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Filter bleeding time: a new in vitro test of hemostasis. I. Evaluation in normal and thrombocytopenic subjects.

Disadvantages of current bleeding time methods include poor reproducibility, limited repeatability, and at times scar formation. We report a new, in vitro technique which circumvents these problems. Venous blood is collected in sodium citrate, stored capped in a siliconized tube at 37 degrees C until use, and made to flow under constant pressure through a filter of Woven Dacron. Flow rate progressively falls as platelet aggregates occlude the filter, as verified by scanning electron microscopy and 111Indium-labeled platelet radioactivity of the filter. Filter bleeding time (FBT) was 4.33 +/- 1.79 (means x +/- SD) min in 23 healthy human volunteers and 3.08 +/- 1.08 min in 14 normal dogs. Bleeding volume (BV) (number of drops) was 29 +/- 12 in the humans and 16 +/- 6 in the dogs. Initial bleeding rate (IBR) (number of drops during first minute) was 14 +/- 3 in citrated blood and 25 +/- 5 drops/min in EDTA blood of the humans (P = 9 X 10(-10)), 8 +/- 2 in citrated blood and 14 +/- 6 drops/min in EDTA blood of the dogs (P = .0004). Percent platelet reduction during passage of blood through the filter (PCR) was 27.5 +/- 7.2 in citrated blood and 4.9 +/- 2.6 in EDTA blood of the humans (P = 2 X 10(-12)), while in dogs it was 25.7 +/- 8.3 in citrated blood and 5.3 +/- 4.2 in EDTA blood (P = 8 X 10(-7)). The results of these parameters using human platelet rich plasmas (PRPs) were similar to those with human whole blood, but significant decrease of drop rate was not observed in canine PRPs during passage of PRP through the filter. FBT, BV, and IBR were correlated (r = -0.91, -0.84 and -0.62 respectively) with platelet count in 12 specimens obtained from 4 dogs in which thrombocytopenia (107 to 4 X 10(3) platelets/microliter) was induced by estradiol. This new test is unaffected by specimen transport by pneumatic tube, is sensitive to platelets, and should be useful for analysis of both quantitative and qualitative platelet abnormalities in man.

Animals↗

Effects of amlodipine and enalapril on platelet function in patients with mild to moderate hypertension.

OBJECTIVE: The aim of this study was to compare the effect of amlodipine and enalapril on platelet aggregation, and platelet production of malondialdehyde in patients with mild to moderate arterial hypertension. PATIENTS AND METHODS: A parallel, double-blind, placebo-controlled study was carried out in 24 patients (2 groups of 12 patients each). Initially all patients received placebo for four weeks; then amlodipine, 5 mg daily or enalapril 20 mg daily taken once a day at 7 am. Dosage was doubled after 4 weeks when diastolic blood pressure was > 90 mmHg in sitting position, the treatment was continued for 12 weeks. At the end of placebo and active phases a platelet aggregation test, using adenosine diphosphate, collagen and adrenaline, and a platelet malondialdehyde production test, either in basal conditions (MDA-basal) and after the stimulation of arachidonic acid pathway by adding ethylmaleimide (MDA-activated) were carried out. RESULTS: Blood pressure was reduced by both agents, enalapril and amlodipine. Enalapril controlled 58.3% of hypertensive patients with an average dosage of 31.7 mg/daily. Amlodipine controlled 75% of patients with a dosage of 7.1 mg/daily. Platelet aggregation was reduced by amlodipine in 15.9% for ADP (10 microM); 17.4% for collagen (2 microg/ml) and 19.9% for adrenaline (2 microM) (p < 0.025). Meanwhile enalapril slightly increased platelet aggregation by 6.7%, 1.3% and 5.6% for the three agents, respectively (p > 0.05, ns). Malondialdehyde was reduced by amlodipine in 45.33% (p < 0.05) for MDA-basal; 3.76% (p > 0.05) for MDA-activated; and the ratio MDA-basal:MDA-activated in 36.79% (p < 0.005). Meanwhile enalapril increased MDA-basal in 2.89%; MDA-activated in 3.58% and reduced the ratio MDA-basal:MDA-activated, in 10.34% (p > 0.05). CONCLUSION: Both agents, enalapril and amlodipine, reduced blood pressure, but only amlodipine reduced platelet aggregation and platelet production of malondialdehyde, indicating its action on the arachidonic acid metabolic pathway.

