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. According to the bioassays of thrombin activity and plasma clotting, γ-radiation (>25 kGy) resulted in a 30-40% loss of the enzymatic activity of the RAPClot™ tubes. However, a visual blood clotting assay revealed that the γ-radiation-sterilized RAPClot™ tubes showed a high capacity for clotting high-dose heparinized blood (8 U/mL) within 5 min. This was confirmed using Thrombelastography (TEG), indicating full clotting efficiency under anticoagulant conditions. The storage of the RAPClot™ tubes at room temperature (RT) for greater than 12 months resulted in the retention of efficient and effective clotting activity for heparinized blood in 342 s. Furthermore, the enzymatic activity of the RAPClot™ tubes sterilized with an electron-beam (EB) was significantly greater than that with γ-radiation. The EB-sterilized RAPClot™ tubes stored at RT for 251 days retained over 70% enzyme activity and clotted the heparinized blood in 340 s after 682 days. Preliminary clinical studies revealed in the two trials that 5 common analytes (K, Glu, lactate dehydrogenase (LD), Fe, and Phos) or 33 analytes determined in the second study in the γ-sterilized RAPClot™ tubes were similar to those in commercial tubes. In conclusion, the findings indicate that the novel RAPClot™ blood collection prototype tube has a significant advantage over current serum or lithium heparin plasma tubes for routine use in measuring biochemical analytes, confirming a promising application of RAPClot™ in clinical medicine.

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 (Echis carinatus) 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). Furthermore, hemolysis was observed with N-Ecarin at relatively high doses in both recalcified citrated and fresh whole blood, while clotting was not observed with rEcarin, providing an important advantage for the recombinant form. In addition, rEcarin effectively clotted both recalcified citrated whole blood and fresh whole blood containing different anticoagulants including heparin, warfarin, dabigatran, Fondaparinux, rivaroxaban and apixaban, forming firm clots in the blood collection tubes. These results demonstrate that rEcarin efficiently clots normal blood as well as blood spiked with high concentrations of anticoagulants and has great potential as an additive to blood collection tubes to produce high quality serum for analyte analysis in diagnostic medicine.

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. The derived parameters, based on the whole course of the clotting reaction are more robust, informative and quantitative than single-point clot time assays. However, the parameters themselves are usually obtained arbitrarily by crude graphical analysis of subjectively selected points of progress curves. The current work aimed to investigate the sensitivity and reproducibility of an MTP clotting assay and examine its suitability for measuring tissue factor (TF) levels in cell culture medium and patient urine. The results demonstrate that progress curves can be analysed by fitting a logistic equation, derived from a simplified autocatalytic clot formation model. The parameters, maximum amplitude (Fm), rate constant (k), time to half-maximum amplitude (tm) and maximum rate of clot formation (vm), fit a power curve showing limiting effects with increasing TF concentration. Log/log plots of tm and k against TF concentration provide standard curves for assessment of unknowns.

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. A simple kinetic analysis of the coupled reactions shows that the early points of such curves should be fitted by second order polynomials, representing the accelerating reaction rate in µmol pNA/min/min. The first derivative of the polynomial then gives the increasing velocity of pNA production in µmol pNA/min over the time course of the assay. We demonstrate here that, with the substrate S2238, these rates can be converted to absolute thrombin concentrations using the Michaelis-Menten equation, substituted with values for kcat and Km. These thrombin concentrations increase linearly over the time course of the assay allowing the activity to be expressed in units, defined as µmol product/min, most commonly used to report enzyme activity.

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 Ki values of 3.03 µM and 0.59 µM, respectively. Another reversible inhibitor of both KARIs, Hoe 704, is more potent than CPD with Ki values of 300 nM and 110 nM for MtKARI and CjKARI, respectively. The most potent inhibitor tested here is N-hydroxy-N-isopropyloxamate (IpOHA). It has a Ki of ~26 nM for MtKARI, but binds rather slowly (kon ~900 M-1s-1). In contrast, IpOHA binds more rapidly (kon ~7000 M-1s-1) to CjKARI and irreversibly.

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. Coagulation was analysed using a visual assay, Hyland-Clotek and thromboelastography. Healthy control blood was spiked with a range of anticoagulants to determine the effectiveness of OsPa-induced clotting. A paired analysis of a Dabigatran patient and a control investigated the effectiveness of the OsPA clotting tubes. Biochemical analytes (N = 31) were determined for 7 samples on chemistry and immunoassay analysers and compared with commercial tubes. RESULTS: Snake venom prothrombin activators efficiently coagulated blood and plasma spiked with heparin and commonly used anticoagulants. Clotting was observed in the presence of anticoagulants whereas no clotting was observed in BDRST tubes containing 3 U/mL of heparin. Snake venom prothrombin activator enhanced heparinised blood clotting by shortening substantially the clotting time and improving significantly the strength of the clot. Comparison of 31 analytes from the blood of five healthy and two anticoagulated participants gave very good agreement between the analyte concentrations determined. CONCLUSIONS: Our results showed that the snake venom prothrombin activators OsPA and PtPA efficiently coagulated recalcified and fresh bloods with or without added anticoagulants. These procoagulants produced high quality serum for accurate analyte measurement.

