during off-pump coronary artery bypass grafting.

BACKGROUND: Hemostatic drugs are widely thought to be unnecessary and potentially detrimental in off-pump coronary artery bypass graft surgery (OPCABG), despite well-established use in on-pump surgery. In a randomized, prospective OPCABG trial, we assessed efficacy and safety of aprotinin through a comprehensive assessment of graft patency and hematologic function. METHODS: Sixty patients were randomly assigned to full-dose aprotinin or placebo. Heparin was titrated to a kaolin-based activated clotting time of greater than 300 seconds. Exclusionary criteria included creatinine greater than 2 mg/dL, conversion to on-pump CABG, and preoperative GPIIb/IIIa inhibition. Hematologic assessments were obtained preoperatively, at the end of surgery, and on days 1 and 3: mean platelet volume, thrombin generation (prothrombin fragment 1.2 assay), and aspirin resistance using a modified thrombelastography, whole blood aggregometry, 11-dehydro-thromboxane B2 levels, and flow cytometry. Thrombotic events were defined as postoperative myocardial infarction by electrocardiography or elevated troponin I, clinical stroke by examination and head computed tomography, and bypass graft failure by multichannel computed tomography angiography on day 5. RESULTS: Aprotinin was associated with a significant reduction in intraoperative and postoperative blood loss compared with placebo but had no effect on transfusion rates. Patients treated with aprotinin had significantly fewer thrombotic events (3% versus 23%, p < 0.05, Fisher's exact test) and less postoperative aspirin resistance (20% versus 46%, respectively, p < 0.05, Fisher's exact test). Postoperative prothrombin fragment 1.2 level was reduced by aprotinin use. CONCLUSIONS: Aprotinin reduced perioperative bleeding after OPCABG. Preserved aspirin sensitivity in the aprotinin group may explain the observed reduction in thrombotic events and might be related to the suppression of perioperative and transmyocardial thrombin formation.

Aprotinin↗
PubMed2006

ADAMTS-13 regulates platelet adhesion under flow. A new method for differentiation between inherited and acquired thrombotic thrombocytopenic purpura.

ADAMTS-13 cleaves large and ultra-large von Willebrand factor multimers normally secreted by endothelial cells. Severe deficiency of this enzyme leads to thrombotic thrombocytopenic purpura (TTP). We applied the Impact-R system [Cone and plate(let) Analyzer, CPA] to determine optimal conditions for ADAMTS-13 function, to assess it's activity in TTP patients and to distinguish inherited TTP (inTTP) from acquired TTP (acTTP). The role of ADAMTS-13 in platelet adhesion under different conditions was investigated applying recombinant forms of VWF and ADAMTS-13. rVWF was first treated by rADAMTS-13 either in solution or when immobilized on the surface of the well, under static or flow conditions, in the presence or absence of BaCl2. The resulting cleaved VWF fragments were then immobilized and served to assess type 3 von Willebrand disease whole blood platelet adhesion under flow. Maximal effect of the rADAMTS-13 (decrease of platelet adhesion in the absence compared to the presence of BaCl2), was observed when the rVWF was pre-immobilized and the cleavage step took place under flow (85%). Mixing plasma ofTTP patients with normal blood (1:3) yielded a 1.6- to 2-fold increase of platelet adhesion under flow compared to mixing normal plasma with normal blood, at shear rate range of 1,800 - 2,500 s(-1) . Maximal increase of platelet adhesion was observed under 2,050 s(-1) . Under these conditions, the extent of adhesion was similarly higher in patients with inTTP and acTTP versus control [surface coverage (SC) 14.5 +/- 2.8% and 14.6 +/- 2.5% vs.7.4 +/- 1.7%, respectively]. ADAMTS-13 activity measured by collagen-binding test was similarly low (4.2 +/- 3.8% and 3.5 +/- 2.4% vs. 72.2 +/- 8.0%, respectively). An inverse correlation between SC and ADAMTS-13 activity was observed in a patient with inTTP assayed eight times during plasma infusion treatment. Addition of BaCl2 to the mixture of TTP plasma and normal whole blood yielded a decrease in platelet adhesion in inTTP (by 51%) but not in acTTP. The lack of reduction of platelet adhesion in acTTP could presumably be due to the presence of ADAMTS-13 inhibitor in these patients. These results suggest that VWF immobilization and high shear flow yielded optimal conditions for ADAMTS-13 activity, and that introduction of BaCl2 in the Impact-R (CPA) test may be useful for differentiation between inherited and acquired TTP.

