Is ischaemia-modified albumin a test for venous thromboembolism?

OBJECTIVE: Patients with symptoms of deep vein thrombosis (DVT) and pulmonary embolism (PE) commonly present to the emergency department (ED). The aim of this study was to assess the role of ischaemia-modified albumin (IMA) testing in the diagnosis of venous thromboembolism (VTE). METHODS: This was a prospective diagnostic cohort study. Inpatients and ED patients >16 years of age investigated for PE or DVT at a single hospital were eligible for study consent. Blinded IMA analysis was performed on the first blood sample taken from each patient. Patients underwent reference standard investigation for PE or DVT, including 3-month follow-up. Receiver operating characteristic (ROC) curves were constructed for IMA and the IMA:albumin ratio in the diagnosis of all VTE, PE and DVT. A sensitivity analysis was performed. RESULTS: 452 patients were consented and investigated for DVT, and 354 patients were consented and investigated for PE (806 in total). 348 patients investigated for PE had IMA testing as did 195 of the first 199 DVT patients. VTE prevalence was 19.7%. The IMA:albumin ratio performed better than IMA alone. The area under the ROC curve (AUC) for IMA:albumin in all VTE was 0.60 (95% CI 0.54 to 0.66), in DVT 0.56 (95% CI 0.46 to 0.65) and in PE 0.63 (95% CI 0.56 to 0.71). In ED patients with symptoms of PE, the AUC for IMA:albumin was 0.69 (95% CI 0.60 to 0.78). CONCLUSIONS: IMA testing cannot be used alone to diagnose DVT or PE, although there is a moderate association with PE in ED patients.

Improving the nutritional value of Golden Rice through increased pro-vitamin A content.

"Golden Rice" is a variety of rice engineered to produce beta-carotene (pro-vitamin A) to help combat vitamin A deficiency, and it has been predicted that its contribution to alleviating vitamin A deficiency would be substantially improved through even higher beta-carotene content. We hypothesized that the daffodil gene encoding phytoene synthase (psy), one of the two genes used to develop Golden Rice, was the limiting step in beta-carotene accumulation. Through systematic testing of other plant psys, we identified a psy from maize that substantially increased carotenoid accumulation in a model plant system. We went on to develop "Golden Rice 2" introducing this psy in combination with the Erwinia uredovora carotene desaturase (crtI) used to generate the original Golden Rice. We observed an increase in total carotenoids of up to 23-fold (maximum 37 microg/g) compared to the original Golden Rice and a preferential accumulation of beta-carotene.

A novel high-throughput method for supported liquid extraction of retinol and alpha-tocopherol from human serum and simultaneous quantitation by liquid chromatography tandem mass spectrometry.

BACKGROUND: Measurement of vitamin A (retinol) and E (alpha-tocopherol) in UK clinical laboratories is currently performed exclusively by high-performance liquid chromatography with ultraviolet detection. We investigated whether retinol and alpha-tocopherol could be measured simultaneously by liquid chromatography tandem mass spectrometry. METHODS: Serum samples (100 µL) were extracted using Isolute + Supported Liquid Extraction plates. Chromatography was performed on a Phenomenex Kinetex Biphenyl 2.6 µm, 50 × 2.1 mm column, and liquid chromatography tandem mass spectrometry on a Waters Acquity TQD. Injection-to-injection time was 4.3 min. The assay was validated according to published guidelines. Patient samples were used to compare liquid chromatography tandem mass spectrometry and high-performance liquid chromatography with ultraviolet detection methods. RESULTS: For retinol and alpha-tocopherol, respectively, the assay was linear up to 6.0 and 80.0 µmol/L, and lower limit of quantification was 0.07 and 0.26 µmol/L. Intra and interassay imprecision were within desirable analytical specifications. Analysis of quality control material aligned to NIST SRM 968e, and relative spiked recovery from human serum, both yielded results within 15% of target values. Method comparison with high-performance liquid chromatography with ultraviolet detection methodology demonstrated a negative bias for retinol and alpha-tocopherol by the liquid chromatography tandem mass spectrometry method. Analysis of United Kingdom National External Quality Assurance Scheme samples yielded mean bias from the target value of +3.0% for retinol and -11.2% for alpha-tocopherol. CONCLUSIONS: We have developed a novel, high-throughput method for extraction of retinol and alpha-tocopherol from human serum followed by simultaneous quantitation by liquid chromatography tandem mass spectrometry. The method offers a rapid, sensitive, specific and cost-effective alternative to high-performance liquid chromatography with ultraviolet detection methodology, and is suitable for routine clinical monitoring of patients predisposed to fat-soluble vitamin malabsorption.

