Effect Of Different Anticoagulants Research Articles

Effect of different anticoagulants and antiplatelets on intraoral bleeding time during professional oral hygiene session

ObjectivePatients with thromboembolic problems, prosthetic valves, or coagulation issues are commonly prescribed anticoagulants and antiplatelets. Anticoagulant and antiplatelet medication might constitute a challenge for dentists and dental hygienists since possible prolonged bleeding might interfere with dental procedures. The aim of the present study was to examine the bleeding durations associated with various anticoagulants and antiplatelets during professional dental hygiene sessions, utilizing a modified Ivy test adapted for the oral context.Materials and methodsNinety-three consecutive patients undergoing professional oral hygiene were recruited. Debridement during oral hygiene was performed using ultrasonic mechanical instrumentation, and bleeding sites were assessed and treated with gentle pressure using sterile gauzes. The time for bleeding cessation was recorded. Patients were categorized into six groups based on their drug intake, Control: no anticoagulants or antiplatelets DTI: direct thrombin inhibitors (dabigatran) AntiXa: directa factor Xa inhibitors (endoxaban, apixaban, rivaroxaban) VKA: vitamin K antagonists (warfarin, acenocoumarol) SAPT: single anti-platelet therapy (acetylsalicylic acid or clopidogrel) DAPT: dual anti-platelet therapy (acetylsalicylic acid and clopidogrel). Bleeding time was measured in seconds and mean values were assessed among the different groups. Differences between groups were investigated with Kruskal-Wallis test followed by Dunn’s post-hoc correction for multiple comparisons or two-way ANOVA followed by Dunnett post-hoc;ResultsControl patients presented the lowest bleeding time 50 s, followed by AntiXa (98), SAPT (105), DTI (120), DAPT (190) and VKA (203). A statistically significant difference was present among control and DTI (p = 0.004), VKA (p < 0.001), DAPT (p < 0.001).ConclusionsBased on the present outcomes, an increased risk of prolonged bleeding emerged in patients taking VKA and DAPT.Clinical significancebleeding did not interfere with the oral hygiene session The optimal period for dental treatment of these patients should be 2–3 h before the next dose, without the need to temporarily suspend the medication.

A Systematic Review and Bayesian Network Meta-Analysis on the Effect of Different Anticoagulants on the Prophylaxis of Post-Thrombotic Syndrome after Deep Venous Thrombosis

Background: Postthrombotic syndrome (PTS) has a major impact on the quality of life after deep venous thrombosis (DVT). From clinical practice and related trials, anticoagulants show potential for reducing the occurrence and alleviating the symptoms of PTS. Methods: A systematic review and Bayesian network meta-analysis (NMA) were conducted by combing the literature from the databases of MEDLINE, Embase, Web of Science, Cochrane Libraries, and ClinicalTrials, through a variety of medical subject headings (Mesh) and PTS keywords. With regard to PTS prophylaxis, all anticoagulant-related randomized controlled trials (RCTs) and observational studies were assessed. The network model was conducted through the R software, and further comparisons were conducted using the Bayesian hierarchical random effects model. The odds ratio (OR) and the corresponding 95% CI were calculated for analysis. Results: Data from two RCTs and nine non-randomized studies meeting the selection criteria were included in the Bayesian analysis model, which incorporated seven anticoagulants. Edoxaban (OR: 0.42, 95% CI: 0.18–1.0) and rivaroxaban (OR: 0.54, 95% CI: 0.38–0.76) were significantly more effective than warfarin in the prevention of PTS (Villalta score ≥ 5). A subgroup analysis based on the severity of PTS showed that rivaroxaban was more effective than warfarin, with OR: 0.59, 95% CI: 0.41–0.84 (Villalta score 5 to 14) and OR: 0.48, 95% CI: 0.22–0.9 (Villalta score ≥ 15, ulceration), respectively. Edoxaban had the highest probability (80.1%) of providing preventive benefits for PTS. For mild/moderate and severe PTS, rivaroxaban provided the highest benefits in preventing PTS (89.3% and 85.6%, respectively). Conclusion: Edoxaban demonstrated a better prophylactic effect on PTS (Villalta score &gt; 5), while rivaroxaban displayed a better effect against mild/moderate (Villalta score 5 to 14) and severe PTS (Villalta score ≥ 15, ulceration).

