Comparison of point-of-care methods for preparation of platelet concentrate (platelet-rich plasma).

PURPOSE: This study analyzed the concentrations of platelets and growth factors in platelet-rich plasma (PRP), which are likely to depend on the method used for its production. MATERIALS AND METHODS: The cellular composition and growth factor content of platelet concentrates (platelet-rich plasma) produced by six different procedures were quantitatively analyzed and compared. Platelet and leukocyte counts were determined on an automatic cell counter, and analysis of growth factors was performed using enzyme-linked immunosorbent assay. RESULTS: The principal differences between the analyzed PRP production methods (blood bank method of intermittent flow centrifuge system/platelet apheresis and by the five point-of-care methods) and the resulting platelet concentrates were evaluated with regard to resulting platelet, leukocyte, and growth factor levels. The platelet counts in both whole blood and PRP were generally higher in women than in men; no differences were observed with regard to age. Statistical analysis of platelet-derived growth factor AB (PDGF-AB) and transforming growth factor ß1 (TGF-ß1) showed no differences with regard to age or gender. Platelet counts and TGF-ß1 concentration correlated closely, as did platelet counts and PDGF-AB levels. There were only rare correlations between leukocyte counts and PDGF-AB levels, but comparison of leukocyte counts and PDGF-AB levels demonstrated certain parallel tendencies. CONCLUSIONS: TGF-ß1 levels derive in substantial part from platelets and emphasize the role of leukocytes, in addition to that of platelets, as a source of growth factors in PRP. All methods of producing PRP showed high variability in platelet counts and growth factor levels. The highest growth factor levels were found in the PRP prepared using the Platelet Concentrate Collection System manufactured by Biomet 3i.

Multicentre evaluation of a second generation point-of-care assay with an extended range for the determination of N-terminal pro-brain natriuretic peptide.

BACKGROUND: In the second generation of the point-of-care (POC) assay Roche CARDIAC proBNP, the upper limit of the measuring range was extended from 3000 to 9000 ng/L. METHODS: A thirteen-site multicentre evaluation was carried out to assess the analytical performance of the POC NT-proBNP assay and to compare it with a laboratory N-terminal pro-brain natriuretic peptide (NT-proBNP) assay. RESULTS: In method comparisons of six lots of POC NT-proBNP with the lab reference method (Elecsys proBNP) mean bias ranged from -10 to +17%. In lot-to-lot comparisons all six investigated lots of POC NT-proBNP showed excellent agreement, with mean bias between -7% and +2%. The majority of all coefficients of variation obtained from ten-fold measurements using 56 native blood samples were below 8%. No interference was observed with hemolytic blood (hemoglobin concentrations up to 0.12 mmol/L), lipemic blood (triglyceride concentrations up to 14.0 mmol/L) nor icteric blood (bilirubin concentrations up to 63 micromol/L). Hematocrit values between 24% and 51% had no influence on the assay result. High NT-proBNP concentrations above the measuring range of POC NT-proBNP did not lead to false low results due to potential high-dose hook effect. Results with POC NT-proBNP were not influenced by different ambient temperatures (18 degrees C to 32 degrees C), the sample material used, nor by over- or underdosing by 15 microL compared to the regular sample volume of 150 microL. CONCLUSIONS: The POC NT-proBNP assay showed an excellent analytical performance including a good agreement with the laboratory method. The assay is therefore suitable for its intended use in point-of-care settings.

Prognostic value of lipoproteins and their relation to inflammatory markers among patients with coronary artery disease.

BACKGROUND: Lipoproteins and their subfractions are associated with the incidence of atherosclerotic diseases. In patients with coronary artery disease (CAD), low serum concentrations of high density lipoprotein (HDL) and high low-density lipoproteins (LDL) are correlated to myocardial infarction and cardiovascular death. There is growing evidence indicating that those lipoprotein factors are related to the inflammatory process in atherogenesis. METHODS: We investigated in a median follow up of 3.9 years the association of HDL, apolipoprotein A-I (apoA-I), LDL, apolipoprotein B (apoB), and triglycerides with the incidence of a combined endpoint (myocardial infarction and cardiovascular death) and their relation to markers of inflammation in 1298 patients with angiographically documented CAD. RESULTS: In univariate analysis, serum concentrations of apoA-I were significantly and inversely related to the combined endpoint, whereas serum concentrations of LDL, apoB, and triglycerides were not. HDL was not significantly related to the endpoint in univariate analyses (p=0.057). Multivariate analyses showed that only apoA-I is an independent predictor. ApoA-I (and HDL) was significantly related to markers of inflammation. CONCLUSION: Serum apoA-I levels were an independent predictor for fatal and non-fatal cardiovascular events in patients with CAD. This may be related to its anti-inflammatory effect.