Amlodipine↗

Template bleeding time and PFA-100 have low sensitivity to screen patients with hereditary mucocutaneous hemorrhages: comparative study in 148 patients.

OBJECTIVES AND PATIENTS: We compared the template bleeding time (BT) and closure time (CT) in the PFA-100 as screening tests in 148 consecutive patients with unequivocal mucocutaneous bleeding and positive family history. EXCLUSION CRITERIA: drug intake, concomitant diseases including minor infections, low platelet count, diseases of secondary hemostasis. RESULTS: Type 1 von Willebrand disease (VWD-1) was diagnosed in 26 patients, primary platelet secretion defect (PSD) in 33, VWD-1 + PSD in nine, whereas 80 patients did not comply with the criteria for known hemostatic disorders (UD, unknown diagnosis). BT and CT were prolonged in 35.8% and 29.7% of all the patients, respectively (P = 0.23). Sensitivity increased to 48% if an abnormality of BT and/or CT was considered. Same comparisons for BT and CT in each diagnostic category were, respectively: 42 vs. 61.5% in VWD-1 (P = 0.18), 42 vs. 24% in platelet secretion defects (P = 0.11), 67 vs. 89% in VWD-1 + PSD (P = 0.50), and 27.5 vs. 15% in UD (P = 0.06). CONCLUSION: Both tests were relatively insensitive and not significantly different in detecting incoming patients with mucocutaneous hemorrhages. In patients with VWD-1, the PFA-100 performed slightly better, whereas the opposite occurred in those patients with platelet secretion defects. In the UD group, both tests lost sensitivity, but the BT detected 1.8 times more patients than the PFA-100. Given the large proportion of undiagnosed bleeders and the overall low sensitivity of these tests, clinical decisions still rely on the medical history and etiological diagnosis of the bleeding disorder.

Bleeding Time↗

The effect of epsilon-aminocaproic acid on HemoSTATUS and kaolin-activated clotting time measurements.

UNLABELLED: New point-of-care assays have been used to identify patients with heparin resistance (i.e. heparin dose response test; Medtronic Blood Management, Parker, CO) and who have platelet dysfunction (i.e. HemoSTATUS; Medtronic Blood Management). We examined the effect of epsilon-aminocaproic acid on results from these two point-of-care tests in patients undergoing cardiac surgery. Twenty patients scheduled for elective cardiac surgical procedures were enrolled in this prospective study. HemoSTATUS clot ratio (% maximal) values in Channels (Ch) 3-6 (Ch 3: 26 +/- 25, Ch 4: 66 +/- 23, Ch 5: 84 +/- 20, Ch 6: 106 +/- 18) obtained after the IV administration of epsilon-aminocaproic acid were similar to values obtained before the administration of this agent (Ch 3: 26 +/- 20, Ch 4: 69 +/- 23, Ch 5: 86 +/- 19, Ch 6: 109 +/- 14). Slope values (86 +/- 23 s x U(-1) x mL(-1)) and projected heparin concentrations (4 +/- 1 U/mL) obtained before the administration of epsilon-aminocaproic acid were similar to slope values (88 +/- 21 s x U(-1) x mL(-1)) and projected heparin concentrations (4 +/- 1 U/mL) values obtained after administration of this agent. Our data indicate that HemoSTATUS clot ratio values and heparin dose response values are not significantly affected after IV dosing of epsilon-aminocaproic acid. IMPLICATIONS: Values from two activated coagulation time-based test systems used to identify significant heparin resistance or platelet dysfunction after cardiopulmonary bypass were not significantly affected by epsilon-aminocaproic acid administered IV.

Aged↗

Hyperprolactinemia of pregnancy is not associated with increased in vivo platelet activity and shortened in vitro bleeding times.