Isolation and molecular characterization of circulating extracellular vesicles from blood of chronic Chagas disease patients

Extracellular vesicles (EVs) are lipid bilayer envelopes that encase several types of molecules. Their contents mostly reflect their cell origin and possible targets at other locations in the organism and can be modified in pathological conditions to interfere with intercellular communication, thus promoting disease establishment and development. These characteristics, in addition to their presence in virtually all body fluids, make such vesicles ideal for biomarker discovery in human diseases. Here, we describe the effect of different anticoagulants and the combination of two purification methods for isolation and characterization of circulating EVs from blood of chronic Chagas disease (CCD) patients. We illustrated this procedure by studying a population of patients with Chagas disease at the indeterminate chronic stage, in which the Trypanosoma cruzi is very scarce in circulation. EVs were harvested from blood collected without or with different anticoagulants. Protein and nanoparticle tracking analysis was used to measure EVs size and concentration. The EVs were purified by ultracentrifugation, followed by size-exclusion chromatography and characterized by chemiluminescent enzyme-linked immunosorbent assay and dot blot using antibodies that recognized parasite-derived EVs, such as hyperimmune sera, polyclonal and monoclonal antibodies against trans-sialidase and mucins. In parallel, antibodies against classical human EV markers CD9, CD63, CD81, and CD82, were also analyzed. The results showed that anticoagulants did not interfere with the analyzed parameters and circulating EVs from CCD patients contain T. cruzi antigens and classical human exosomal markers. Overall, our protocol is adequate for the isolation of the total circulating EVs and can serve as an important basis for further studies on biomarker discovery in Chagas' disease.

Performance of complete blood count (CBC) upon use of different anticoagulants in rats

Complete blood count (CBC) is used as an index of health status of human and different animals as well as to diagnose a variety of diseases. Therefore, there is a growing need of using the most suitable anticoagulant to obtain the most appropriate hemogram. The present study was designed to assess the effect of different anticoagulants viz. Heparin, Sodium Citrate and EDTA on complete blood count (CBC) in rat with a view to choosing the best suitable candidate among the common anticoagulants. A total of 30 samples out of which 10 were for each type anticoagulant were collected from 10 apparently healthy rats of Long Evans strain. From each rat 6 ml of blood was drawn and subsequently divided into three different test tubes with three different anticoagulants. The samples were analyzed for their complete blood count (TEC, TLC, Hb, Hct, DLC, absolute leukocyte count, Red Cell Indices, RDW-SD, RDWCV, Platelet, MPV, PCT and PDW) using Sysmex XT-1800i Auto hematological analyzer. Results showed a significant (p&lt;0.05) decrease in TLC, absolute neutrophil, and platelet count, and a significant (p&lt;0.05) higher level of relative lymphocyte count in Sodium Citrate treated sample than with Heparin and EDTA. No significant changes were observed for RBC or erythrocyte indices among the three different groups. It can be concluded that samples treated with Sodium Citrate results in significant change in blood parameters. Therefore, it is better to use Heparin and EDTA than Sodium Citrate for complete blood count (CBC).&#x0D; Asian J. Med. Biol. Res. March 2020, 6(1): 16-21

Effect of Different Anti-Coagulants on the Accuracy of Glycated Haemoglobin Results

Background: Most manufacturers of glycated haemoglobin kits advocate for the use of EDTA bottles for sample collection. Other manufacturers even when using the same glycated haemoglobin assay method, advocate for the use of any of these anticoagulant: EDTA, heparin and fluoride oxalate as any of these anticoagulants for sample collection.&#x0D; Aim: This study was therefore designed to evaluate the effect of different anticoagulants on the accuracy of glycated haemoglobin value using the same method.&#x0D; Methods: Thirty subjects were selected by purposive sampling method and 2ml of blood was collected from each subject into sodium heparin, EDTA and fluoride oxalate bottles and stored for three days at 4ºC. Fifteen subjects’ samples were analysed daily for the next two days then all the samples were analysed on the third day. All samples were analysed using the boronate affinity chromatographic method by Clover.&#x0D; Results: The mean of the values of glycated haemoglobin of samples for each anticoagulants were about the same for the first, second and third day. The differences in the mean values for each anticoagulant were not statistically significant, indicating fairly good stability.&#x0D; Conclusion: From this study, it could be concluded that blood sample in EDTA, fluoride oxalate and heparin bottles can be used for glycated haemoglobin estimation without affecting the accuracy of the result. These samples in these containers were found to be stable for at least three days.