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. Blood clotting was visually observed, and thromboelastography was also performed to determine the important parameters of coagulation. Thrombin generation was assayed using the chromogenic substrate S-2238, and biochemical analytes in the serum were determined on chemistry and immunoassay analysers. Fibrinogen was determined by either ELISA or Clauss fibrinogen assay. Results We initially showed that OsPA had strong coagulation activity in clotting not only recalcified citrated plasma and recalcified citrated whole blood, but also fresh whole blood in a clinical setting. The use of TEG clearly showed improved speed of clotting and generation of a firmer clot. We also showed that the use of OsPA to produce serum did not interfere with the determination of commonly measured biochemical analytes. The underlying clotting mechanism involves a burst of thrombin production at the initial stages of the clotting process upon contact with prothrombin in blood. Conclusions These results demonstrate rapid generation of high-quality serum, contributing to faster turnaround times with standardised quality samples, for accurate analyte determinations in normal individuals.

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. This best practice guideline has been developed with reference to the Centers for Disease Control and Prevention (CDC) "Laboratory Medicine Best Practices: Developing an Evidence-Based Review and Evaluation Process". The CDC document cites an evaluation framework for generating best practice recommendations that are specific to laboratory medicine. These 50 recommendations proposed herein, were generated from a comprehensive literature search and the extensive combined experience of the AACB Vitamins Working Party members. They were formulated based on comparison between an impact assessment rating and strength of evidence and were classified as either: (1) strongly recommend, (2) recommend, (3) no recommendation for or against, or (4) recommend against. These best practice recommendations represent the consensus views, in association with peer reviewed evidence of the AACB Vitamins Working Party, towards best practice for the collection, analysis and interpretation of vitamins A, E and carotenoids in blood.

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. METHODS: Patients with a self-reported history of sustained heavy alcohol consumption were recruited from the hepatology outpatient department or medical wards. Each patient was interviewed with a validated structured questionnaire of alcohol consumption and CDT analysis using the standardized reference measurement technique with high performance liquid chromatography was performed on serum collected at time of interview. RESULTS: 52 patients were recruited: 19 from the hepatology outpatient department and 33 from general medical wards. Median alcohol intake was 1013 (range 366-5880) g/week over the preceding two week period. 26 patients had a diagnostic CDT based on a threshold value of %CDT > 1.7 indicating heavy alcohol consumption, yielding a sensitivity of 50%. Overweight/obesity (defined as body mass index (BMI) ≥ 25 kg/m2 in Caucasians and ≥ 23.0 kg/m2 in Asians), female gender and presence of cirrhosis were independently associated with non-diagnostic %CDT (≤ 1.7). CONCLUSIONS: CDT has limited sensitivity as a biomarker of heavy alcohol consumption. Caution should be applied when ordering and interpreting %CDT results, particularly in women, patients with cirrhosis and those with an elevated BMI.

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. This study was designed to determine whether alcohol-independent factors influence CDT levels in patients with chronic liver disease (CLD). METHODS: The relationship between serum %CDT and self-reported history of alcohol consumption was examined in 254 patients referred for evaluation of liver disease. CDT analysis was performed on serum collected at time of liver biopsy. RESULTS: CDT levels were not affected by severity or etiology of nonalcoholic liver disease. Thirteen of 254 subjects had a %CDT >1.7, predictive of heavy alcohol intake, 6 of whom did not acknowledge heavy drinking. Twelve of these 13 subjects were suspected heavy drinkers on review of their medical records and clinical results. Conversely, not all acknowledged heavy drinkers had %CDT >1.7. Heavy drinkers with a body mass index (BMI) in the overweight or obese range had significantly lower %CDT than lean heavy drinkers. This persisted even when lean body weight was used as an approximation of the EtOH volume of distribution. CONCLUSIONS: An elevated BMI reduces the diagnostic utility of CDT at higher alcohol intake in subjects with CLD using the standardized method. In a hepatology outpatient setting, this assay is likely to be useful to confirm suspicion of heavy drinking in subjects who are not overweight, but cannot reliably identify moderate drinkers or heavy drinkers who are overweight.