Comparison of the platelet concentrate collection system with the plasma-rich-in-growth-factors kit to produce platelet-rich plasma: a technical report.

PURPOSE: The aim of this study was to compare a new method for the production of platelet-rich plasma (PRP), the plasma-rich-in-growth-factors kit (PRGF kit; G.A.C. Medicale San Antonio, Vitoria, Spain), with an established method, the Platelet Concentrate Collection System (PCCS; 3i/Implant Innovations, Palm Beach Gardens, FL) with respect to resulting cellular and growth factor contents. MATERIALS AND METHODS: Whole blood was drawn from 51 healthy donors (20 men, 31 women) aged 19 to 59 years (mean +/- SD 35.12 +/- 9.65 years), and PRP was prepared by both methods. RESULTS: Platelet counts differed significantly (signed rank test, P < .001 for all) between the donor blood (274,200 +/- 54,050/microL), the PCCS PRP preparation (1,641,800 +/- 426,820/microL), and the PRGF kit PRP preparation (513,630 +/- 139,470/microL). The PCCS concentrated leukocytes (whole blood, 6,992 +/- 2,011/microL; PCCS PRP, 14,153 +/- 7,577/microL), while the PRGF kit produced a leukocyte-poor PRP (65 +/- 108/microL). Higher concentrations of transforming growth factor beta1 (TGF-beta1) and platelet-derived growth factor AB (PDGF-AB) were found in the PCCS PRP (TGF-/beta1, 290 +/- 95 ng/mL; PDGF-AB, 157 +/- 62 ng/mL) than in the Anitua PRGF kit PRP (TGF-beta1, 73 +/- 26 ng/mL; PDGF-AB, 47 +/- 21 ng/mL). Statistical analysis showed significant differences (P < .001 for TGF-beta1 and P < .01 for PDGF-AB). DISCUSSION: The results of this study and some data in the literature indicate that the content of growth factors in PRP can vary tremendously, depending on the system used for the preparation of PRP. CONCLUSION: PCCS collects more platelets and leukocytes than the PRGF kit. This results in significantly higher growth factor levels. Further in vivo studies are needed to determine whether this results in a clinically different biologic effect.

Safety and efficacy of single bolus anticoagulation with enoxaparin for chronic hemodialysis. Results of an open-label post-certification study.

BACKGROUND: Low-molecular-weight heparin (LMWH) is supposed to be advantageous compared to unfractionated heparin for chronic hemodialysis (HD) with respect to lipid and bone metabolism, polymorphonuclear cell stimulation, induction of antibody-mediated thrombocytopenia, and aldosterone suppression. Due to longer biological half-life, LMWH offers the possibility of single bolus administration. METHODS: To assess safety and efficacy of single bolus anticoagulation with enoxaparin for chronic HD, 781 stable HD patients from 79 German dialysis centers (mean age 62 years; 31% ESRD due to diabetes mellitus) were monitored by clinical and laboratory parameters for 32 weeks. Additionally, in a single dialysis center, 22 chronic HD patients were investigated by molecular markers of coagulation during chronic HD under conditions of single bolus or continuous anticoagulation regimens. Anti-Xa activity and the thrombin- antithrombin-III complex (TAT) were determined before the enoxaparin bolus, after 15 min, 2 h, and at the end of HD in venous and arterial blood lines. RESULTS: Chronic HD was performed in 24,117 HD treatments with enoxaparin at a median dose of 70.1 IU/kg (5,000 IU median total dose) for single bolus anticoagulation. In 83.0% of HD treatments, enoxaparin was given as single bolus. In 98.3% of patients no adverse event was reported. No drug-related severe adverse event occurred. Significant clotting problems were observed in only 0.3% of HD treatments with single bolus anticoagulation. As assessed in 257 HD treatments, essentially identical anti-Xa levels were detected at the end of HD with single bolus (50 IU/kg) or continuous (mean total dose 43 IU/kg) anticoagulation regimens. Bolus anticoagulation resulted in higher TAT generation at the end of HD. However, this was not associated with increased macroscopic clot formation. CONCLUSION: Single bolus anticoagulation with enoxaparin was safe and effective for chronic HD. For a duration of 4 h HD, a median dose of 70 IU/kg can be recommended for regular use, which is in accordance with the manufacturer's instructions for use of enoxaparin recommending a range of 50-100 IU/kg.