ADAM Proteins↗
PubMed2001

Platelet storage in PAS-2 or autologous plasma: impact on functional parameters.

Currently, several platelet additive solutions for long-term platelet storage have been introduced. The aim of this study was to compare the deterioration of functional status of platelets stored for up to 5 days in autologous plasma (AP) only, with platelet stored in PAS-2, a salt solution containing acetate, citrate and sodium chloride. Change in platelet adhesion, aggregation and activation was measured by flow cytometric technique. In addition, beta-Thromboglobulin (beta-TG), lactate and glucose were determined. After 5 days of storage, the expression of P-Selectin was significantly higher, the production of lactate and the consumption of glucose were significantly lower, in platelets stored in PAS-2 than in autologous plasma. No significant differences were detected on day 5 between the two groups with regard to fibrinogen, von Willebrand factor binding capacity, or to beta-TG release. It can be concluded that neither storage medium was consistently better for the parameters tested. However, it must be emphasized that platelets stored in autologous plasma exhibited less lesion, in terms of P-Selectin expression compared with platelets stored in PAS-2.

Acetates↗
PubMed1995

Quality control of platelet concentrates by the Thrombostat 4000.

Quality control of platelet concentrates (PC) is an important prerequisite for good transfusion praxis. However, direct measurement of platelet function is complex, since available methods (e.g. aggregometry, serotonin release) are time consuming and require special equipment. Therefore a test system is needed, which is easy to handle, fast, and achieves reliable results. The present paper compares the results of conventional platelet function tests with those of a modified in-vitro bleeding test (IVBT) (Thrombostat 4000) in liquid-stored and cryopreserved PCs. A high correlation between aggregometry, serotonin release, GMP 140 expression upon stimulation, and IVBT was demonstrated. Therefore IVBT seems to be a good alternative to the conventional platelet function tests for quality control of PCs. In addition, a good correlation between the results of IVBT of patients' blood after PC transfusion and IVBT of patients blood before transfusion supplemented with platelets of the respective PC could be found. Therefore IVBT seems to be able to predict PC transfusion success. However, since these data were obtained in a small sample undergoing bone marrow transplantation, further studies are needed to verify this hypothesis.

Bleeding Time↗
PubMed1991

Preoperative hemostatic activity and excessive bleeding after cardiopulmonary bypass.

The rationale for predicting the risk of excessive postoperative bleeding by assessing the hemostatic status of a patient before cardiopulmonary bypass was investigated. A novel, rapid, overall test (hemostatometry) consisting of a physiologically relevant test of platelet function (shear-induced hemostasis) and coagulation was performed using nonanticoagulated blood and compared with the routine coagulation screen. Two hundred five patients undergoing elective coronary revascularization were studied 3 to 4 days before operation. Forty-nine bled excessively for nonsurgical reasons; none were predicted by the routine coagulation tests. Using a stepwise discriminant analysis, hemostatometry correctly predicted 31 of 49 (63%). Thirty of 156 predicted as bleeders by hemostatometry did not bleed. Thus, preoperative hemostatometry predicted 77% of the true outcome. The false predictions suggest, however, that certain bleeding abnormalities probably acquired during cardiopulmonary bypass cannot be predicted. These findings do not justify the routine use of preoperative tests in assessing the bleeding risk in patients undergoing cardiopulmonary bypass.

Blood Coagulation↗