Comparative Evaluation of Two NGAL Automated Immunoassays in Urine and Plasma.

BACKGROUND: Acute kidney injury (AKI), a frequent and serious complication of hospitalized patients, is associated with increased mortality and morbidity. Neutrophil gelatinase-associated lipocalin (NGAL) is a biomarker for the early identification of AKI. We report a comparative laboratory verification of the Abbott Diagnostics (ARCHITECT® urine NGAL) and BioPorto Diagnostics (NGAL TestTM) assays including an assessment of the Abbott assay's performance in EDTA plasma. METHODS: Intra-/interbatch imprecision, linearity, recovery, and limit of quantitation (LoQ) were assessed and an interassay comparison performed (n = 51). Between-laboratory agreement was assessed against other laboratories using the Abbott (n = 48) and BioPorto (n = 94) assays. Plasma NGAL (pNGAL) levels were measured in non-AKI patients with a range of estimated glomerular filtration rates (n = 80). RESULTS: Coefficients of variation (CVs) for intra- and interbatch imprecision were 0.7%-12.4% and 1.9%-27.5% for the BioPorto assay, respectively, and 1.4%-6.3%/3.4%-6.8%, respectively, for the Abbott assay. The BioPorto assay exhibited a higher LoQ (27.5 ng/mL vs 1.2 ng/mL). Both assays were linear over the range 5-6000 ng/mL. Recovery of recombinant NGAL was 113.1 ± 7.1% and 96.5 ± 7.8% for the Abbott and BioPorto assays, respectively. On average, the Abbott assay gave results 9.2% lower than the BioPorto assay. Mean differences of 0.2% (Abbott) and 20.2% (BioPorto) were observed in the between-laboratory comparison. In patients without AKI, pNGAL levels were inversely proportional to eGFR. CONCLUSIONS: Performance of the Abbott and BioPorto assays was similar although the latter performed less well at lower NGAL concentrations. The Abbott assay tended to yield lower results, exhibited a lower LoQ and over-recovered NGAL. Although only Conformité Européenne-marked and marketed for use in urine, the Abbott assay demonstrated equivalent performance to the BioPorto assay with EDTA plasma.

Do reflex comments on laboratory reports alter patient management?

INTRODUCTION: Laboratory comments appended on clinical biochemistry reports are common in the UK. Although popular with clinicians and the public, there is little evidence that these comments influence the clinical management of patients. METHODS: We provided reflex automated laboratory comments on all primary care lipid results including, if appropriate, recommendation of direct referral to the West Midlands Familial Hypercholesterolaemia service (WMFHS). Over a two-year period, the number GP referrals from the Wolverhampton City Clinical Commissioning Group (CCG) to the WMFHS were compared with four comparator CCGs of similar population size, who were not provided with reflex laboratory comments. RESULTS: Over the study period, the WMFHS received more referrals from Wolverhampton GPs (241) than any other comparator CCG (range 8-65) and greater than the combined referrals (172) from all four comparator CCGs. CONCLUSION: Targeted reflex laboratory comments may influence the clinical management of patients and may have a role in the identification of individuals with familial hypercholesterolaemia.

kEDTA Sample Contamination: A Reappraisal.

BACKGROUND: Potassium EDTA (kEDTA) contamination of serum samples is common, causing spurious hyperkalemia, hypozincemia, and hypocalcemia that if unrecognized may adversely affect patient care. Gross kEDTA contamination is easy to detect, but identification of spurious electrolytes due to small amounts of contamination requires measurement of serum EDTA. We validated an EDTA assay on the Abbott Architect and reassessed its value in identifying kEDTA contamination and in studying mechanisms for contamination. METHODS: Within- and between-batch imprecision, linearity, recovery, interference, and carryover were assessed. Serum supplemented with k2EDTA plasma, to mimic sample contamination, was used to study its effect on potassium, calcium, zinc, magnesium, and alkaline phosphatase. Our current laboratory protocol for identification of kEDTA contamination, based on measurement of serum calcium, was compared to that of EDTA measurement. RESULTS: The EDTA assay displayed acceptable performance characteristics. Hemoglobin was a positive interferent. EDTA was detectable in serum contaminated with 1% (v:v) k2EDTA plasma. An increase in serum potassium of 0.54 mmol/L (11.9%) was observed at a measured EDTA concentration of 0.19 mmol/L, equivalent to 3.2% (v:v) contamination. At this EDTA concentration reductions were also observed in zinc (71%), calcium (1%), alkaline phosphatase (ALP) (4%), and magnesium (2.4%). The serum EDTA assay detected contamination in 31/106 patient samples with hyperkalemia (potassium ≥6.0mmol/L), 20 of which were undetected by the current laboratory protocol. CONCLUSIONS: The EDTA assay displayed acceptable performance, with the ability to reliably measure EDTA at low concentrations. Only a small amount of kEDTA causes significant spurious hyperkalemia and is only reliably detected with EDTA measurement.