Genetic and environmental influences on the fibrinolytic system: a twin study.

The determination of heritability is a key issue to assess the predictive power of polymorphisms for disease in clinical studies. The aim of this study was to determine the heritability of proteins and activation markers of the fibrinolytic system in a large cohort of healthy twins. Heritability was calculated as 0.76 for thrombin activatable fibrinolysis inhibitor (TAFI), 0.44 for plasminogen activator inhibitor-1 (PAI-1), and 0.43 for tissue plasminogen activator. No significant genetic influence was observed for alpha2-antiplasmin-plasmin-complex and D-dimer. Heritability explained by single gene polymorphisms was 25.2% for TAFI 505G>A, 31.5% for 1542C>G, and 50.0% for combination of both. The influence on TAFI levels of 1542C>G (CC-->GG, median: -80.5%) was considerably stronger than that of 505G>A (GG-->AA, median: +49.3%) and in both cases there seems to be a dose-response relationship. Significant environmental influences on TAFI levels were observed for combined interaction terms (age*sex and bmi*sex). The PAI-1 4G/5G polymorphism explained 56.4% of the calculated heritability. The genetic variables accounting for the 43% heritability of tPA remain unknown. Our data show that the production of several key components of the fibrinolytic system is strongly genetically determined. This genetic influence is accounted for in large part but not completely by a limited number of polymorphisms within the respective genes associated with plasma levels of the gene products.

Release of choline in the isolated heart, an indicator of ischemic phospholipid degradation and its protection by ischemic preconditioning: no evidence for a role of phospholipase D.

The release of choline as a water-soluble product of phospholipid hydrolysis was measured in the perfusate of rat hearts to monitor ischemic membrane degradation and its protection by ischemic preconditioning (IPC). Hearts were subjected to global ischemia (GI; 30 min of no-flow) followed by 60 min of reperfusion. To induce IPC, GI was preceded by four no-flow episodes of 5 min each. Deleterious consequences of GI and reperfusion, namely coronary flow reduction, incidence of arrhythmias and release of cardiac troponin T, were significantly attenuated by IPC. The release of choline increased during reperfusion in a biphasic manner: a first phase peaked immediately after GI and was followed by a second, delayed phase indicating choline release caused during reperfusion. Only the second phase was blocked by both IPC and by AACOCF3 (5 microM), an inhibitor of cytosolic phospholipase A2. The activity of phospholipase D (PLD) was unchanged after GI or IPC or GI plus IPC. In conclusion, choline release into heart perfusate was found to be a useful real-time indicator of phospholipid degradation caused by GI and by reperfusion and its protection by IPC. The results supplement previous observations on the accumulation of fatty acids in the phospholipid pool. There was no evidence for PLD activation by GI or IPC.

Comparative analysis of procoagulatory activity of haemodialysis, haemofiltration and haemodiafiltration with a polysulfone membrane (APS) and with different modes of enoxaparin anticoagulation.

