The impact of intravenous aspirin administration on platelet aspirin resistance after on-pump coronary artery bypass surgery.

Aspirin resistance continues to be a major challenge in patients after coronary artery bypass grafting (CABG). We investigated the impact of intravenous aspirin on platelet function in this clinical setting. Forty-two patients received 100 mg of oral aspirin once daily, beginning on day 1 after the operation. Between day 6 and 8 post operation one oral dose was replaced by an intravenous dose of 300 mg. Platelet function analyzer (PFA-100) closure times (CT), turbidimetric platelet aggregation (TPA) and impedance platelet aggregation (IPA) induced by arachidonic acid (AA), collagen and ADP were measured prior to and 1 h and 24 h after intravenous aspirin. Results obtained prior to the intravenous aspirin were compared with respective values from 120 healthy individuals. Despite the postoperative oral aspirin that was given once daily, ADP-induced TPA (ADPTPA) and IPA values induced by AA, ADP or collagen were significantly greater in patients than in controls, while PFA-100 CT were significantly shorter. Intravenous aspirin induced a significant reduction of platelet aggregability as measured by collagen/epinephrine (CEPI) CT, TPA and IPA induced by AA and collagen 1 h and 24 h after administration. Intravenous aspirin was not found to influence collagen/ADP (CADP) CT and IPA induced by ADP. Concomitantly, the number of patients with laboratory aspirin resistance as measured by CEPI-CT and TPA but not by IPA induced by AA or collagen dropped significantly. Agreement in the detection of aspirin responders and non-responders among platelet function tests was poor. Our findings indicate that the intravenous aspirin appears to be a promising approach for reducing laboratory aspirin resistance during the postoperative phase of CABG.

Variables influencing Multiplate(TM) whole blood impedance platelet aggregometry and turbidimetric platelet aggregation in healthy individuals.

We investigated the relationship between impedance platelet aggregometry (IPA) as measured by the Multiplate system and turbidimetric platelet aggregation (TPA) induced by ADP, arachidonic acid (AA), and collagen; blood cell counts; platelet function analyzer (PFA-100) closure times (CT), and von Willebrand factor (VWF) in 120 well-characterized healthy individuals. Pre-analytical and analytical conditions were standardized comprehensively. Analytical reliability of IPA and TPA and the influence of pre-analytical variables on assay results were also examined. IPA and TPA did not change significantly between 0.5 and 5 hours after blood collection when samples were stored at room temperature. TPA and IPA showed significantly greater intra-assay imprecision than respective TPA induced by the same agonists. Intra-individual variation did not differ significantly between IPA and TPA. The lower limits of reference range (2.5th percentiles) of AAIPA, ADPIPA and collagen IPA determined AM were 37, 20 and 40 AU, respectively. ADPIPA showed significantly lower maximum aggregation values than AAIPA and collagen IPA (P < 0.0001). There were no significant differences in any parameter between males and females. No significant differences between blood group 0 and non-0 individuals were noted with respect to IPA and TPA. IPA did not change significantly during the day. In contrast, TPA measured PM was significantly lower than corresponding values determined a.m. (p < 0.0001). CEPI-CT, CADP-CT and leukocyte counts increased significantly from a.m. to p.m. (P = 0.008 and P > 0.0001, respectively). Donors had significantly greater IPA induced by any agonist than non-donors (P-values < 0.0001, 0.0001 and 0.001, respectively), whereas TPA was not significantly different between donors and non-donors. IPA did not correlate significantly with TPA nor with PFA-100 CT. ADPIPA and collagen IPA correlated significantly with platelet count. TPA was not associated with platelet count. An inverse significant correlation was observed between TPA induced by any agonist and leukocyte count, whereas leukocyte count did not influence IPA. CEPI-CT and CADP-CT correlated significantly with VWF:CBA and with each other but not with TPA. We concluded that IPA and TPA measure different aspects of platelet function. IPA results reflect interactions between platelets, red and white cells, while TPA does not. This explains discrepancies in associations of IPA and TPA with cell counts, time of day and blood donation. The clinical significance of IPA determined using the Multiplate device remains to be determined in studies on patients with platelet dysfunction and under treatment with antiplatelet agents.

