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Platelet storage solution affects on the accuracy of laboratory tests for platelet function: a multi-laboratory study.

BACKGROUND AND OBJECTIVES: Extent of shape change (ESC) and hypotonic shock response (HSR) have been widely used to characterize the in vitro function of platelets and have been shown to correlate with in vivo viability. These assays have been routinely performed using platelet-poor plasma (PPP) as the test sample diluent. Because of the increasing popularity of storing platelets in synthetic media, it is important to understand the effects of using these synthetic media as test diluents for ESC and HSR measurements. The objective of this study was to determine the effect of using platelet storage solutions vs. plasma for the in vitro testing of ESC and HSR. MATERIALS AND METHODS: Six laboratories participated in this study. Platelets were prepared by apheresis, the platelet-rich plasma (PRP) method, or derived from buffy-coats. Each platelet preparation was divided, half being stored in plasma and the other half in storage solution. ESC and HSR testing were performed in duplicate on days 1 and 5, using each of three diluents: autologous plasma; fresh-frozen plasma; or storage solution. RESULTS: For both ESC and HSR, dilutions made in each of the three diluents yielded significantly different results. Dilutions made in storage solutions were more than 30% lower for ESC and HSR than those made in autologous plasma (P < 0.0001). Dilutions made in thawed fresh-frozen plasma were more than 16% lower for ESC and HSR than those made in liquid autologous plasma (P < 0.0005). CONCLUSIONS: ESC and HSR test results are significantly affected by the test diluent. Platelets should be diluted in plasma (preferably autologous) for the in vitro testing of ESC and HSR, regardless of the media in which they are stored.

Blood Platelets↗

Reproducibility of two in vivo tests of platelet function.

We correlated repeat determinations of platelet aggregate ratios (PAR) and plasma beta-thromboglobulin (BTG) in the right and left arms of thirty-four subjects in order to measure test reproducibility and to evaluate the effects of venipuncture. The reproducibility of PAR on the same blood samples was relatively low but similar to that obtained when the right arm and left arm values were compared, thus indicating little variation secondary to venipuncture. For BTG, the reproducibility on identical blood samples was quite high, but fell significantly when plasma samples from the two sides were correlated. This finding and increased BTG values when obvious blood collection problems occurred, indicated that plasma BTG levels are quite sensitive to venipuncture technique.

Humans↗

Design criteria: tests used to assess platelet function.

A brief overview of platelet function tests used in the past to assess relations between dietary fatty acids and thrombogenicity reveals problems that need to be recognized and addressed before planning future studies. Implementation of new strategies that integrate new technologies with measures of two or more markers of platelet activity may be more successful in predicting the thrombotic potential of dietary fatty acids.

Animals↗

Platelet function analysis.

Since the last guidelines for BCSH platelet function testing were written in the late 1980s, many new tests have become available. Previously most platelet function tests were traditionally utilized to aid in the diagnosis and management of patients with platelet and haemostatic disorders. Most traditional tests were also largely restricted to the specialized laboratory or centre. However, nowadays there is also much renewed interest in monitoring the efficacy of anti-platelet therapy and measuring platelet hyper-function. A number of dedicated platelet function instruments have now become available that are much simpler to use and are beginning to be utilized as point of care instruments. These can now provide measurement of platelet function within whole blood without the requirement of sample processing. Some are also beginning to be incorporated into routine clinical use and can be utilized as not only as general screening tests of platelet function but to monitor anti-platelet therapy and to potentially assess both risk of bleeding and/or thrombosis. Modern flow cytometric-based platelet function analysis now also provides a wide variety of specific tests that can assess different aspects of platelet biology that are useful for diagnostic purposes. This review will highlight some of these of new tests/instruments and discuss their potential utility both within the haemostasis laboratory but also as potential point of care instruments.