BACKGROUND: Treatment modalities of renal replacement therapy differ in their diffusive and convective mass transfer characteristics. It was the goal of this study to clarify whether an increase in convective mass transfer as performed with haemofiltration (HF) and haemodiafiltration (HDF) in comparison with high-flux haemodialysis (HD) is associated with an alteration in procoagulatory activity or with complement activation. METHODS: Ten stable chronic HD patients were monitored during 120 treatments in a randomized cross over design. A high-flux polysulfone dialyser (APS 900) was used for high-flux HD, pre-dilution HF and pre-dilution HDF. Constant flow of on-line substitution fluid for HF and HDF was 200 ml/min. The low molecular weight heparin (LMWH) enoxaparin was used for anticoagulation (i) as single bolus (50 IU/kg body weight, median 3700 IU) and (ii) as bolus of 1200 IU followed by a median continuous dose of 400 IU/h. Blood samples were collected before the LMWH bolus, after 10 min, 60 min, 120 min and at the end of treatment in venous and arterial blood lines to determine antiXa activity, thrombin-antithrombin-III complex (TAT), D-dimer and C5a generation. RESULTS: Net ultrafiltration did not significantly differ between HD, HF and HDF but total ultrafiltration in HF and HDF far exceeded total ultrafiltration in HD. With conditions of single bolus, or bolus and continuous anticoagulation with enoxaparin, after comparable treatment times (median duration 4.25 h), TAT and D-dimer generation at identical anti-Xa levels revealed significantly higher coagulation activity during HF and HDF, compared with high-flux HD as assessed by comparative area under the curve (AUC) analysis. Plasma concentration of C5a in venous bloodlines did not significantly differ during HD, HF and HDF. CONCLUSION: A higher convective mass transfer during HF and HDF, in comparison with high-flux HD caused by a greater total ultrafiltration volume was associated with increased procoagulatory activity in the extracorporeal circuit. Molecular markers assessing the activation of coagulation are appropriate to adjust the anticoagulation regime to high UF volumes in order to minimize bleeding risk and optimize patency of the extracorporeal circuit.

Glutathione peroxidase 1 activity and cardiovascular events in patients with coronary artery disease.

BACKGROUND: Cellular antioxidant enzymes such as glutathione peroxidase 1 and superoxide dismutase have a central role in the control of reactive oxygen species. In vitro data and studies in animal models suggest that these enzymes may protect against atherosclerosis, but little is known about their relevance to human disease. METHODS: We conducted a prospective study among 636 patients with suspected coronary artery disease, with a median follow-up period of 4.7 years (maximum, 5.4) to assess the risk of cardiovascular events associated with base-line erythrocyte glutathione peroxidase 1 and superoxide dismutase activity. RESULTS: Glutathione peroxidase 1 activity was among the strongest univariate predictors of the risk of cardiovascular events, whereas superoxide dismutase activity had no association with risk. The risk of cardiovascular events was inversely associated with increasing quartiles of glutathione peroxidase 1 activity (P for trend <0.001); patients in the highest quartile of glutathione peroxidase 1 activity had a hazard ratio of 0.29 (95 percent confidence interval, 0.15 to 0.58; P<0.001), as compared with those in the lowest quartile. Glutathione peroxidase 1 activity was affected by sex and smoking status but retained its predictive power in these subgroups. After adjustment for these and other cardiovascular risk factors, the inverse association between glutathione peroxidase 1 activity and cardiovascular events remained nearly unchanged. CONCLUSIONS: In patients with coronary artery disease, a low level of activity of red-cell glutathione peroxidase 1 is independently associated with an increased risk of cardiovascular events. Glutathione peroxidase 1 activity may have prognostic value in addition to that of traditional risk factors. Furthermore, increasing glutathione peroxidase 1 activity might lower the risk of cardiovascular events.

Impact of pathogen burden in patients with coronary artery disease in relation to systemic inflammation and variation in genes encoding cytokines.

The number of infectious pathogens to which an individual has been exposed (pathogen burden) has been linked to the development and the prognosis of coronary artery disease (CAD). The interaction among infection, genetic host susceptibility, and CAD remains unclear. This study was aimed at evaluating the modulation of the association between CAD and pathogen burden, by serum levels of inflammatory markers and polymorphisms of the interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha genes. Immmunoglobulin (Ig) G or IgA antibodies to 8 pathogens were determined in 991 patients with CAD and 333 control subjects. Serum levels of high-sensitivity C-reactive protein, fibrinogen, IL-6, and TNF-alpha were also measured. All subjects were genotyped for the IL-6/G-174C, the TNF/C-851T, and the TNF/G-308A polymorphisms. Analysis of single pathogens demonstrated a positive relation to the presence of CAD for some (Chlamydia pneumoniae, cytomegalovirus, Helicobacter pylori, and herpes virus simplex type 1), but not all pathogens. A strong association between increasing pathogen burden and CAD was confirmed, even after adjustment for risk factors. The prevalence of a high pathogen burden (>/=4 pathogens) was 50% in patients and 21% in controls (p <0.0001). A high pathogen burden was associated with decreased high-density lipoprotein cholesterol levels (p <0.001). The association between CAD and pathogen burden was modulated by the IL6/G-174C polymorphism, the odds ratio being higher in heterozygotes than in both types of homozygotes (p <0.05). This interaction appeared to be mediated by variations in serum IL-6 levels. No such interaction was detected with any of the 2 TNF-alpha polymorphisms.