Is there any value in measuring faecal calprotectin in Clostridium difficile positive faecal samples?

Markers of intestinal inflammation have been proposed for inclusion in Clostridium difficile diagnostic algorithms. Faecal calprotectin (f-Cp), a sensitive marker of intestinal inflammation, was evaluated for utility in C. difficile diagnosis in the hospital setting. One hundred and twenty C. difficile positive and 99 C. difficile negative faecal samples of hospital-acquired diarrhoea were analysed for f-Cp using a quantitative ELISA. C. difficile positivity was confirmed using ELISAs for either toxins (n = 45) or glutamate dehydrogenase (GDH) with toxin gene confirmation (n = 75). Non-parametric ANOVA (Kruskal-Wallis) was used for data analysis. C. difficile positive samples had higher (P<0.05) median (interquartile range) f-Cp levels; 336 µg g(-1) (208-536) for toxin and 249 µg g(-1) (155-498) for GDH and toxin gene positive compared with 106 µg g(-1) (46-176) for C. difficile and culture-negative faecal samples. Five C. difficile positive samples were f-Cp negative (<50 µg g(-1)). A f-Cp concentration >50 µg g(-1) was 96 % sensitive and 26 % specific for C. difficile, with area under the ROC curve of 0.82. There is no role for f-CP alone in predicting C. difficile infection in hospital-acquired diarrhoea due to its low specificity.

A combined laboratory and field evaluation of the Cholestech LDX and CardioChek PA point-of-care testing lipid and glucose analysers.

BACKGROUND: Combined lipid and glucose point-of-care testing (POCT) devices could facilitate widespread population screening for cardiovascular disease (CVD) and diabetes as part of the NHS Vascular Risk Assessment and Management Program (NHS Health Checks). An evaluation of the Cholestech LDX and CardioChek PA POCT analysers was performed in collaboration with the Wolverhampton City Primary Care Trust (PCT). METHODS: Intra-/inter-batch imprecision, between-analyser variation and the effect of haematocrit and ascorbic acid assay interference were investigated. Accuracy of the POCT capillary whole blood total cholesterol (TC), high-density-lipoprotein cholesterol (HDL-C) and glucose measurements was estimated by comparison with those from the laboratory analysis of paired venous samples. POCT usability and clinical governance were also assessed. RESULTS: The LDX exhibited lower intra- and inter-batch imprecision and external quality assessment (EQA) scheme between-analyser variation for the measurement of TC, HDL-C and glucose when compared to the CardioChek. Ascorbic acid negatively interfered in all three assays on both POCT analysers and results reported by the CardioChek were influenced by the specimen haematocrit. The LDX displayed closer agreement with the laboratory methods for the measurement of TC and HDL-C but both the LDX and the CardioChek displayed positive bias for the measurement of glucose. CONCLUSIONS: POCT has clear advantages for delivering NHS Health Checks over the laboratory-based approach although device performance does differ. Users should also be aware of the potential clinical governance and interference issues associated with these devices.

Effect of order of draw of blood samples during phlebotomy on routine biochemistry results.

AIM: To investigate whether incorrect order of draw of blood samples during phlebotomy causes in vitro potassium ethylenediaminetetraacetic acid EDTA (kEDTA) contamination of blood samples. METHODS: Serum kEDTA, potassium, calcium, magnesium, alkaline phosphatase, zinc and iron concentrations were measured in blood samples drawn before and after collecting blood into kEDTA containing sample tubes by an experienced phlebotomist using the Sarstedt Safety Monovette system. RESULTS: EDTA was undetectable in all samples. The concentrations of other analytes were similar in blood samples drawn before and after collection of the EDTA blood sample. CONCLUSION: Order of draw of blood samples using the Sarstedt Safety Monovette system has no effect on serum biochemistry results, when samples are taken by an experienced phlebotomist.

X-ray absorption studies of Zn(2+)-binding sites in Escherichia coli transhydrogenase and its betaH91K mutant.