Blood Platelet Disorders↗

Design of a modified platelet immunofluorescence test to assess platelet-reactive antibody burden and its association with platelet functional exhaustion and clinical features in chronic immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by platelet destruction and dysfunction associated with anti-platelet antibodies. This study evaluated the relationship between total anti-platelet antibody burden, measured using a modified platelet immunofluorescence test (PIFT), platelet functional responses and clinical features in chronic ITP. Thirty-one patients with chronic ITP and 20 healthy controls were included. Bleeding severity was assessed, and platelet function was analysed in peripheral blood by measuring P-selectin expression, PAC-1 binding (antibody against active conformation of GPIIb/IIIa)&#xa0;and reactive oxygen species (ROS) generation at baseline and following agonist stimulation. Relative platelet-reactive antibody burden was assessed using a ratio-based PIFT assay. ITP patients demonstrated significantly higher antibody burden compared with controls. Increased platelet-reactive immunoglobulin G (IgG) signals were associated with reduced platelet responsiveness to agonist stimulation. Antibody burden correlated with bleeding severity (p&#x2009;<&#x2009;0.01) but not with platelet count. Non-responders exhibited significantly higher antibody levels than responders. receiver operator characteristic (ROC) analysis demonstrated discrimination between responder groups at a PIFT cut-off &#x2265;3.85 (area under the curve [AUC] 0.877, sensitivity 80%; specificity 87%). Patients above this threshold showed attenuated platelet functional responses. Taken together, this study concluded that quantitative assessment of total antibody burden against platelets using modified PIFT is associated with platelet dysfunction, bleeding severity and treatment response status in chronic ITP.

Humans↗

Görög Thrombosis Test: a global in-vitro test of platelet function and thrombolysis.

This is the first laboratory evaluation of a new instrument, designed to test both platelet function and thrombolytic activity from a native blood sample, in vitro. The inventor assumed that the reduction and arrest of blood flow was due to activation, aggregation and stabilized thrombus formation by shear-activated platelets, and that re-establishment of flow was due to thrombolysis. Morphologic and functional studies presented here confirm these mechanisms. In vitro tests provided incontestable evidence for the principal role of platelets in the obstruction of flow (occlusion time) and for thrombolysis as the principal mechanism underlying the restoration of blood flow (lysis time). In addition to aggregation, it is the explosive generation of thrombin by shear-activated platelets that results in the formation of an occlusive haemostatic thrombus. Anticoagulation of blood completely prevented occlusion. Platelet-rich thrombus formation (occlusion time) was dose-dependently inhibited by monoclonal antibody against platelet glycoprotein (GP) Ib (6B4 and 12E4), aurin tricarboxylic acid, monoclonal antibody against platelet GPIIb/IIIa (MA-16N7C2 and abciximab), a synthetic GPIIb/IIIa antagonist (TAK-029), thrombin inhibitor (argatroban), and anti-von Willebrand factor, but not by anti-fibrinogen. Plasminogen activator streptokinase (Varidase) and tissue-type plasminogen activator (Monteplase) dose-dependently enhanced thrombolysis (lysis time) without affecting platelet function (occlusion time). The test is specific for thrombolysis. The plasmin inhibitor tranexamic acid prevented plasminogen activator-induced thrombolysis, while inhibition of clot retraction by cytochalasin B did not affect the lysis time. This rapid and sensitive global test of platelet function and thrombolytic activity could be of great value both in research and in clinical practice.

Adult↗

Effect of ionizing radiation on platelet function in vitro.

The effect of ionizing radiation on platelet function was investigated in vitro. Platelet-rich plasma (300 x 10(9/1)) was irradiated with doses of 1, 4, 10, 20 and 50 Gy. Platelet function tests were performed on both irradiated and control (non-irradiated) platelet samples. The platelet function tests were (1) platelet aggregation by ADP (1, 2, 4 mumol final concentration), adrenaline and collagen, (2) ADP-release from platelets, (3) clot retraction and (4) platelet factor-3 availability. It was found that roentgen irradiation of platelets in vitro did not affect these platelet functions tests.

Adenosine Diphosphate↗

Thrombohaemorrhagic complications in 101 cases of myeloproliferative disorders: relationship to platelet number and function.

A series of 101 consecutive patients with chronic myeloproliferative disorders including polycythaemia vera, chronic myelogenous leukaemia, idiopathic myelofibrosis and essential thrombocythaemia have been studied. The aim was to establish the incidence of thrombotic and haemorrhagic complications and the possible role played by platelet number and function. The total incidence of haemostatic complications was 21% and the platelet functional tests investigated (platelet aggregation, generation of malondialdehyde, endogenous serotonin, beta-thromboglobulin and platelet coagulant activity) were of little help for predicting these clinical complications.

Adolescent↗

Use of native or platelet count adjusted platelet rich plasma for platelet aggregation measurements.