The aim of the study is to investigate whether platelet activity is increased by hyperprolactinemia during pregnancy as reflected by beta-thromboglobulin level. Forty-eight healthy, pregnant, and 30 healthy, non-pregnant women were investigated with respect to platelet count, collagen/ADP and collagen/epinephrine closure times, beta-thromboglobulin and prolactin levels. The comparison of the variables between the two groups was made by Mann-Whitney U test. The correlation analyses were performed by Spearman's rank correlation test. Our results revealed that platelet counts, collagen/ADP and collagen/epinephrine closure times and beta-thromboglobulin showed no statistically significant differences between pregnant and non-pregnant women. We found no significant correlation between prolactin and collagen/ADP closure time (r = 0.175), between prolactin and collagen/epinephrine closure time (r = -0.112) and between prolactin and beta-thromboglobulin (r = 0.220) in pregnant women. Our findings suggest that platelet activity is comparable during pregnant and non-pregnant states and there is no significant effect of prolactin on platelet function in vivo as reflected by beta-thromboglobulin level.

Adenosine Diphosphate↗

The values of synthetic substrates in the improvement of diagnostic tests for haemostatic function.

The accumulated evidence suggests that synthetic substrates measure a similar biological expression as their natural counterpart with, in some cases, a better precision and simplicity of operation. Proteolytic enzymes are involved in a variety of physiological and pathological processes extending from blood coagulation to the release of specific enzymes from certain cells and bacteria. The variation in the level of these proteases, and their inhibitors in plasma or in cells, will be an excellent indicator of any malfunction in the particular system. Excellent agreement between these new generation tests and other conventional methods particularly in measuring fibrinolytic components is seen, and the assay time is cut from hours to a matter of minutes using a reaction rate analyser. Some discrepancy in the estimated potency was observed between methods used for the assay of thrombin, FVIII and several other clotting factors and their inhibitors. There has been methodological improvement and, in some cases, the cause of discrepancy has been revealed. Nevertheless, greater emphasis should still be given to standardization problems. It is, therefore, suggested that until the problems of standardization of various assay methods are resolved and the complications due to non-specificity in chromogenic methods and non-selectivity in clotting tests are overcome, the ratio of amidolytic to clotting activities should be included in the request for potency estimations and in the diagnostic assessment. Whether or not the use of substrates should be complementary or supplementary is left to individual centres according to their particular circumstances.

Aniline Compounds↗

New methods showing in vitro changes of platelets.

INTRODUCTION: In vitro methods for assessing stored platelets are time consuming and variably correlate with in vivo survival. We used the ADVIA 120 and the Thromboelastogram (TEG) to assess changes during storage to determine a correlation of the results with those of other commonly used measures. METHODS: Platelet concentrates were tested at 1 and 5 days of storage. In vitro test included platelet count, aggregation response, the hypotonic shock response (HSR), The Kunicki morphology score (KMS) and platelet activation using CD62. The ADVIA was used to measure the platelet density and the granule content. The TEG measured the kinetics of clot formation. RESULTS: The platelet count and HRS did not change during storage. Aggregation decreased 50% by day 5. The KMS dropped from 307 at day 2-170. The MPC fell below 200g/l. There was slight platelet activation; the rate of clot formation decreased. CONCLUSIONS: Standard measures of in vitro platelet function indicated alterations during storage. A MPC of less than 200 corresponded to a Kunicki morphology score of less than 200. The TEG results correlated will with the KMS. These measurements may prove useful in predicting platelet survival and recovery.

Blood Platelets↗

Platelet consumption in cardiovascular disease.

Many tests have been devised to investigate the role of platelets in arterial or venous thromboembolism. The mechanisms of platelet reactivity that the tests have measured have been variable and the early studies have been of little value in determining the contribution of platelet consumption in thromboembolic diseases. More recently, tests of in vivo platelet release and of platelet survival and turnover have been introduced and thought to be of great potential in the investigation of thromboembolic disorders. However, the data published thus far using more tests are far from conclusive that platelet activation and consumption occurs in thromboembolic disorders associated with arteriosclerosis. It may be that they are neither sensitive nor specific enough to detect minor changes in platelet activation. There is, however, some consistency that in conditions involving the larger vessels, prosthetic surfaces, or in association with active and recurrent venous thromboembolism, platelet release and platelet consumption measured by platelet survival time may occur. In particular, the abnormal results are consistently found in acute and recurrent venous thromboembolism, cardiac valve replacement with the older prosthetic devices, and arteriosclerosis of the larger arteries with clinical episodes of thromboembolism. However, in coronary artery disease, cerebrovascular disease, and peripheral vascular disease, the data are inconclusive, probably because the tests lack sensitivity. In addition, the value of the tests in the management of patients with thromboembolic disorders appears to be limited.