Preclinical Studies of RUC-4, a Novel Platelet αIIbβ3 Antagonist, in Non-Human Primates and With Human Platelets.

We are developing the novel αIIbβ3 antagonist, RUC-4, for subcutaneously (SC)-administered first-point-of-medical-contact treatment for ST Segment Elevated Myocardial Infarction (STEMI). We studied the: 1. pharmacokinetics (PK) of RUC-4 at 1.0, 1.93, and 3.86 mg/kg IV, IM, and SC in non-human primates (NHPs); 2. impact of aspirin on RUC-4 IC50 in human platelet-rich plasma (PRP); 3. effect of different anticoagulants on the RUC-4 IC50 in human PRP; and 4. relationship between αIIbβ3 receptor blockade by RUC-4 and inhibition of ADP-induced platelet aggregation. 1. All doses of RUC-4 were well tolerated, but animals demonstrated variable temporary bruising. IM and SC RUC-4 reached dose-dependent peak levels within 5-15 min, with T½ s between 0.28 and 0.56 hrs. Platelet aggregation studies in NHPs receiving IM RUC-4 demonstrated >80% inhibition of the initial slope of ADP-induced aggregation with all 3 doses 30 minutes post-dosing, with subsequent dose-dependent loss of inhibition over 4-5 hours. 2. The RUC-4 IC50 for ADP-induced platelet aggregation was unaffected by aspirin treatment (40±9 nM vs. 37±5 nM; p=0.39). 3. The RUC-4 IC50 was significantly higher in PRP prepared from PPACK-anticoagulated blood compared to citrate-anticoagulated blood using either TRAP (122±17 vs. 66±25 nM; p=0.05; n=4) or ADP (102±22 vs. 54±13; p<0.001; n=5). 4. There was a close correspondence between receptor blockade and inhibition of ADP-induced platelet aggregation, with aggregation inhibition beginning with ~40% receptor blockade and becoming nearly complete at >80% receptor blockade. Based on these results and others, RUC-4 has now progressed to formal preclinical toxicology studies.

EFFECT OF DIFFERENT ANTICOAGULANTS IN DETERMINING METFORMIN HYDROCHLORIDE LEVELS IN HUMAN PLASMA USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Objective: This study aimed to develop and validate an analytical method and to determine the effect of different anticoagulants for analyzingmetformin HCl level in human plasma.Methods: Analysis was performed with high-performance liquid chromatography method using a photodiode array detector set at 234 nm, a C18SunFire™ column (5 μm, 250 mm×4.6 mm), a temperature of 40°C, a mobile phase comprising 10 mM sodium dodecyl sulfate and 10 mM phosphatebuffer in water-acetonitrile (60:40 v/v) at pH 7.0, with a flow rate of 1.0 mL/min, and atorvastatin calcium as the internal standard. Plasmaextraction was performed through protein precipitation using human plasma (300 μL) and acetonitrile (600 μL; 1:2 v/v). The method was linear at aconcentration range of 20.0–5000.0 ng/mL, with r&gt;0.9998.Results: The stability and recovery of metformin HCl in human plasma were not different between citrate with ethylenediaminetetraacetic acid(EDTA) and heparin with EDTA as anticoagulants. However, a significant difference in the peak area response ratio (p&lt;0.05) was observed betweenthe three anticoagulants.Conclusion: The validation results for the three types of plasma anticoagulants met validation requirements based on the European MedicinesAgency bioanalytical guidelines of 2011 for accuracy and precision, selectivity, calibration curve linearity, dilution integrity, carry-over, and stability.