BACKGROUND: It is still not clear whether native or platelet count adjusted platelet rich plasma (PRP) should be used for platelet aggregation measurements. AIM: To evaluate the necessity of using adjusted PRP in platelet function testing. METHODS: Platelet aggregation with native PRP and adjusted PRP (platelet count: 250/nl, obtained by diluting native PRP with platelet poor plasma) was performed on the Behring Coagulation Timer (BCT(R)) using ADP, collagen, and arachidonic acid as agonists. Healthy subjects, patients on antiplatelet treatment, and patients with thrombocytosis (platelet counts in PRP > 1250/nl) were investigated. RESULTS: No significant differences in the maximum aggregation response were seen when using either native or adjusted PRP from healthy subjects and patients on antiplatelet treatment. Nevertheless, some patients taking aspirin or clopidogrel showed reduced inhibition of ADP and arachidonic acid induced aggregation in adjusted PRP but not in native PRP. The maximum velocity of healthy subjects and patients on antiplatelet treatment varied significantly as a result of the degree of dilution of the adjusted PRP. Surprisingly, the BCT provided good results when measuring platelet aggregation of native PRP from patients with thrombocytosis, whereas commonly used aggregometers could not analyse platelet aggregation of native PRP in these patients. CONCLUSION: The time consuming process of PRP adjustment may not be necessary for platelet aggregation measurements. Moreover, using adjusted PRP for monitoring aspirin or clopidogrel treatment may falsify results. Therefore, it may be better to use native PRP for platelet aggregation measurements, even in patients with thrombocytosis.

Adult↗

Young male smokers have altered platelets and endothelium that precedes atherosclerosis.

BACKGROUND: Cigarette smoking results in abnormalities of platelets and endothelium with platelet dysfunction implicated in vascular complications. Healthy endothelium plays a pivotal role in regulating hemostasis via the inhibition of platelet activation and aggregation. Thus, we examined if platelet dysfunction correlated with serum vWF levels-a circulating marker of endothelial dysfunction. MATERIALS AND METHODS: We tested this hypothesis in young male smokers. The parameters of platelet function tested included: CD62/CD63; Glycoprotein (GP) IIb/IIIa; Platelet function analzyer (PFA) studies, platelet aggregometry, flow assessment of platelet microparticles, platelet-leukocyte interactions and receptor numbers. Endothelial dysfunction was assessed with serum von Willebrand Factor (vWF). RESULTS: There was a significant increase in platelet CD62 receptor expression and aggregation with an associated delay in time to aggregation using PFA. Endothelial dysfunction was represented by higher serum levels of von Willebrand Factor. All other platelet parameters tested were within the standardized reference range. CONCLUSIONS: These initial data suggests that anti-platelet therapy may have a role in reduction of platelet activation and aggregation in young smokers and possibly alter vascular endothelial abnormalities.

Adenosine Diphosphate↗

The laboratory diagnosis of platelet disorders.

OBJECTIVE: To provide both a detailed description of the laboratory tests available in the diagnosis of platelet disorders and a testing algorithm, based on platelet count, that can be used to direct the evaluation of platelet disorders. DATA SOURCES: A literature search was conducted using the National Library of Medicine database. STUDY SELECTION: The literature on laboratory testing of platelet function was reviewed. DATA EXTRACTION AND DATA SYNTHESIS: Based on the literature review, an algorithm for platelet testing was developed. CONCLUSIONS: A history of mucocutaneous bleeding often indicates abnormal platelet function that can be associated with a normal, increased, or decreased platelet count. Multiple laboratory procedures can now be used to determine the underlying pathologic condition of platelet dysfunction when other deficiencies or defects of the coagulation cascade or fibrinolysis are ruled out. Simple procedures, such as platelet count, peripheral blood smear, and a platelet function screening test, will often lead the investigator to more specific analyses. Although platelet function testing is often limited to larger medical centers with highly trained technologists, newer technologies are being developed to simplify current procedures and make platelet function testing more accessible. This review provides an algorithm for platelet testing that may be of benefit to pathologists and physicians who deal with hemostatic disorders.

Algorithms↗

Association of reduced factor VIII with impaired platelet reactivity to adrenalin and collagen after total thyroidectomy.