Can platelet function tests predict the clinical efficacy of aspirin?

"Aspirin resistance" and "aspirin nonresponsiveness" are terms used both to describe both the failure of aspirin to protect subgroups of individuals from severe vascular events and to evoke an appropriate inhibition of platelet function. Several studies utilizing a broad range of platelet function tests have shown that some subgroups of individuals exhibit a reduced or completely missing antiplatelet response to aspirin. The clinical significance of aspirin nonresponsiveness for the prediction of clinical endpoints remains, however, to be determined. Thus far, only three prospective clinical trials have demonstrated a possible relationship between aspirin nonresponsiveness and subsequent vascular events. Most platelet function tests used in respective clinical studies cannot be reliably performed in clinical routine and are not interchangeable for monitoring antiplatelet treatment. There is a need for a simple and reliable assay for predicting the clinical efficacy of antiplatelet therapy. Recent data demonstrate that none of the currently developed assays, including the PFA-100 system, are presently able to accomplish these objectives.

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

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

Relationship between factor XIII activity, fibrinogen, haemostasis screening tests and postoperative bleeding in cardiopulmonary bypass surgery.

We investigated the relationship between factor XIII, fibrinogen, blood coagulation screening tests and postoperative bleeding in 98 patients undergoing cardiopulmonary bypass (CPB) surgery. All patients received aprotinin. Blood samples were collected preoperatively (T1),after termination of CPB (T2),12 h (T3) and 24 h (T4) after surgery to determine FXIII activity, fibrinogen, platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT) and D-dimers (DD). Laboratory results were correlated with the chest tube drainage 24 h after surgery and compared between patients with 24-hour chest tube drain volumes in the lower (Group 1) with those in the upper tertile (Group 3). Median FXIII and fibrinogen levels dropped by 33.9% and 34.2%, respectively, during CPB. No association between FXIII activity and the extent of postoperative bleeding was found. However, chest tube bleeding was significantly correlated with preoperative and postoperative fibrinogen. This was confirmed by comparing Groups 1 and 3. Group 3 patients had significantly lower fibrinogen levels than Group 1 at T1 - T4, although most fibrinogen values were within or above the reference range (medians, g/l: 3.5 vs. 4.0, p = 0.043 at T1; 2.3 vs. 2.7, p = 0.015 at T2; 2.9 vs. 3.3, p = 0.008 at T3; 4.2 vs. 5.2, p = 0.002 at T4). There was also a significant relationship of platelet count, PT and APTT, as measured after CPB (T2), with postoperative chest tube drainage. In conclusion, plasma FXIII activity does not influence postoperative bleeding in patients undergoing CPB surgery. There is however an inverse association between preoperative or postoperative plasma fibrinogen levels and postoperative bleeding. These findings indicate a modulation of postoperative bleeding by fibrinogen levels.

The quality of plasma collected by automated apheresis and of recovered plasma from leukodepleted whole blood.

BACKGROUND: There exists a current lack of information about the composition of the different types of plasma. No direct comparisons between apheresis plasma (AP) and recovered plasma (RP) derived from in-line-filtered whole blood (WB) have been published to date. STUDY DESIGN AND METHODS: Sixty AP units, 100 RP units from in-line-filtered WB held for 3 hours at 20 degrees C between donation and freezing, and an additional 100 RP units held for 15 hours at 20 degrees C before freezing were analyzed for coagulation factors and inhibitors, total protein, immunoglobulin G (IgG), and hemostasis and proteolysis activation markers. The influence of twice freezing and thawing on clotting factors V, VIII, and XI was also examined. RESULTS: AP contains substantially greater activities of factor (F) V, FVIII, F IX, and FXI than RP frozen within 3 hours after WB donation. Prolonged holding of RP at 20 degrees C for more than 15 hours caused an additional reduction in FVIII, FXI, and protein S activities. Significantly greater levels of prothrombin fragments 1 and 2, platelet factor 4, and neutrophil elastase were found in RP compared with AP. IgG was lower in AP compared with RP. Twice freezing and thawing caused a marked drop in FV, FVIII, and FXI activity. CONCLUSION: Higher FVIII and F IX potencies in AP compared with RP can be expected to result in greater yields when used for purification of these clotting factors. AP is presumably more efficient than RP for treating coagulopathies. RP, however, may contain higher IgG levels than AP.