Comparison of platelet, leukocyte, and growth factor levels in point-of-care platelet-enriched plasma, prepared using a modified Curasan kit, with preparations received from a local blood bank.

The potential use of autologous thrombocytic growth factors to accelerate bone regeneration requires improved methods of isolating platelet-rich plasma (PRP). In addition to discontinuous cell separation, a second method by which PRP is produced at the point-of-care has now become available. In this study, growth factor levels in PRP from these two sources were compared. Whole blood was drawn from 115 healthy donors (73 males, 42 females) aged 21 - 62 years (mean 36, SD 10). The PRP was separated by the blood bank (BB) using the discontinuous cell separation method or at the 'point-of-care' by the so-called 'buffy coat' method (analogous to the Curasan PRP Kit). Growth factor content differed significantly for TGF-beta1 (BB 268.65+/-70.77 ng/ml, Curasan 95.02+/-60.67 ng/ml (sign test P<0.001)) and PDGF-AB (BB 133.59+/-46.26 ng/ml, Curasan 233.70+/-111.86 ng/ml (P<0.001)), while the content of IGF-I (BB 85.37+/-25.58 ng/ml, Curasan 101.72+/-47.7 ng/ml (P<0.160)) showed no significant difference. The higher thrombocyte count in the BB PRP (BB 1434300+/-351960/ microl, Curasan 908.500+/-492.30/microl) seems to result in higher TGF-beta1 levels, while the higher leukocyte count in the Curasan PRP (BB 160+/-320/ microl, Curasan 30130+/-12500/microl) seems to result in higher PDGF-AB levels. The similar IGF-I levels in the two preparations might merely reflect similar amounts of plasma in the PRP produced by each approach.

Plasma concentrations and genetic variation of matrix metalloproteinase 9 and prognosis of patients with cardiovascular disease.

BACKGROUND: Matrix metalloproteinase (MMP)-9 secretion by macrophages and other inflammatory cells accelerates atherosclerotic progression and destabilizes vulnerable plaque in animal models. However, epidemiological data evaluating the prognostic impact of circulating concentrations and functional genetic variations of MMP-9 are lacking. METHODS AND RESULTS: In a prospective study of 1127 patients with documented coronary artery disease, we measured baseline plasma MMP-9 levels and determined the MMP-9/C-1562T and MMP-9/R279Q genotypes. During the follow-up period (mean of 4.1 years), 97 patients died from cardiovascular (CV) causes. Median concentrations of MMP-9 were significantly higher among patients who experienced a fatal CV event than among those who did not (62.2 versus 47.8 ng/mL; P<0.0001). The crude hazard risk ratio of CV death associated with increasing quartiles of MMP-9 was 1.4 (95% CI, 1.2 to 1.8; P<0.0001), and after adjustment for clinical and therapeutic confounders, it was 1.3 (95% CI, 1.1 to 1.6; P=0.005). Additional adjustment for highly sensitive CRP, interleukin-6, fibrinogen, and interleukin-18 revealed a hazard risk ratio to 1.2 (95% CI, 0.9 to 1.6; P=0.15). The T allele of the C-1562T polymorphism was associated with increased MMP-9 levels in a fairly codominant fashion (P=0.004). Although none of the polymorphisms was significantly related with future CV death, there was a significant association (P=0.02) between the R279Q polymorphism and CV events in patients with stable angina. CONCLUSIONS: Plasma MMP-9 concentration was identified as a novel predictor of CV mortality in patients with coronary artery disease. Whether it provides independent prognostic information compared with other inflammatory markers will have to be additionally assessed.

Nickel release after implantation of the Amplatzer occluder.