Twenty-one thyroprival patients, previously submitted to total thyroidectomy for tumours, were investigated during stabilized L-thyroxine supplementation and at the end of a 20-25 day "no-therapy" period, necessary for a 131I total body scintigraphy. During supplementation therapy a lower than normal mean beta-thromboglobulin (beta-TG) release level was found, the other blood clotting and platelet function tests being normal. After substitution therapy withdrawal, platelet function tests showed reduced adrenalin aggregation, increased collagen threshold aggregating concentrations, decreased beta-TG release values and reduced aggregation to ristocetin, whereas blood clotting tests showed prolonged aPTT values and reduced levels of factor VIII:C and vWf:Ag. We conclude that in acquired hypothyroidism the lowering of factor VIII:C and vWf:Ag (acquired von Willebrand disease) is associated with impaired platelet reactivity not only to ristocetin but also to collagen and especially adrenalin. In the patients investigated these changes were almost completely corrected by substitutive therapy with L-thyroxine at clinically effective doses.

Adult↗

Clopidogrel and bleeding in patients undergoing elective coronary artery bypass grafting.

OBJECTIVE: In an effort to minimize transfusions in patients undergoing elective coronary artery bypass grafting operations after recent clopidogrel exposure, we studied laboratory tests predictive of platelet dysfunction and used a strict algorithm-driven treatment of bleeding. METHODS: Forty-five patients receiving clopidogrel within 6 days of the operation and 45 control subjects were studied. Prothrombin time, activated partial thromboplastin time, platelet count, and platelet function test results were measured before heparinization, after protamine administration, and then every 2 hours. No transfusions were administered unless a patient met both laboratory and clinical criteria. RESULTS: Algorithm-driven treatment of bleeding significantly reduced the mean units of all blood components transfused by about one third, as shown by comparison with current control and historical data. Compared with current control subjects, clopidogrel recipients required significantly more transfusions of platelets (9.0 +/- 1.7 vs 1.2 +/- 0.5 U; P <.0001) and packed red blood cells (4.3 +/- 0.6 vs 2.3 +/- 0.5 U; P =.01) and required longer periods of controlled ventilation (12.4 +/- 1.3 vs 8.6 +/- 0.8 hours; P =.02). Preoperative platelet dysfunction before heparin administration for cardiopulmonary bypass, as measured by using adenosine diphosphate aggregometry (response <40%), predicted all but 1 case of severe coagulopathy requiring multiple transfusions (16.6 +/- 2.8 U of platelets and 5.8 +/- 1.0 U of packed red blood cells). CONCLUSIONS: A strict transfusion algorithm can reduce the transfusion requirement for all blood components. Preheparin testing of platelet function with adenosine diphosphate aggregometry can identify patients at highest risk for perioperative bleeding and transfusions and might further reduce the perioperative transfusion requirement.

Algorithms↗

Thrombocytopenia-adapted in vitro bleeding test assesses platelet function in thrombocytopenic patients.

Platelet counts do not always reflect the true bleeding risk in chronically thrombocytopenic patients, and the posttransfusion platelet increments do not necessarily demonstrate that therapeutic efficacy. There are no easy and reliable tests yet permitting the determination of platelet function in thrombocytopenic patients. The in-vitro bleeding test (IVBT) with the Thrombostat 4000 proved to be a very sensitive and specific test for the detection of platelet disorders. In order to become suitable for the investigation of thrombocytopenic blood with platelet count between 5 x 10(9)/L and 50 x 10(9)/L, special modifications were necessary. We report on the evaluation of two thrombocytopenia-adapted modifications (TP-IVBT 150/120), first with blood of healthy donors made thrombocytopenic (three experiments with six blood samples each of different platelet concentrations and identical hematocrit) and then in a clinical study on 77 thrombocytopenic patients (69 with bone marrow hypoplasia, eight with autoimmune thrombocytopenia) receiving 267 platelet transfusions. The patients were followed over 15 days on average (1-67 days) by daily examinations (total 1,285 observation days). Most TP-IVBT measurements were carried out in triplicate, using the modification with the 120-microns filter (TP-IVBT 120) because it proved to be superior to the other modification. Additionally, cell counts, hematocrit, body temperature, platelet volume, platelet distribution width, expression of CD 36, 41a, 42b on platelets, Simplate bleeding time, and detailed analysis of bleeding signs were performed for the calculation of a bleeding score. There was a close correlation between TP-IVBT and platelet counts with thrombocytopenic normal blood (r2 = 0.81-0.94). This indicated the suitability of this test modification to examine platelet function in thrombocytopenic patients. The clinical study showed that the TP-IVBT helped at least to determine the platelet-related bleeding risk in thrombocytopenic patients. It allowed differentiation between hypoplastic and autoimmune thrombocytopenia in most cases. In addition, significant differences in platelet function of various diseases and of different bone marrow regeneration could be demonstrated. The TP-IVBT is well-suited for the control of platelet transfusion efficacy and may replace the in-vivo bleeding time in most cases. On the other hand, the test still shows too much of a variation and involves too much labor and cost for routine application.