Therapeutic drug monitoring of ciclosporin A and tacrolimus in heart lung transplant patients using dried blood spots.

BACKGROUND: Therapeutic drug monitoring of ciclosporin A (CsA) and tacrolimus is traditionally performed using venous whole blood sampling. A number of reports have described development of ultra high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) methods for the quantitation of CsA and tacrolimus from dried blood spots (DBS), which may offer a convenient alternative. As yet, no such reports have validated this methodology using fingerprick capillary DBS samples collected from transplant patients. METHODS: Capillary fingerprick DBS were collected from heart and lung transplant patients in a specialist cardiothoracic transplant centre. We utilized our previously published method for the extraction and simultaneous quantitation of CsA and tacrolimus from DBS using UPLC-MS/MS. Drug concentrations measured from DBS were compared to concentrations measured in venous whole blood by our routine clinical UPLC-MS/MS assay. RESULTS: In total, 91 heart or lung transplant patients were enrolled onto the study; 46 patients were on CsA therapy and 45 on tacrolimus therapy. Passing-Bablock analysis demonstrated excellent agreement between capillary fingerprick DBS samples and venous whole blood samples. There was a mean positive bias of 2.6 µg/L (95% confidence interval (CI) -2.2 to 7.5 µg/L) for CsA (n = 45) and mean negative bias of -0.7 µg/L (95% CI -1.1 to -0.3 µg/L) for tacrolimus (n = 42). CONCLUSIONS: We demonstrate utility of DBS for serial monitoring of CsA and tacrolimus using UPLC-MS/MS in heart and lung transplant patients. This may offer significant advantages for these patients including the ability to take capillary DBS samples in the community prior to clinic visits.

Quantitation of aldosterone in human plasma by ultra high performance liquid chromatography tandem mass spectrometry.

Aldosterone is a mineralocorticoid steroid hormone whose measurement in the clinical laboratory is principally performed for the investigation of primary hyperaldosteronism. Traditionally measurement of aldosterone has been performed by radioimmunoassay, however these assays lack specificity as they are prone to interference from structurally related steroid hormones. Herein, we have developed a novel, sensitive and specific method utilising solid phase extraction and quantitation of aldosterone from human plasma by UPLC-MS/MS. Standards, quality controls and samples (250µL) were extracted using Oasis(®) HLB 96-well plates. Extract (30µL) was injected onto a Krudcatcher UPLC In-Line Filter, 0.5µm guard column, coupled to a Kinetex PFP, 100mm×2.1mm, 2.6µm column with methanolic mobile phase gradient elution. Eluant was connected to a Waters(®) Xevo TQS tandem mass spectrometer operating in electrospray negative mode. We detected multiple reaction monitoring (MRM) transitions of m/z 359.0>189.1 for aldosterone and 366.0>194.1 for d7-aldosterone respectively, which co-eluted at 2.65min. Ion suppression was negligible. Mean recovery was 89.6%, limit of detection and lower limit of quantitation were 26pmol/L and 30pmol/L respectively. The assay was linear up to 3200pmol/L (r(2)=0.9999). Mean intra- and inter-assay imprecision and bias were all <10%. Comparison of the UPLC-MS/MS method with an immunoassay in routine clinical use in the UK yielded the equation UPLC-MS/MS=0.789(RIA)-41.7, linear regression r(2)=0.88, n=54. We have developed a sensitive and specific method for the extraction and measurement of aldosterone from human plasma. The method features a simple 96-well plate solid phase extraction procedure, highly selective column chemistry and short chromatographic run times.

Predicting short term mortality after investigation for venous thromboembolism.

