Generation of Rapid and High-Quality Serum by Recombinant Prothrombin Activator Ecarin (RAPClot™).

We recently reported the potential application of recombinant prothrombin activator ecarin (RAPClot™) in blood diagnostics. In a new study, we describe RAPClot™ as an additive to develop a novel blood collection prototype tube that produces the highest quality serum for accurate biochemical analyte determination. The drying process of the RAPClot™ tube generated minimal effect on the enzymatic activity of the prothrombin activator.

Potential Application of Recombinant Snake Prothrombin Activator Ecarin in Blood Diagnostics.

We describe here the purification and cloning of a codon-optimized form of the snake prothrombin activator ecarin from the saw scaled viper () expressed in mammalian cells. Expression of recombinant ecarin (rEcarin) was carried out in human embryonic kidney cells (HEK) cells under conditions for the development and performance of a novel and scalable recombinant snake ecarin to industry standards. Clotting performance of the rEcarin was established in recalcified citrated whole blood, plasma, and fresh whole blood and found to be comparable to native ecarin (N-Ecarin).

Progress curve analysis of microtitre plate plasma clotting assays. Assessment of tissue factor levels.

MTP plasma clotting assays monitor the time course of fibrin formation in re-calcified plasma by absorbance measurements and are increasingly used as alternatives to traditional one-point clot time assays employed in clinical laboratories to detect thrombotic disorders. The parameters derived from these analyses are analogous to thromboelastography viz. time, rate and maximum extent of clot formation.

Progress Curve Analysis of the one stage chromogenic assay for ecarin.

Snake venom prothrombin activators such as Ecarin are readily assayed by continuous spectrophotometric monitoring of p-nitroaniline production in a one step assay containing prothrombin and a p-nitroanilide peptide substrate for thrombin. The coupled reactions result in accelerating p-nitroaniline (pNA) production over the course of the assay giving non-linear progress curves, from which initial velocities are not readily obtained. Most studies therefore resort to approximate estimates of activity, based on the absorbance reached at an arbitrary time.

Inhibition studies of ketol-acid reductoisomerases from pathogenic microorganisms.

Ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid (BCAA) biosynthesis pathway, is an emerging target for the discovery of biocides. Here, we demonstrate that cyclopropane-1,1-dicarboxylate (CPD) inhibits KARIs from the pathogens Mycobacterium tuberculosis (Mt) and Campylobacter jejuni (Cj) reversibly with K values of 3.03 μM and 0.

Rapid serum tube technology overcomes problems associated with use of anticoagulants.

Introduction: Failure to obtain complete blood clotting in serum is a common laboratory problem. Our aim was to determine whether snake proth-rombin activators are effective in clotting blood and producing quality serum for analyte measurement in anticoagulated patients.

Materials And Methods: Whole blood clotting was studied in a total of 64 blood samples (41 controls, 20 Warfarin patients, 3 anticoagulated patients using snake venom prothrombin activator (OsPA)) with plain tubes.

Next-generation rapid serum tube technology using prothrombin activator coagulant: fast, high-quality serum from normal samples.

Background Incomplete blood clotting or latent clotting in serum is a common laboratory problem, especially for patients on anticoagulant therapy or when serum tubes are centrifuged before clotting is completed. We describe a novel approach to producing high-quality serum using snake venom prothrombin activator complex (OsPA) as an additive in blood collection tubes for non-anticoagulated (normal) individuals. Methods Plasma clotting assays were performed using a Hyland-Clotek instrument.

Laboratory medicine best practice guideline: vitamins a, e and the carotenoids in blood.

Despite apparent method similarities between laboratories there appear to be confounding factors inhibiting uniform reporting and standardisation of vitamin assays. The Australasian Association of Clinical Biochemists (AACB) Vitamins Working Party, in conjunction with The Royal College of Pathologists of Australasia Quality Assurance Programs, has formulated a guideline to improve performance, reproducibility and accuracy of fat-soluble vitamin results. The aim of the guideline is to identify critical pre-analytical, analytical and post-analytical components of the analysis of vitamins A, E and carotenoids in blood to promote best practice and harmonisation.

Diagnostic sensitivity of carbohydrate deficient transferrin in heavy drinkers.