Pharmacokinetic (PK) and Pharmacodynamic (PD) Studies of RUC-4 Succinate, a Novel αIIbβ3 Pure Antagonist for First Point of Contact Treatment of ST Segment Elevated Myocardial Infarction (STEMI)

We are developing the novel αIIbβ3 pure antagonist, RUC-4, for first point of contact treatment of ST Segment Elevated Myocardial Infarction (STEMI) in combination with aspirin. We have chosen the subcutaneous (SC) route to facilitate its delivery by ambulance and emergency room personnel, and potentially by self-administration. In preparation for initiating human studies, we have conducted preclinical studies of: 1. the pharmacokinetics (PK) and pharmacodynamics (PD) of RUC-4 succinate administered IV, IM, and SC to non-human primates (NHP); 2. the impact of in vitro aspirin on the RUC-4 IC50 in human platelet-rich plasma (PRP); and 3. the effect of different anticoagulants on the IC50 of RUC-4 in human PRP.Non-human primates (Macaca fascicularis; NHPs) received RUC-4 at 1.0, 1.96, and 3.86 mg/kg IV, IM, and SC. Blood samples for IV PK studies were collected at 0, 1, 5, 15, 30, 120, and 270 min; samples for IM and SC PK studies were collected at 0, 5, 15, 30, 45, 120, and 270 min and ~27 hrs, the latter only for animals that received 3.86 mg/kg IM. There were 2 animals per group and 2 groups per dose, so no more than 4-5 samples were collected from a single animal. Platelet aggregation was performed with 5 µM ADP on citrated PRP from the NHPs that received RUC-4 IM. All doses of RUC-4 administered IM and SC were well tolerated, but animals demonstrated variable temporary bruising. None of the animals developed thrombocytopenia despite receiving up to 4 doses of RUC-4 over 3 months. The PK data are shown in the Table; both IM and SC RUC-4 reached dose-dependent peak levels within 5-15 min, with T1/2 s between 0.28 and 0.56 hrs. The bioavailability of RUC-4 IM and SC ranged between 0.60-0.88 at doses of 1.93 and 3.86 mg/kg; there was greater variability in bioavailability at 1 mg/kg (1.2 IM and 0.55 SC). Platelet aggregation studies demonstrated >80% inhibition of the initial slope of aggregation in response to ADP in all samples in which the whole blood RUC-4 concentration was ≥ ~8 nM, with partial inhibition at lower concentrations.Since aspirin is the standard-of-care for initial treatment of STEMI, we plan to administer RUC-4 + aspirin. To assess aspirin-RUC-4 interactions, we compared the RUC-4 IC50 in untreated human citrated PRP to the RUC-4 IC50 in PRP pre-treated with 0.83 mM aspirin for 10 min at 22°C. This aspirin dose completely inhibited platelet aggregation to 1.5 mM arachidonic acid in each donor, indicating inhibition of thromboxane A2 production. In studies on 5 donors using 5 μM ADP as the agonist, the RUC-4 IC50 was 40 ± 9 nM in untreated PRP and 37 ± 5 nM in aspirin-treated PRP (p=0.50).Previous studies demonstrated that citrate anticoagulation can variably enhance the antiplatelet effect of some αIIbβ3 antagonists (Phillips et al., Circulation 96:1488, 1997: Kereiakes et al., J Thromb Thrombolysis 12:123, 2001). We therefore compared the IC50 of RUC-4 in human blood anticoagulated with citrate (0.38%), heparin (15.8 U ml), and PPACK (300 nM) when stimulated with a thrombin receptor activating peptide (T6; TRAP) at 20 µM. The resulting IC50 values were 66 ± 25, 111 ± 7, and 122 ± 17, respectively (n=4; p=0.05 for citrate vs PPACK and p=0.04 for heparin vs citrate). With 20 µM ADP activation, the IC50 values for blood anticoagulated with citrate and PPACK (100 µM) were 54 ± 13 and 102 ± 12 nM (p=0.001).In summary: 1. In accord with our studies with RUC-4 free base (Li et al., ATVB 34: 2321, 2014), IM RUC-4 succinate achieves peak blood levels in NHP within 5 min, resulting in >80% inhibition of platelet aggregation that persists for several hrs at a dose of 3.86 mg/kg. 2. SC RUC-4 achieves peak blood levels within 5-15 min; while the peak concentrations are lower than those with IM RUC-4 at the same doses, the blood levels are more sustained, with levels equivalent to the IM values producing ≥80% inhibition of initial slope persisting for several hrs. 3. Aspirin at a concentration that eliminates arachidonic acid-induced platelet aggregation does not affect the IC50 for RUC-4; 4. Assaying RUC-4 IC50 in human PRP from blood anticoagulated with citrate yields a value ~50% of that obtained with PRP prepared from blood anticoagulated with agents that do not affect divalent cation levels. Thus, the values in the presence of citrate may overestimate the in vivo antiplatelet effect. We conclude that the PK and PD of SC RUC-4 are favorable for short-term, high-potency inhibition of platelet aggregation as part of first point of care treatment of STEMI. [Display omitted] DisclosuresNedelman:Biomere: Employment, Equity Ownership. Coller:Centocor: Patents & Royalties: Abciximab; CeleCor: Consultancy, Equity Ownership; Rockefeller University: Patents & Royalties: RUC-4; Platelet Biogenesis: Consultancy; Accumetrics: Patents & Royalties: VerifyNow Assays; Scholar Rock: Consultancy, Equity Ownership.