INTRODUCTION: Deaths following diagnosis of venous thromboembolism (VTE) often result from another concurrent illness. The specificity of mortality markers predicting death from pulmonary embolism is unknown. The aim of this analysis was to compare blood predictors of death in patients with confirmed VTE to patients with negative investigations for VTE. MATERIALS AND METHODS: Consecutive patients investigated for VTE were prospectively consented from a single hospital over 9months. VTE was diagnosed and excluded with a standard diagnostic algorithm. Blood was drawn for biomarker analysis and analyzed in batches for NT-proBNP, high sensitivity troponin T, C-reactive protein (CRP), fatty acid binding protein (FABP) and ischemia modified albumin (IMA). Participants were followed for 3months. The cohort was analyzed in two groups: those diagnosed with VTE and those who had thrombosis excluded. Regression analysis for 3-month mortality was performed for each group. RESULTS: 16/153 patients diagnosed with VTE died within three months (10.5%) as did 23/606 patients who had negative investigations for VTE (3.8%). Predictors for death following VTE included cancer, NT-proBNP, troponin T, FABP, and Hb<95g/L. NT-proBNP>500pg/ml in acute cancer associated VTE predicted death with C-statistic of 0.89 (0.80-0.99). Cancer, NT-proBNP and troponin T also predicted death in patients with negative investigations for VTE. CONCLUSION: Several blood markers are not specific for death from PE and may be surrogate markers of global declining health.

Simultaneous measurement of cyclosporin A and tacrolimus from dried blood spots by ultra high performance liquid chromatography tandem mass spectrometry.

Cyclosporin A (CsA) and tacrolimus are immunosuppressant drugs principally used in solid organ transplant recipients. Therapeutic drug monitoring (TDM) of both drugs is essential to avoid toxicity related to overdosage, and transplant rejection from underdosage. This necessitates frequent hospital visits to phlebotomy services. Capillary blood sampling onto dried blood spots (DBS) provides numerous advantages to venous whole blood sampling, including the ability for patients to send DBS to the laboratory by post, significantly reducing the number of unnecessary hospital visits. We have developed a novel, simple and rapid method for the extraction and simultaneous UPLC-MS/MS measurement of both CsA and tacrolimus from DBS. The extraction method involved a simple 30 min hot solvent extraction with ultrasonication. Extract (10 µL) was injected onto a Waters Acquity UPLC column filter unit security frit, coupled to a Waters Acquity BEH C18 UPLC column, with methanolic mobile phase gradient elution. Eluant was connected to a Waters Quattro Premier XE tandem mass spectrometer operating in ES+ mode. We detected multiple reaction monitoring (MRM) transitions of m/z 1220>1203 and 1231.9>1215.1 for CsA and d12 CsA respectively which co-eluted at 1.30min, and 821.6>768.5 and 809.6>756.5 for tacrolimus and ascomycin respectively which co-eluted at 1.17 min. Ion suppression was negligible. Mean recovery was 95.5% for CsA and 92.8% for tacrolimus. Limit of detection and limit of quantitation were both 8.5 µg/L for CsA, and 0.5 and 2.3 µg/L respectively for tacrolimus. The assay was linear up to 1500µg/L for CsA (r(2)=0.9999), and up to 50 µg/L for tacrolimus (r(2)=0.9994). Mean intra assay imprecision, inter assay imprecision and bias were all <10% for both CsA and tacrolimus. DBS were stable for at least 14 days at room temperature. Comparison of the DBS UPLC-MS/MS method and the routine venous whole blood LC-MS/MS assay demonstrated good agreement between the two methods for both drugs. We have developed a simple and robust method for the extraction and simultaneous measurement of CsA and tacrolimus from DBS. The method will allow TDM of transplant recipients to proceed at home using capillary blood sampling.

Three isoforms of isoamylase contribute different catalytic properties for the debranching of potato glucans.

Isoamylases are debranching enzymes that hydrolyze alpha-1,6 linkages in alpha-1,4/alpha-1,6-linked glucan polymers. In plants, they have been shown to be required for the normal synthesis of amylopectin, although the precise manner in which they influence starch synthesis is still debated. cDNA clones encoding three distinct isoamylase isoforms (Stisa1, Stisa2, and Stisa3) have been identified from potato. The expression patterns of the genes are consistent with the possibility that they all play roles in starch synthesis. Analysis of the predicted sequences of the proteins suggested that only Stisa1 and Stisa3 are likely to have hydrolytic activity and that there probably are differences in substrate specificity between these two isoforms. This was confirmed by the expression of each isoamylase in Escherichia coli and characterization of its activity. Partial purification of isoamylase activity from potato tubers showed that Stisa1 and Stisa2 are associated as a multimeric enzyme but that Stisa3 is not associated with this enzyme complex. Our data suggest that Stisa1 and Stisa2 act together to debranch soluble glucan during starch synthesis. The catalytic specificity of Stisa3 is distinct from that of the multimeric enzyme, indicating that it may play a different role in starch metabolism.