Transhydrogenase couples hydride transfer between NADH and NADP(+) to proton translocation across a membrane. The binding of Zn(2+) to the enzyme was shown previously to inhibit steps associated with proton transfer. Using Zn K-edge X-ray absorption fine structure (XAFS), we report here on the local structure of Zn(2+) bound to Escherichia coli transhydrogenase. Experiments were performed on wild-type enzyme and a mutant in which betaHis91 was replaced by Lys (betaH91K). This well-conserved His residue, located in the membrane-spanning domain of the protein, has been suggested to function in proton transfer, and to act as a ligand of the inhibitory Zn(2+). The XAFS analysis has identified a Zn(2+)-binding cluster formed by one Cys, two His, and one Asp/Glu residue, arranged in a tetrahedral geometry. The structure of the site is consistent with the notion that Zn(2+) inhibits proton translocation by competing with H(+) binding to the His residues. The same cluster of residues with very similar bond lengths best fits the spectra of wild-type transhydrogenase and betaH91K. Evidently, betaHis91 is not directly involved in Zn(2+) binding. The locus of betaHis91 and that of the Zn-binding site, although both on (or close to) the proton-transfer pathway of transhydrogenase, are spatially separate.

Inhibition of proton-transfer steps in transhydrogenase by transition metal ions.

Transhydrogenase couples proton translocation across a bacterial or mitochondrial membrane to the redox reaction between NAD(H) and NADP(H). Purified intact transhydrogenase from Escherichia coli was prepared, and its His tag removed. The forward and reverse transhydrogenation reactions catalysed by the enzyme were inhibited by certain metal ions but a "cyclic reaction" was stimulated. Of metal ions tested they were effective in the order Pb(2+)>Cu(2+)>Zn(2+)=Cd(2+)>Ni(2+)>Co(2+). The results suggest that the metal ions affect transhydrogenase by binding to a site in the proton-transfer pathway. Attenuated total-reflectance Fourier-transform infrared difference spectroscopy indicated the involvement of His and Asp/Glu residues in the Zn(2+)-binding site(s). A mutant in which betaHis91 in the membrane-spanning domain of transhydrogenase was replaced by Lys had enzyme activities resembling those of wild-type enzyme treated with Zn(2+). Effects of the metal ion on the mutant were much diminished but still evident. Signals in Zn(2+)-induced FTIR difference spectra of the betaHis91Lys mutant were also attributable to changes in His and Asp/Glu residues but were much smaller than those in wild-type spectra. The results support the view that betaHis91 and nearby Asp or Glu residues participate in the proton-transfer pathway of transhydrogenase.

Structures of the dI2dIII1 complex of proton-translocating transhydrogenase with bound, inactive analogues of NADH and NADPH reveal active site geometries.

Transhydrogenase couples the redox reaction between NADH and NADP+ to proton translocation across a membrane. The enzyme comprises three components; dI binds NAD(H), dIII binds NADP(H), and dII spans the membrane. The 1,4,5,6-tetrahydro analogue of NADH (designated H2NADH) bound to isolated dI from Rhodospirillum rubrum transhydrogenase with similar affinity to the physiological nucleotide. Binding of either NADH or H2NADH led to closure of the dI mobile loop. The 1,4,5,6-tetrahydro analogue of NADPH (H2NADPH) bound very tightly to isolated R. rubrum dIII, but the rate constant for dissociation was greater than that for NADPH. The replacement of NADP+ on dIII either with H2NADPH or with NADPH caused a similar set of chemical shift alterations, signifying an equivalent conformational change. Despite similar binding properties to the natural nucleotides, neither H2NADH nor H2NADPH could serve as a hydride donor in transhydrogenation reactions. Mixtures of dI and dIII form dI2dIII1 complexes. The nucleotide charge distribution of complexes loaded either with H2NADH and NADP+ or with NAD+ and H2NADPH should more closely mimic the ground states for forward and reverse hydride transfer, respectively, than previously studied dead-end species. Crystal structures of such complexes at 2.6 and 2.3 A resolution are described. A transition state for hydride transfer between dihydronicotinamide and nicotinamide derivatives determined in ab initio quantum mechanical calculations resembles the organization of nucleotides in the transhydrogenase active site in the crystal structure. Molecular dynamics simulations of the enzyme indicate that the (dihydro)nicotinamide rings remain close to a ground state for hydride transfer throughout a 1.4 ns trajectory.

Zinc ions selectively inhibit steps associated with binding and release of NADP(H) during turnover of proton-translocating transhydrogenase.

Transhydrogenase couples the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. In membrane vesicles from Escherichia coli and Rhodospirillum rubrum, the transhydrogenase reaction (measured in the direction driving inward proton translocation) was inhibited by Zn(2+) and Cd(2+). However, depending on pH, the metal ions either had no effect on, or stimulated, "cyclic" transhydrogenation. They must, therefore, interfere specifically with steps involving binding/release of NADP(+)/NADPH: the steps thought to be associated with proton translocation. It is suggested that Zn(2+) and Cd(2+) bind in the proton-transfer pathway and block inter-conversion of states responsible for changing NADP(+)/NADPH binding energy.