Stability of glucose in plasma with different anticoagulants

People with hyperglycemia, especially those who are pregnant, depend on accurate blood glucose measurements for correct diagnosis of diabetes and monitoring. Glycolysis after blood draw, however, decreases glucose concentrations in blood that is collected at room temperature in the absence of a stabilizer. Cold temperatures (4°C) inhibit glycolysis; but prompt cooling and processing of each blood sample in the cold is difficult to achieve in routine clinical practice. Therefore, preservatives are used to stabilize glucose during blood collection and processing procedures that are performed at room temperature. This study examined the effect of different anticoagulants (EDTA, heparin, oxalate), with or without glycolysis inhibitors (NaF, citrate), on the stability of glucose in plasma samples - obtained from blood that was collected and stored at room temperature for up to 24 h. Venous blood was collected from 60 volunteers; each donor blood sample was divided into six tubes, each one containing a different anti-glycolysis-anticoagulant composition. Terumo VENOSAFE™ Glycemia tubes contained NaF/citrate buffer)/Na2EDTA; NaF/Na-heparin; and NaF/K2oxalate. Sarstedt tubes contained NaF/citrate; NaF/Na2EDTA; and K2EDTA. At 0, 2, 8 and 24 h, plasma was obtained for glucose measurements using the Glucose Hexokinase and Glucose Oxidase methods, and the ADVIA® 1800 Clinical Chemistry System. Both methods demonstrated minimal glycolysis by 24 h (<3.8%) for the three Terumo VENOSAFE™ Glycemia tubes, and the Sarstedt S-Monovette GlucoEXACT tube that contained NaF/citrate. Glycolysis was higher in tubes containing NaF/Na2EDTA-alone (11.7%) and K2EDTA-alone (85%). Terumo VENOSAFE™ Glycemia tubes (containing NaF/citrate buffer/Na2EDTA; NaF/Na-heparin; and NaF/K2oxalate) and the Sarstedt S-Monovette® GlucoEXACT tubes (containing NaF/citrate) are suitable for shipping venous whole blood samples to the testing laboratory within 24 h at room temperature.

Comparative Study of the Effect of Different Anticoagulants on Blood Cell Morphology in Common Carp ( C. carpio ) and Rainbow Trout ( O. mykiss )

Abstract. Hematological assessment is an important tool in evaluating the fish health status. This tool corroborated with biochemical, parasitological and bacterial examinations aids the researchers in monitoring fish welfare status. For relevant blood samples using the right harvesting methodology with the right anticoagulant is imperative. This study presents a comparison between two different anticoagulants like Li-Heparin and ethylenediaminetetraacetic acid (EDTA) in common carp ( Cyprinus carpio ) and rainbow trout ( Oncorhynchus mykiss ). Two common carps and two rainbow trouts were submitted for blood harvesting using Li-Heparin and EDTA containers. Blood smears were prepared using a Romanowsky staining procedure and examined via light microscopy. The results of the study revealed significant in vitro hemolysis and red blood cell morphologic alterations in the common carp blood samples when harvested on EDTA in contrast with the Li-Heparin anticoagulant. The white blood cells showed no significant changes induced by neither of the two anticoagulants. No significant anticoagulant induced cell morphological alterations were noticed in the rainbow trout. It is concluded that EDTA is not the appropriate anticoagulant for hematologic evaluation in common carp.