Laboratory monitoring of heparin and the combination of heparin and the platelet glycoprotein IIb/IIIa receptor antibody fragment abciximab (c7E3) in patients undergoing percutaneous transluminal coronary angioplasty (PTCA).

BACKGROUND: Previous studies in patients undergoing percutaneous transluminal coronary angioplasty (PTCA) have restricted laboratory monitoring to the activated clotting time (ACT). It remains unknown whether the ACT-prolonging effect of abciximab is clinically equivalent to a comparable degree of anti-coagulation by heparin. PATIENTS AND METHODS: 30 patients undergoing PTCA received 100 IU of heparin/kg body weight. Another 30 patients received an initial bolus of 70 IU of heparin/kg + abciximab. We determined ACT, laboratory and onsite activated partial thromboplastin time (APTT) and plasma heparin levels. RESULTS: Despite markedly different preintervention heparin dosing, the ACTs were not significantly different between groups. After termination of PTCA, the median ACTs of both study groups were nearly equivalent (267 vs. 272 s; p = 0.79). The median ACT-prolonging effect of abciximab could be equated with 0.68 IU/ml heparin. Both APTT assays were not suitable for monitoring the anticoagulant effects during PTCA due to their high sensitivity. By contrast, the plasma heparin levels clearly reflected the different heparin doses. The weak correlation (r = 0.23) between ACTs and heparin levels in patients receiving heparin + abciximab was due to excessively prolonged ACTs in six patients (540-1,245 s). These data could be attributed to an unusually high response to abciximab. By contrast, the ACT was a reliable measure of the anticoagulant effect of heparin in patients receiving exclusively heparin. CONCLUSIONS: ACT reflects both heparin and abciximab therapy, whereas heparin levels reflect only heparin dose. The APTT assays were not suitable for monitoring the anticoagulant effects during PTCA.

The impact of intensive serial plasmapheresis and iron supplementation on iron metabolism and Hb concentration in menstruating women: a prospective randomized placebo-controlled double-blind study.

BACKGROUND: Iron (Fe) depletion is common among regular whole-blood donors, but can be prevented through regular oral Fe supplementation. Little is known, however, about the Fe metabolism of donors undergoing intensive plasmapheresis. These donors lose considerable amounts of blood drawn for laboratory analyses and remaining in the disposable plastic sets. STUDY DESIGN AND METHODS: Menstruating women were enrolled in a prospective placebo- controlled double-blind study. One hundred women were randomly allocated to receive either 100 mg of elemental Fe per day or placebo over 24 weeks and asked to donate plasma at 1-week intervals. Hb was determined before each plasmapheresis. Ferritin, transferrin, and Fe concentration and reticulocyte count were measured every 4 weeks. RESULTS: Thirty donors in the placebo group and 29 receiving Fe completed the study. The total mean blood loss was 526 mL in the placebo group and 546 mL in the Fe arm (p=0.271). The number of donations with Hb values lower than 12.5 g per dL requiring prolongation of the time interval until the next plasmapheresis was significantly greater in the placebo arm. In the placebo group (n=30), ferritin levels began to decline significantly 4 weeks after entry. When the study was completed, Hb concentration and reticulocyte count also were found to be significantly lower in the placebo group than in the Fe study arm (p=0.028 and p=0.036, respectively). Hb, ferritin, and transferrin levels and reticulocyte counts did not change significantly in the Fe group during the observation period. CONCLUSION: Menstruating women undergoing regular plasmapheresis at short intervals are prone to develop Fe depletion. This can be prevented by regular Fe intake. Laboratory analyses in product plasma instead of serum gained from whole-blood samples could be an alternative to reduce blood loss.