BACKGROUND: Transcatheter closure of atrial septal defects is a new and less traumatic technique than open heart surgery. In recent years, patients with a patent foramen ovale sustaining potential paradoxical embolism have also become candidates for interventional closure devices. One of the more popular occluding devices is the Amplatzer septal occluder, which, like many other occluders, is made of nitinol. Nitinol-based alloys are widely used in medical products, for example, in orthopedics and orthodontics. However, the clinical use of nitinol, which contains 55% nickel, is still controversial because of concerns about its biocompatibility. Therefore, we examined the systemic nickel release after implantation of the Amplatzer occluder. METHODS AND RESULTS: In 67 patients with no history of nickel sensitivity, blood samples were taken 24 hours before and 24 hours, 1, 3, and 12 months after occluder implantation. Nickel serum concentrations were measured by atomic absorption spectrometry; a value of <2 ng/mL of nickel was considered to be normal. A rise in mean serum levels of nickel was observed, from 0.47 ng/mL before implantation to 1.27 ng/mL (24 hours after), to a maximum of 1.50 ng/mL 1 month after implantation, which was statistically significant (P =.008 and P = 0.022, Wilcoxon Test). During follow-up, the values decreased to those measured before implantation. CONCLUSIONS: Nickel seems to be released from the device, causing a systemic rise in serum levels of nickel, possibly until a calcium-phosphate layer has formed on the passive oxide film of the device or until endothelialization is complete. Possible biological effects should be considered, particularly in young patients or patients with nickel hypersensitivity.

Prognostic value of troponin T, troponin I, and CK-MBmass in patients with chronic renal failure.

BACKGROUND: In patients with chronic renal failure undergoing long-term hemodialysis, ischemic heart disease accounts for up to 50% of mortality. Cardiac troponins T (TnT) and I (TnI) are frequently elevated in this patient group, but data on the prognostic relevance of these markers, especially TnI, are controversial. The aim of this study was to investigate the prognostic power of a new, sensitive TnI assay in comparison with TnT and other cardiac markers. PATIENTS AND METHODS: 104 ambulatory and in-hospital patients (41 women, median age: 65 years, interquartiles: 48-72.5 years; 63 men, median age: 63 years, interquartiles: 51.3-72.5 years) undergoing long-term hemodialysis were investigated. Patients were followed up for 6 months for all kinds of fatal events and nonfatal cardiac events. Serum levels of cardiac TnT, two TnI assays (ACS:180 and Stratus II), CK (creatine kinase), and CK-MB were measured pre- and post-dialysis. RESULTS: Pre- and post-dialysis results were not different for TnT and TnI, while CK and CK-MB levels were significantly lower post-dialysis. Elevated (pre-)dialysis levels were found in 65.7% of patients for TnT (> 0.1 microg/l), in 32.4% for TnI (ACS:180, > 0.15 microg/l), in 2.9% for TnI (Stratus II, > 0.4 microg/l), in 5.7% for CK (> 170 U/l), and in 2.8% for CK-MB (> 1.0 microg/l). 6-month events were observed in 42 patients (40.4%; 20 fatal, 22 nonfatal). The relative risk for 6-month events was calculated to be 16.0-fold for TnT and 8.5-fold for a known coronary heart disease, while both TnI and CK-MB did not predict 6-month events independently. CONCLUSIONS: TnT and a known coronary heart disease are relevant, independent risk factors in patients with chronic renal failure undergoing long-term hemodialysis.

The Harvest Smart PRePTM system versus the Friadent-Schütze platelet-rich plasma kit.