Effect of Different Anticoagulants on HbA1c Estimation and its Stability

Effect of Different Anticoagulants on HbA1c Estimation and its Stability

The effect of selected pre‐analytical phase variables on plasma thromboxane A2 measurements in humans

Platelet function testing is often affected by the existence of different pre-analytical variables that can cause platelet activation. The aim of this study was to assess the effect of such variables that are present when samples are taken (different anticoagulants, incubation temperature, and storage conditions) to select those which enable to reach optimal range of measured plasma concentrations of the two stable thromboxane A₂ metabolites, that is, thromboxane B₂ (TxB₂) and 11-dehydrothromboxane B₂ (11-dTxB₂). For the purpose of this study, whole blood samples obtained from 20 volunteers were screened for TxB₂ and 11-dTxB₂ concentrations using commercial EIA kits (Cayman Chemicals™; Neogen™) in relation to the effect of different anticoagulants, using different incubation temperatures and storage conditions. Trisodium citrate has been shown not to be affecting the TxB₂ and 11-dTxB₂ concentrations compared with the values measured in the serum. Incubation of the samples for 1 h at 37 °C and freezing at -20 °C or -80 °C give the most suitable concentration range of both thromboxanes in the used EIA measurement. This study describes the setup of such pre-analytical phase conditions that enable the screening of platelet function in terms of the plasma concentrations of TxB₂ and 11-dTxB₂ in selected EIA measurement.

Effect of different anticoagulants on multiple electrode platelet aggregometry after clopidogrel and aspirin administration in patients undergoing coronary stent implantation: A comparison between citrate and hirudin

Outcomes of platelet function tests are highly dependent on the type of blood anticoagulant used. The primary objective of this study was to clinically evaluate the platelet function after dual antiplatelet therapy using two different types of anticoagulant (citrate and hirudin). We compared data obtained from multiple electrode platelet aggregometry (MEA) with reference to light transmission aggregometry (LTA) and VerifyNow (VN) assays. Blood samples were obtained from 119 patients on dual antiplatelet therapy at the time of PCI (PCI) and the following morning (post-PCI). The platelet function tests were performed using two anticoagulated (citrate or hirudin) blood types for MEA as well as citrated blood for LTA and VerifyNow assays. ADP-induced MEA values at PCI for citrated and hirudinated anticoagulants were 36.5 ± 14.3 AUC and 41.4 ± 18.2 AUC (p = 0.021) and post-PCI values were 28.2 ± 11.9 AUC and 28.3 ± 12.8 AUC (p = 0.95). Additionally, AA-induced MEA values at PCI by citrated and hirudinated blood was 13.4 ± 7.3 AUC and 17.6 ± 13.4 AUC (p < 0.01). Post-PCI AA-induced MEA values were 12.0 ± 6.7 AUC and 13.5 ± 8.5 AUC (p = 0.12), respectively. Significant correlations were observed between the two anticoagulants used for MEA and LTA or VN values under ADP-induced platelet stimulation. Citrate tubes are clinically adequate for MEA assays and provide a more economical alternative to hirudin for early and/or delayed phases after clopidogrel-loading doses.

Methodological considerations for the assessment of ADP induced platelet aggregation using the Multiplate® analyser

Factors affecting the Multiplate® assay's analytical precision have not been well defined. We investigated the effect of methodological factors on the measurement of ADP-induced platelet aggregation using the Multiplate® assay. ADP-induced platelet aggregation was analysed in whole blood using the Multiplate® assay. We tested the reproducibility of measurement, the effect of different anticoagulants (hirudin, citrate and heparin) and the effect of time delay (15, 30, 45, 60, 120 and 180 minutes) between sampling and analysis in patients. The use of a manual calibrated pipette with the Multiplate® analyser was also tested. The mean coefficient of variation (CV) using the manufacturers recommended methods was 10.8 ± 8.7% (n = 30). When compared to hirudin (359.5 ± 309 AU*min) the use of heparin (521.0 ± 316 AU*min, p = 0.0015) increased platelet aggregation, while the use of sodium citrate (245.0 ± 209 AU*min, p = 0.003) decreased the platelet aggregation (n = 20). The addition of CaCl2 to the citrate-anticoagulated blood resulted in platelet aggregation levels similar to hirudin. Platelet aggregation varied with time delay (n = 20). When compared to platelet aggregation at 30 minutes (391.1 ± 283 AU*min), platelet aggregation was reduced at 60 minutes (335.2 ± 251.6 AU*min, p < 0.05), 120 minutes (198.8 ± 122.9 AU*min, p < 0.001) and 180 minutes (160.7 ± 92 AU*min, p < 0.001). The use of a manual calibrated pipette did not significantly reduce the mean CV in the assay (n = 20). Methodological factors such as the anticoagulant used and the time delay should be standardised where possible to reduce variability, and allow thresholds derived from one study to be comparable across multiple studies.