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, ß-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. DESIGN AND METHODS: A questionnaire was sent to thirteen laboratories. Eleven laboratories were returning QAP results for vitamin B1 (thiamine diphosphate) and five were returning results for vitamin B6 (pyridoxal-5-phosphate). RESULTS: All nine respondents provided a clinical service for vitamins B1 and B6. HPLC with fluorescence detection was the most common method principle. For vitamin B1, six respondents used a commercial assay whilst three used in-house methods; whole blood was the matrix for all. For vitamin B6, five respondents used commercial assays and four used in-house assays. The choice of matrix for vitamin B6 varied with three respondents using whole blood and five using plasma for analysis. Sample preparation incorporated protein precipitation and derivatization steps. An internal standard was employed in sample preparation by only one survey respondent. CONCLUSIONS: The immediate result of this survey was the incorporation of plasma vitamin B6 into the RCPA QAP vitamin program. The absence of an internal standard in current vitamin B1 and B6 assays is a likely contributor to the wide dispersion of results seen in this program. We recommend kit manufacturers and laboratories investigate the inclusion of internal standards to correct the variability that may occur during processing.

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. RESULTS: Chromatography time was 3 min with excellent linearity, precision and low detection limits (IS of 0.02 µmol/L and pCS of 0.05 µmol/L). Both IS and pCS increased with a decrease in renal function and were moderately correlated with eGFR (R(2) 0.65 and 0.33 respectively). The serum reference ranges were (µmol/L): total IS of 0.7-6.3; free IS of 0.0-0.2; total pCS of 0.0-38.4; and free pCS of 0.1-2.4. The intra individual biological variation was estimated at 35.9% and 50.5% with a critical difference of 3.9 µmol/L (100%) and 20.7 µmol/L (141%) for total IS and pCS respectively. CONCLUSION: We describe a robust analytical method with a short chromatography time that quantifies both IS and pCS. The data on reference ranges and intra-individual biological variation need to be considered in clinical studies that investigate these uremic toxins.

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. The crystal structures of the human microplasmin-textilinin-1 and the trypsin-textilinin-1 complexes have been determined to 2.78 Å and 1.64 Å resolution respectively, and show that textilinin-1 binds to trypsin in a canonical mode but to microplasmin in an atypical mode with the catalytic histidine of microplasmin rotated out of the active site. The space vacated by the histidine side-chain in this complex is partially occupied by a water molecule. In the structure of microplasminogen the χ(1) dihedral angle of the side-chain of the catalytic histidine is rotated by 67° from its "active" position in the catalytic triad, as exemplified by its location when microplasmin is bound to streptokinase. However, when textilinin-1 binds to microplasmin the χ(1) dihedral angle of this amino acid residue changes by -157° (i.e. in the opposite rotation direction compared to microplasminogen). The unusual mode of interaction between textilinin-1 and plasmin explains textilinin-1's selectivity for human plasmin over plasma kallikrein. This difference can be exploited in future drug design efforts.

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. scutellatus inhibitor, termed TSPI, was subsequently expressed and purified. TSPI was investigated for inhibition against a panel of 12 enzymes involved in haemostasis and estimates of the K(i) value determined for each enzyme. TSPI was found to be a broad spectrum inhibitor with most potent inhibitory activity observed against plasma kallikrein that corresponded to a K(i) of 0.057 ± 0.019 nM. TSPI also inhibited fibrinolysis in whole blood and prolonged the intrinsic clotting time. These inhibitors are also unique in that they appear to be found only in Oxyuranus sp. venoms.

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. One highly conserved isoform, which displayed close sequence identity with family 2 cystatins, was detected in each elapid snake. Crude Austrelaps superbus (Australian lowland copperhead) snake venom inhibited papain, and a recombinant form of A. superbus cystatin inhibited cathepsin L â‰… papain > cathepsin B, with no inhibition observed for calpain or legumain. While snake venom cystatins have truncated N-termini, sequence alignment and structural modelling suggested that the evolutionarily conserved Gly-11 of family 2 cystatins, essential for cysteine protease inhibition, is conserved in snake venom cystatins as Gly-3. This was confirmed by mutagenesis at the Gly-3 site, which increased the dissociation constant for papain by 10(4)-fold. These data demonstrate that elapid snake venom cystatins are novel members of the type 2 family. The widespread, low level expression of type 2 cystatins in snake venom, as well as the presence of only one highly conserved isoform in each species, imply essential housekeeping or regulatory roles for these proteins.

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. The activated partial thromboplastin time for plasma was markedly increased by aprotinin but unaffected by textilinin-1. In a mouse tail-vein bleeding model, intravenous textilinin-1 and aprotinin caused similar decreases in blood loss but time to haemostasis in the textilinin-treated animals was significantly shorter than in aprotinin-treated mice. Based on these data, textilinin-1 merits further investigation as a therapeutic alternative to aprotinin.

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%. Whole blood clot lysis was inhibited strongly by both aprotinin and textilinin-1, as shown by thrombelastography. At 2 microM inhibitor lysis initiated by t-PA was greater than 99% inhibited by aprotinin (LY60 = 0.4 +/- 0.1) whereas textilinin-1, inhibited lysis by 91% (LY60 = 8.9 +/- 0.7). The same trend was found with the lysis of euglobulin fractions. From these data textilinin-1 appears to be a more specific plasmin inhibitor than aprotinin but aprotinin inhibits clot lysis to a greater extent.