An important reason to improve methods for isolating platelet-rich plasma (PRP) is the potential use of autogenous platelet growth factors. In addition to the Curasan PRP kit (Curasan, Kleinostheim, Germany) and the platelet concentrated collection system (PCCSTM) system, two new methods for the preparation of PRP by the surgeon are now available. This study compared the suitability of these new methods for the preparation of PRP. Whole blood was drawn from 54 healthy donors (33 men and 21 women) aged 23-79 years (38.0 +/- 17.7 years). PRP was prepared from each donor's blood using both the Smart PRePTM system (Harvest Technologies Corporation, Munich, Germany) and the Friadent-Schütze method (PRP kit; Friadent-Schütze, Vienna, Austria). The platelet count in donor whole blood was 276 810 +/- 59 440 /microl. Platelet counts differed significantly between the Smart PRP preparation (1227 890 +/- 312 440 platelets/microl) and the Friadent-Schütze PRP preparation (1440 500 +/- 501 700 platelets/microl) (sign test, P < 0.001). The Smart PRePTM system had a significantly higher collection efficiency (63.4 +/- 7.9%) than the Friadent-Schütze kit (49.6 +/- 13.6%) (sign test, P < 0.001). The leukocyte contents in the two platelet concentrates were similar (Smart PRePTM, 19 261 +/- 8082 platelets/microl; Friadent-Schütze, 21 691 +/- 16 430). Transforming growth factor (TGF)-beta1 and platelet-derived growth factor (PDGF)-AB were higher in the Friadent-Schütze PRP (TGF-beta1, 196.8 +/- 109.6 ng/ml; PDGF-AB, 251.6 +/- 115.4 ng/ml) than in the Smart PRePTM (TGF-beta1, 77.2 +/- 54.8 ng/ml; PDGF-AB, 208 +/- 85.2 ng/ml). The sign test indicated significant differences between the two methods in the concentrations of TGF-beta1 (P < 0.001) and PDGF-AB (P < 0.01). Insulin-like growth factor (IGF)-1 levels in the two PRP preparations were similar (Friadent-Schütze PRP, 72.8 +/- 22.3 ng/ml; Smart PRePTM, 91.4 +/- 21.3 ng/ml). The Smart PRePTM system was superior with respect to ease of handling and preparation time. It also had a significantly higher platelet collection efficiency than the Friadent-Schütze PRePTM kit. The Friadent-Schütze PRP kit offers a slight advantage in the resulting PRP platelet concentration. However, this is easily compensated for in the Smart PRePTM system by reducing the volume of the resulting PRP.

Methods for the monitoring of direct thrombin inhibitors.

Direct thrombin inhibitors are available for prophylactic as well as therapeutic purposes. Application of hirudin in therapeutic doses has been shown to require drug monitoring. Currently, most experience is available for recombinant hirudin, but the principle aspects of drug monitoring are the same for all direct thrombin inhibitors. Most frequently, activated partial thromboplastin time (aPTT) and modifications of the activated clotting time (ACT) have been used for the monitoring of hirudin therapy. However, these methods are insensitive at plasma levels higher than 0.6 mg/L of hirudin, so that overdoses may be missed despite monitoring. Correlations between ecarin clotting time (ECT), enzyme immunoassays, and chromogenic substrate assays on one side and global tests on the other side are poor. Fully automated chromogenic substrate-based assays, also available as point-of-care tests (POCT), are more precise and sensitive and are not disturbed by interferents such as heparin and antithrombin. Good correlations can be observed between chromogenic assays and the ECT performed in plasma or whole blood samples. ECT can also be determined with POCT systems. Test characteristics such as imprecision and measuring range are comparable to those of the chromogenic assays. In conclusion, therapy with direct thrombin inhibitors should be monitored with chromogenic assays or ECT.

Impact of infectious burden on progression of carotid atherosclerosis.

BACKGROUND AND PURPOSE: Recent findings suggest a causative role of infections in the pathogenesis of atherosclerosis. The extent of atherosclerosis and the prognosis of patients with atherosclerosis seem to be increased by the number of infections to which an individual has been exposed. In a prospective study, we evaluated the effect of 8 pathogens and the aggregate pathogen burden on the progression of carotid atherosclerosis. METHODS: In 504 patients (74.9% men; age, 62.9+/-10 years), we measured intima-media thickness and prevalence of carotid artery stenosis. Follow-up measurements after a mean of 2.5 years were available in 427 patients (85%). Blood samples were taken, and IgG or IgA antibodies to Chlamydia pneumoniae, Helicobacter pylori, Haemophilus influenzae, Mycoplasma pneumoniae, cytomegalovirus, Epstein-Barr virus, and herpes simplex virus types 1 and 2 were measured. Statistical evaluation was performed with logistic regression procedures. RESULTS: Elevated IgA antibodies against C pneumoniae (P<0.04) and IgG antibodies against Epstein-Barr virus (P<0.01) and herpes simplex virus type 2 (P<0.04) were associated with progression of atherosclerosis (increase of intima-media thickness > or =0.1 mm/y or progression of carotid stenosis) after adjustment for age, sex, cardiovascular risk factors, highly sensitive C-reactive protein, and statin intake. Infectious burden, divided into 0 to 3, 4 to 5, and 6 to 8 seropositivities, was significantly associated with progression of atherosclerosis, with odds ratios of 1.8 (95% confidence interval, 1.1 to 2.9) for 4 to 5 and 3.8 (95% CI, 1.6 to 8.8) for 6 to 8 compared with 0 to 3 seropositivities after adjustment. CONCLUSIONS: Our results support the hypothesis that the number of infectious pathogens to which an individual has been exposed independently contributes to the progression of carotid atherosclerosis.