Banking of Biological Fluids for Studies of Disease-associated Protein Biomarkers

With the increasing demand of providing personalized medicine the need for biobanking of biological material from individual patients has increased. Such samples are essential for molecular research aimed at characterizing diseases at several levels ranging from epidemiology and diagnostic and prognostic classification to prediction of response to therapy. Clinically validated biomarkers may provide information to be used for diagnosis, screening, evaluation of risk/predisposition, assessment of prognosis, monitoring (recurrence of disease), and prediction of response to treatment and as a surrogate response marker. Many types of biological fluids or tissues can be collected and stored in biorepositories. Samples of blood can be further processed into plasma and serum, and tissue pieces can be either frozen or fixed in formalin and then embedded into paraffin. The present review focuses on biological fluids, especially serum and plasma, intended for study of protein biomarkers. In biomarker studies the process from the decision to take a sample from an individual to the moment the sample is safely placed in the biobank consists of several phases including collection of samples, transport of the samples, and handling and storage of samples. Critical points in each step important for high quality biomarker studies are described in this review. Failure to develop and adhere to robust standardized protocols may have significant consequences as the quality of the material stored in the biobank as well as conclusions and clinical recommendations based on analysis of such material may be severely affected.

Isolation and activation of human neutrophils in vitro. The importance of the anticoagulant used during blood collection

Objectives To assess the effect of different anticoagulants (EDTA, citrate and heparin) on the isolation procedure of human neutrophils and in the subsequent alterations of calcium levels and respiratory burst induced by phorbol myristate acetate (PMA). Design and methods Isolation of human neutrophils from whole blood was performed by the gradient density centrifugation method. PMA-induced neutrophil burst was measured by chemiluminescence. Intracellular calcium ([Ca 2+] i) was measured using Fluo-3 AM, a calcium-sensitive dye. Results EDTA provided the highest number of isolated neutrophils/mL of blood (1.7 × 10 6 ± 1.5 × 10 5) when compared with citrate (0.46 × 10 6 ± 0.95 × 10 5) and heparin (0.66 × 10 6 ± 0.15 × 10 5). EDTA originated less degree of PMA-induced activation (370 ± 30%) relatively to citrate (830 ± 98%) and heparin (827 ± 77%). [Ca 2+] i was lower with EDTA (122 ± 11 nM) when compared with citrate and heparin (150 ± 13 and 230 ± 30 nM). Conclusion The anticoagulant used during blood collection interfered differently with the yield of isolated neutrophils as well as on their calcium levels and reactivity to PMA.

Comparison of the inhibition effect of different anticoagulants on vitamin K epoxide reductase activity from warfarin-susceptible and resistant rat

Anti-vitamin K drugs are widely used as anticoagulant in human thromboembolic diseases. Similar compounds have also been used as rodenticides to control rodent population since 1950s. Massive use of first generation anticoagulants, especially warfarin, has lead to the development of genetic resistances in rodents. Similar resistances have been reported in human. In both cases, polymorphisms in VKORC1 (Vitamin K epoxide reductase subunit 1), the subunit 1 of the VKOR (Vitamin K epoxide reductase) complex, were involved. In rats ( Rattus norvegicus), the Y139F mutation confers a high degree of resistance to warfarin. Little is known about the in vitro consequences of Y139F mutation on inhibitory effect of different anticoagulants available. A warfarin-susceptible and a warfarin-resistant Y139F strain of wild rats ( Rattus norvegicus) are maintained in enclosures of the Lyon College of Veterinary Medicine (France). Using liver microsomes from susceptible or resistant rats, we studied inhibition parameters by warfarin ( K i = 0.72 ± 0.1 μM; 29 ± 4.1 μM), chlorophacinone ( K i = 0.08 ± 0.01 μM; 1.6 ± 0.1 μM), diphacinone ( K i = 0.07 ± 0.01 μM; 5.0 ± 0.8 μM), coumachlor ( K i = 0.12 ± 0.02 μM; 1.9 ± 0.2 μM), coumatetralyl ( K i = 0.13 ± 0.02 μM; 3.1 ± 0.4 μM), difenacoum ( K i = 0.07 ± 0.01 μM; 0.26 ± 0.02 μM), bromadiolone ( K i = 0.13 ± 0.02 μM; 0.91 ± 0.07 μM), and brodifacoum ( K i = 0.04 ± 0.01 μM; 0.09 ± 0.01 μM) on VKOR activity. Analysis of the results leads us to highlight different anticoagulant structural elements, which influence inhibition parameters in both susceptible and Y139F resistant rats.