Bleeding Time↗

Decreased platelet function in aortic valve stenosis: high shear platelet activation then inactivation.

OBJECTIVE: To elucidate the mechanism of the bleeding tendency observed in patients with aortic valve stenosis. DESIGN: A prospective study of high and low shear platelet function tests in vitro in normal controls compared with that in patients with severe aortic valve stenosis with a mean (SD) systolic gradient by Doppler of 75 (18) mm Hg before and at least 4 months after aortic valve replacement. SETTING: District general hospital. RESULTS: The patients showed reduced retention in the high shear platelet function tests. (a) Platelet retention in the filter test was 53.6 (12.6)% in patients with aortic valve stenosis and 84.8 (9.6)% in the controls (P < 0.001). (b) Retention in the glass bead column test was 49.8 (19.2) in the patients and 87.4 (8.7) in the controls (P < 0.001). (c) The standard bleeding time was longer in the patients (P < 0.06). Results of the high shear tests (a, b, and c) after aortic valve replacement were within the normal range. The platelet count was low but within the normal range before surgery and increased postoperatively (P < 0.01). There were no differences in the results of standard clotting tests, plasma and intraplatelet von Willebrand's factor, or in 15 platelet aggregation tests using five agonists between patients with aortic valve stenosis and controls. CONCLUSIONS: The high shear haemodynamics of aortic valve stenosis modify platelet function in vivo predisposing to a bleeding tendency. This abnormality of platelet function is detectable only in vitro using high shear tests. The abnormal function is reversed by aortic valve replacement. High shear forces in vitro activate and then inactivate platelets. By the same mechanisms aortic valve stenosis seems to lead to high shear damage in vivo, resulting in a clinically important bleeding tendency in some patients.

Aged↗

Biological assessment of aspirin efficacy on healthy individuals: heterogeneous response or aspirin failure?

BACKGROUND AND PURPOSE: The widespread use of aspirin requires clarification of the aspirin resistance phenomenon. Most studies on this field are focused on patients which may affect the action of aspirin. METHODS: We evaluated the biological efficacy of aspirin in healthy subjects. RESULTS: Agonist-induced platelet aggregation was fully abrogated by 100 mg of aspirin in all individuals. By contrast, with the platelet function analyzer-100 device, 33.3% of the subjects displayed no response. This failure was overcome by 500 mg or by in vitro treatment of blood with 30 mumol/L acetylsalicylic acid. Intake of 100 mg of aspirin efficiently reduced by 75% the level of 11-dehydro thromboxane B2 (11-dTxB2) in all cases. However, variability on the pre-aspirin level (range 72.4 to 625.9 ng/mmol creatinine) led to substantial differences in the residual amount of the metabolite between subjects treated with aspirin (range 12.9 to 118.0 ng/mmol creatinine). Finally, there was no influence of platelet glycoprotein IIb/IIIa (Pro33Leu), platelet glycoprotein Ia/IIa, (C807T), and FXIII (Val34Leu) polymorphisms on the efficacy of aspirin. However, the cyclooxygenase (Cox)-1 50T allele associated with higher level of 11-dTxB2, both before and after aspirin. Moreover, the Cox-2 -765C variant displayed a slightly higher reduction in 11-dTxB2 level on treatment with aspirin. CONCLUSIONS: Our findings suggest that full resistance of healthy subjects to aspirin is rather unlikely. However, differences in aspirin absorption, or pharmacokinetic, or other unrecognized factors may lead to lack of effect of low dose of aspirin in some subjects when using tests like platelet function analyzer-100. Whether Cox polymorphisms are thrombotic risk factor for patients under aspirin will require further research.

Absorption↗