Annexin V does not represent a diagnostic alternative to myoglobin for early detection of myocardial infarction.

Annexin V is a calcium binding protein, which is widely present in various cells and tissues. Due to an early release reaction after myocardial injury the determination of annexin V might be useful for the rapid diagnosis of acute myocardial infarction. An enzyme-linked immunosorbent assay was used to measure annexin V in comparison to myoglobin in samples from healthy individuals, patients suffering from acute or chronic liver, renal, and pulmonary diseases as well as acute coronary syndromes and aortocoronary bypass surgery. Increased myoglobin and annexin V concentrations were observed 80 and 140 (maximum) minutes after myocardial ischemia induced by percutaneous transluminal coronary angioplasty. For the diagnosis of myocardial infarction annexin V (cutoff-level: 5.9 microg/L) showed a slightly higher sensitivity than myoglobin (annexin V: 74.5%; myoglobin: 59.6%), but specificity was much lower (annexin V: 39%; myoglobin: 82.5%). The area under the curve of a ROC analysis demonstrated that annexin V cannot be used as an early marker for the diagnosis of acute coronary syndromes. Increased annexin V levels are induced by several diseases, leading to a low specificity for the diagnosis of a myocardial injury.

Quantification of thrombocyte growth factors in platelet concentrates produced by discontinuous cell separation.

Platelet concentrates (PC) are increasingly used to increase bone regeneration in pre-prosthetic surgery. Although it is generally appreciated that certain growth factors (PDGF, TGF, EGF, and ECGF) are present in thrombocyte preparations, relatively little is known about these components in quantitative terms. The study reported here analysed the amounts of growth factors in PC produced under standard conditions from healthy volunteers. All the blood samples (237 in total) were analysed using Quantikine ELISA kits (R and D). The mean +/- SD platelet count in whole blood from these donors was 262,000+/-58,000/microl, while in PC produced by discontinuous cell separation it was 1.419,000+/-333,000/microl. The mean growth factor concentrations in PC preparations in ng/ml were as follows: PDGF-AB 125+/-55 ng/ml; TGF-beta1 221+/-92 ng/ml; IGF-I 85+/-25 ng/ml; PDGF-BB 14+/-9 ng/ml; TGF-beta2 0.4+/-0.3 ng/ml. These growth factor concentrations typically covered a 3-10 fold range: PDGF-AB 29-277ng/ml; PDGF-BB 2-33ng/ml; TGF-beta1 32-397ng/ml; TGF-beta2 0.1-1.2 ng/ml; IGF-I 40-138 ng/ml. Platelet counts in PC were slightly higher for women (Mann-Whitney Test all p < 0.001) than for men, while the concentrations of growth factors in PC exhibited no gender-related difference of any statistical significance.

An improved assay for the determination of C-reactive protein.

C-reactive protein as the most important acute phase reactant in clinical use provides information about acute as well as chronic inflammatory processes. This application requires an improvement of traditional assays that have been available to the clinical laboratory regarding lipemic interference, precision especially at the lower end, assay and calibration stability as well. In the present study the improved Olympus turbidimetric assay for the determination of C-reactive protein (CRP) was evaluated on the Olympus AU640 analytical system. The assay has a lower detection limit of 1.57 mg/l CRP and is linear from 5 mg/l to 200 mg/l. Prozone hook effect did not occur until 300 mg/l with increased prozone sample detection to 3500 mg/l. Imprecison CV values for within-run of less than 2.25% and between-day of lower than 3.1% were found. On-board stability was extended to 60 days, and calibration stability to 28 days. Method comparison to another automated CRP assay yielded a correlation coefficient of r=0.999. Endogenous substances did not interfere with the test results. There was satisfactory recovery of target values according to CRM 470 standardization. In comparison to the previous assay the improved assay offers more accuracy, precision, and linearity and is free from any known interference, which meets user needs, for rapid and convenient determination of CRP on automated analyzers.