Angina Pectoris↗

A study of platelet aggregation and adhesion in gout.

Platelet aggregation and adhesion were measured in twelve male patients with gout. Tests were performed with the patients off all treatment for six weeks and after the serum uric had been lowered with allopurinol. Results of aggregation and adhesion tests were normal and not influenced by the uric acid level. The increased cardiovascular disease risk in gout is unlikely to be due to an increased thrombotic tendency.

Adult↗

[Maternal mortality in HELLP syndrome in Bavaria 1983-1992].

150 cases of maternal death were registered in Bavaria from 1.1.1983-31.12.1992. 15 women (10%) died in the puerperal period due to preeclampsia. 14 of these patients either had single or complete laboratory findings in HELLP-Syndrom. Anamnesis, age, parity, nationality, way of delivery and causes of death are analysed. To improve the prognosis of patients with HELLP-Syndrom early diagnosis and immediate delivery are necessary. First signs of pre-eclampsia require regular liver-enzymes and platelet controls. In the occurrence of right upper abdominal pain in the second half of pregnancy precise diagnostics and close supervision in hospital is mandatory.

Cause of Death↗

Modified in vivo platelet retention test for evaluating primary haemostasis.

We present a modified in vivo platelet retention test for evaluating platelet plug formation in a standardized incision applied to the skin (template bleeding time). In contrast to previously published similar procedures we determined platelet retention at defined time intervals. The investigation of 20 healthy subjects with this method revealed a characteristic biphasic pattern of platelet consumption at the wound surface, which could be correlated with primary and secondary platelet aggregation. In 10 patients with congenital platelet dysfunction and 2 patients with von Willebrand's disease the pattern of platelet retention was affected principally in its early phase, whereas aspirin, when given to 2 healthy subjects, inhibited platelet retention predominantly in its late phase. Our modified platelet retention test provides information about dynamic events associated with platelet plug formation in both normal and disordered primary haemostasis and may also be useful for evaluating in vivo effects of antithrombotic drugs.

Adolescent↗

Comparison of PFA-100 and bleeding time testing in pediatric patients with suspected hemorrhagic problems.

BACKGROUND: The bleeding time test is difficult to perform, standardize, and interpret in children. In this study the authors evaluated the sensitivity, specificity, and efficiency of the bleeding time test and the PFA-100 in a series of children referred for possible bleeding problems. PATIENTS AND METHODS: Between February 2000 and August 2001 patients aged more than 6 months and less than 18 years of age who were referred to the authors' institution for a hemostatic evaluation were included in the study if residual blood was available for testing on the PFA-100 instrument. Fifty-two children had platelet count, prothrombin time, partial thromboplastin time, bleeding time, and PFA-100 testing performed as well as an evaluation by a hematologist. For PFA-100 testing, 52 patients had Col/Epi measurements; adequate sample remained for Col/ADP testing on 32. Additional testing for diagnostic purposes was at the discretion of the treating physician. RESULTS: Use of the Col/Epi cartridge in the PFA-100 instrument offered 100% sensitivity and 97% specificity for detection of qualitative platelet abnormalities, compared with 37% and 88%, respectively, for bleeding time testing. For pediatric patients with von Willebrand disease, the sensitivity was 100% using the Col/Epi cartridge, compared with 17% for the bleeding time test. The sensitivities for combined qualitative platelet defects and von Willebrand disease using the Col/Epi or Col/ADP cartridges were 100% and 87%, respectively, compared with 37% for the bleeding time test. CONCLUSIONS: The PFA-100 is a more efficient test; it can replace the bleeding time test as a component of the laboratory evaluation of children with a potential bleeding problem.

Adolescent↗

Micromethod for bleeding time in the newborn.

A new method of measuring bleeding time using an Autolet device is described. Normal ranges of bleeding time and volume of blood used in the test have been defined in a population including preterm babies. Abnormal values have been shown in newborns with a variety of problems, and an abnormality of bleeding time has been found to precede intraventricular haemorrhage.

Bleeding Time↗

Attenuated platelet sensitivity to collagen in patients with neurofibromatosis type 1.