The effect of different anticoagulants on thrombin generation

Decrease in thrombin generation is the key effect in anticoagulation. The aim of the present study was to investigate the effect of anticoagulants on thrombin generation and the relation to platelet count. Plasma samples from 10 healthy volunteers (mean age 43.0 +/- 9 years) were incubated at preset platelet counts with different doses of the anticoagulants lepirudin, fondaparinux and low molecular weight heparins. Thrombin generation was measured in a tissue factor-mediated assay using a fluorometer and a slow-reacting fluorogenic substrate. The endogenous thrombin potential, the lag phase, the maximum reaction velocity (Vmax) and the concentration of a given anticoagulant required for 50% inhibition of thrombin generation (IC50) are presented. All three anticoagulants decreased endogenous thrombin potential and prolonged the lag phase in a dose-dependent manner. Fondaparinux and low molecular weight heparins, but not hirudin, decreased Vmax in a concentration-dependent manner. With increasing platelet count, the IC50 increased but the extent of this increase was not uniform for the three anticoagulants and the three variables investigated. The influence of anticoagulants on thrombin generation is variable, depending on their basic mechanism of action. In defining and comparing their effects, the endogenous thrombin potential, the lag phase and the maximum reaction velocity should be considered together. Platelets have a considerable influence on the magnitude of thrombin generation.

Effect of anticoagulants on plasma homocysteine determination

Background: Waiting temperature before centrifugation and anticoagulants used, markedly effect total homocysteine concentrations. The aim of this study was to investigate the effect of different anticoagulants and temperature on plasma homocysteine levels. Methods: We studied total homocysteine concentrations in 23 healthy subjects. Blood was drawn in K 3EDTA, sodium citrate- or sodium fluoride-containing tubes, and kept at 0°C or 22°C for 3 h. Total homocysteine measurements were performed with fluorescence polarization immunoassay (FPIA) method. We compared all results with baseline EDTA values (samples put on crushed ice and centrifuged immediately) recommended in literature for reference handling. Results: At 22°C, the tubes containing sodium citrate and sodium fluoride showed significantly higher total homocysteine concentrations than their respective baseline values ( p=0.000). However, sodium fluoride tubes were not significantly different than baseline EDTA levels. Waiting 3 h at 0°C did not effect sodium citrate and EDTA plasma total homocysteine concentrations when compared to baseline EDTA, but sodium fluoride-containing plasma levels were significantly decreased ( p=0.000). Conclusions: According to our results, the most available and practical temperature and anticoagulant for total homocysteine determination is sodium fluoride at room temperature up to 3 h.

Effect of anticoagulants and storage temperatures on stability of plasma and serum hormones

Objectives: To determine the effect of different anticoagulants and storage conditions on the stability of hormones in plasma and serum. Design and methods: Human blood samples were collected from volunteers into EDTA, lithium heparin, sodium fluoride/potassium oxalate, or tubes without anticoagulant, plasma and serum left at −20°C, 4°C or 30°C for 24 and 120 hours then assayed for ACTH, aldosterone, α-subunit, AVP, CRH, C-peptide, estradiol, FSH, glucagon, GH, IGF-1, IGFBP-3, insulin, leptin, LH, PPP, PTH, prolactin and VIP, or at room temperature for 0 to 72 hours (BNP, NT-BNP)( n = 6 per condition). Results: The anticoagulant altered the measured concentrations for 9 hormones when compared to EDTA. All hormones except ACTH were stable for >120 hours in EDTA or fluoride at 4°C, but only 13 hormones were stable in all anticoagulants. At 30°C, 8 hormones were stable for >120 hours in EDTA, and 3 hormones in all anticoagulants. BNP and NT-BNP were stable for <24 hours when stored in EDTA or heparin at room temperature. Discussion: Storage of samples in EDTA plasma at 4°C is suitable for most hormones (except ACTH) for up to 120 hours.