Background And Aim: Carbohydrate deficient transferrin (CDT) is the most specific serum biomarker of heavy alcohol consumption, defined as ≥ 350-420 g alcohol/week. Despite introduction of a standardized reference measurement technique, widespread use of CDT remains limited due to low sensitivity. The aim of this study was to determine the factors that affect diagnostic sensitivity in patients with sustained heavy alcohol intake.

BMI but not stage or etiology of nonalcoholic liver disease affects the diagnostic utility of carbohydrate-deficient transferrin.

Background: A reliable biomarker is required in hepatology clinics for detection and follow-up of heavy alcohol consumption. Carbohydrate-deficient transferrin (CDT) increases with sustained heavy alcohol consumption and is the most specific biomarker of ethanol (EtOH) consumption. Recent introduction of a standardized method for measuring CDT has improved its clinical application.

Reference ranges and biological variation of free and total serum indoxyl- and p-cresyl sulphate measured with a rapid UPLC fluorescence detection method.

Introduction: The uremic toxins indoxyl sulphate (IS) and p-cresyl sulphate (pCS) are absorbed bacterial metabolites of tryptophan and tyrosine respectively and may be predictive of clinical outcome. Long chromatography times, incomplete data on the reference ranges of the free and total fractions and the biological variation limit wider clinical application.

Methods: An UPLC method with fluorescence detection was developed and reference ranges and biological variation were investigated in healthy volunteers.

Vitamin B1 and B6 method harmonization: comparison of performance between laboratories enrolled in the RCPA Quality Assurance Program.

Objectives: The RCPA Quality Assurance Program (RCPA QAP) offers monthly proficiency testing for vitamins A, B1, B6, β-carotene, C and E to laboratories worldwide. A review of the results submitted for the whole blood vitamin B1/B6 sub-program revealed a wide dispersion. Here we describe the results of a methodology survey for vitamins B1 and B6.

The structure of human microplasmin in complex with textilinin-1, an aprotinin-like inhibitor from the Australian brown snake.

Textilinin-1 is a Kunitz-type serine protease inhibitor from Australian brown snake venom. Its ability to potently and specifically inhibit human plasmin (K(i) = 0.44 nM) makes it a potential therapeutic drug as a systemic anti-bleeding agent.

Identification and characterisation of Kunitz-type plasma kallikrein inhibitors unique to Oxyuranus sp. snake venoms.

As part of a wider study on Australian snake venom components, we have identified and characterised Kunitz-type protease inhibitors from the venoms of Oxyuranus scutellatus and Oxyuranus microlepidotus (Australian taipans) with plasma kallikrein inhibitory activity. Each inhibitor had a mass of 7 kDa and was purified from the venom as part of a protein complex. Mass spectrometry and N-terminal sequencing was employed to obtain amino acid sequence information for each inhibitor and a recombinant form of the O.

Cloning and characterisation of novel cystatins from elapid snake venom glands.

Snake venoms contain a complex mixture of polypeptides that modulate prey homeostatic mechanisms through highly specific and targeted interactions. In this study we have identified and characterised cystatin-like cysteine-protease inhibitors from elapid snake venoms for the first time. Novel cystatin sequences were cloned from 12 of 13 elapid snake venom glands and the protein was detected, albeit at very low levels, in a total of 22 venoms.

Textilinin-1, an alternative anti-bleeding agent to aprotinin: Importance of plasmin inhibition in controlling blood loss.

Aprotinin has been used widely in surgery as an anti-bleeding agent but is associated with a number of side effects. We report that textilinin-1, a serine protease inhibitor from Pseudonaja textilis venom with sequence relatedness to aprotinin, is a potent but reversible plasmin inhibitor and has a narrower range of protease inhibition compared to aprotinin. Like aprotinin, textilinin-1 at 5 micromol/l gave almost complete inhibition of tissue plasminogen activator-induced fibrinolysis of whole blood clots.

Comparison of textilinin-1 with aprotinin as serine protease inhibitors and as antifibrinolytic agents.

Textilinin-1 (Q8008) was isolated from the venom of the Pseudonaja textilis and has a 47% sequence identity to the antihaemorrhagic therapeutic agent aprotinin. When equimolar concentrations of enzyme and aprotinin were pre-incubated, plasmin was inhibited 100%, plasma kallikrein 58%, and tissue kallikrein 99%. Under the same conditions, textilinin-1 inhibited plasmin 98%, plasma kallikrein 16% and tissue kallikrein 17%.