Haemostatis has not previously been studied in patients with neurofibromatosis 1 (NF-1), despite case reports of an association with von Willebrand disease and reported excessive bleeding in those undergoing surgery for neurofibromas. Platelets from NF-1 patients (n = 28) were tested for aggregation and ATP release with agonists including ADP, arachidonic acid, thrombin and collagen. Mepacrine staining of platelets and three different assays for von Willebrand factor (VWF) were also performed. In response to collagen as the platelet agonist, tested at both 2 and 1 micrograms/ml, NF-1 patients had an attenuated rate of aggregation (P < 0.007), aggregation lag phase (P < 0.005) and ATP release (P < 0.045), as well as requiring higher collagen concentrations to attain threshold aggregation response (P = 0.041). Normal platelets resuspended in selected NF-1 plasma exhibited significantly reduced platelet aggregation and release compared to controls, which was not corrected by mixing 1:1 with normal plasma. Collagen binding activity was reduced in NF-1 patients compared with controls (127% v 161%, P = 0.05). As a group, patients with NF-1 display defective platelet function characterized by in vitro evidence of impaired responsiveness to collagen. It is suggested that a plasma factor, present in a significant proportion of NF-1 patients, may interfere with the ability of collagen to interact with other proteins such as von Willebrand factor and the platelet collagen receptor.

Adenosine Triphosphate↗

Neonatal alloimmune thrombocytopenia due to an antibody against a labile component of human platelet antigen-3b (Bak b).

We report the second case of neonatal alloimmune thrombocytopenia due to anti-human platelet antigen (HPA)-3b (Bak(b)) antibody. The infant was severely affected with intracranial haemorrhage. Most importantly, we were able to detect this antibody only by using fresh, unfixed platelets as antigen. This antibody was detectable neither by conventional mixed passive haemagglutination, platelet immunofluorescence test using fixed platelets, nor by monoclonal antigen immobilization of platelet antigen assay using solubilized platelets. We assume that this antibody reacts only with fresh platelets and that the antigen is a labile component of HPA-3b (Bak(b)).

Antigens, Human Platelet↗

[Platelet hyperaggregation premonitory of myocardial infarct? A clinical case report].

In a patient, treated with antiplatelet drugs because of two previous TIA episodes, an occasional control in apparent welfare, just 4 hours before the onset an IMA, showed a sudden considerable increase of spontaneous aggregation. Two weeks after the infarction all haematological findings, including aggregation, were back to normal. Platelet adhesiveness, evaluated for the first time, was nevertheless considerably increased. A treatment with an association of low doses of acenocoumarine and ASA was useful to take aggregation and adhesiveness firmly back to normal values that persist 22 month after the episode. Spontaneous platelet hyperadhesion, occasionally recorded a few hours before IMA, bears out the importance of the role played by platelets in vascular coronary accident. Authors suggest that it is therefore advisable to evaluate platelet adhesiveness too, regardless of the specific meaning of this test; it is worth investigating whether this monitoring should lead to some attempt whatsoever enabling to prevent recurrent crisis, adding to antiaggregant therapy, a management with anticoagulating drugs, administered with low dosage.

Anticoagulants↗

Coagulation and bleeding disorders: review and update.

Hemostasis is initiated by injury to the vascular wall, leading to the deposition of platelets adhering to components of the subendothelium. Platelet adhesion requires the presence of von Willebrand factor and platelet receptors (IIb/IIIa and Ib/IX). Additional platelets are recruited to the site of injury by release of platelet granular contents, including ADP. The "platelet plug" is stabilized by interaction with fibrinogen. In this review, I consider laboratory tests used to evaluate coagulation, including prothrombin time, activated partial thromboplastin time, thrombin time, and platelet count. I discuss hereditary disorders of platelets and/or coagulation proteins that lead to clinical bleeding as well as acquired disorders, including disseminated intravascular coagulation and acquired circulating anticoagulants.

Blood Coagulation Disorders↗

An in vitro test of platelet viability.

The survival of transfused platelets is inversely related to the time of storage of platelets in vitro. The effects of storage on the ability of platelets to produce retraction of clots and on the morphologic alterations of platelets within the clots has been studied. The results suggest that clot retraction, using low concentrations of platelets, may be useful as an in vitro method for evaluating the viability of platelets.

Blood Platelets↗