Recurrent pregnancy loss and its relation to FV Leiden, FII G20210A and polymorphisms of plasminogen activator and plasminogen activator inhibitor.

Thrombophilic disorders and hypofibrinolysis were demonstrated to be risk factors in a majority of women with recurrent pregnancy loss (RPL) and infertility. We investigated the association of FV G1691A mutation, F II G20210A gene polymorphism (PM), 4G/5G PAI-1 and Alu I/D tPA PM in 32 women with infertility and 49 women with at least 2 unexplained early abortions. FV Leiden mutation was significantly more common in women with RPL (10%, p = 0.02) and infertility (19%, p = 0.0005) compared with controls (2%). PAI-1 4G PM and t-PA Alu I PM, alone or in combination, were not associated with RPL or infertility. 9/49 women with RPL showed coagulation disorders with heterozygous FV Leiden mutation (5), FXII (1), protein C (1) or protein S (2) deficiency. However, due to the small number of patients studied, no definite conclusion can be drawn.

Indications for plasma in massive transfusion.

The initial aim in massive transfusion (MT) is to supply crystalloids, colloids, and plasma-poor red cell concentrates (RCCs) to maintain normovolemia and oxygen supply. This frequently leads to dilution coagulopathy, which is frequently aggravated and accelerated by hypothermia, acidosis, shock-induced impairment of liver function and disseminated intravascular coagulation (DIC), and increased consumption of clotting factors and platelets at extensive wound sites. Disorders of hemostasis deteriorate the prognosis of massively transfused patients dramatically. Therefore, the second therapeutic objective is the timely administration of plasma and platelet concentrates as required to halt the microvascular bleeding (MVB) induced by impaired hemostasis. Close laboratory monitoring, to include as a minimum platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen, is essential to identify hemostatic disorders requiring therapeutic intervention. Coagulopathy promoting microvascular bleeding can be assumed when PT or APTT values exceed 1.5 times mean controls and/or when fibrinogen levels fall below 1.0 g/l. Repeated rapid infusion of 10-15 ml plasma per kg of body weight will be required to raise clotting factor levels significantly and to achieve adequate hemostasis. The turnaround time for obtaining laboratory results and for readying plasma for transfusion must be taken into particular consideration in cases of rapid blood loss.

Manufacture and composition of fresh frozen plasma and virus-inactivated therapeutic plasma preparations: correlation between composition and therapeutic efficacy.

The clinical efficacy of the therapeutic plasma used in the treatment of congenital and acquired severe coagulopathy depends on the potency of clotting factor and inhibitor activities. The composition of plasma strongly depends on the conditions under which it is produced. A low citrate anticoagulant-to-blood ratio, short intervals between donation and plasma separation and rapid freezing markedly improve the preservation of unstable coagulation factors. The influence of different leukocyte reduction filters on plasma quality still requires clarification. Recent trials on long-term storage conditions suggest that keeping plasma at -30 degrees C or colder over a period of 24-36 months prevents substantial decrease in clotting factor activities including factor VIII (FVIII). Three types of therapeutic plasma are currently available. Quarantine-stored fresh frozen plasma (FFP) contains physiological activities of therapeutically relevant plasma proteins, but carries a risk of transmitting blood-borne viruses that cannot be detected by human immunodeficiency virus (HIV) and hepatitis B and C screening. In contrast, solvent/detergent-treated plasma (SDP) and methylene blue/light-treated plasma (MBP) is virtually free of HIV and hepatitis C virus (HCV) subtypes. Virus inactivation procedures can have the consequence of reducing several clotting factors and inhibitors in SDP and MBP to varying degrees. However, pooling of plasma units before solvent/detergent (SD) treatment results in well-standardized protein levels of SDP. At least five prospective trials and four observational studies covering different clinical settings suggest that SDP and FFP do not substantially differ in their clinical efficacy or in their tolerance. By way of contrast, there is a lack of data about the clinical efficacy and tolerance of MBP compared to FFP.