Maria Fitzgibbon

Establishment of a reference interval for urinary neutrophil gelatinase-associated lipocalin

Introduction Neutrophil gelatinase-associated lipocalin (NGAL) is emerging as a promising new biomarker for the early identification of acute kidney injury (AKI). We have determined a reference range in a large healthy population. In addition, as NGAL is a neutrophil-related protein, we investigated whether the presence of leukocyturia has the potential to significantly alter the specificity of NGAL in the diagnosis of AKI. Methods One hundred and seventy-four subjects (100 men, 74 women ranging from 19 to 88 y) were included in the reference population. Urinary NGAL was analysed on the Abbott ARCHITECT and results expressed in mass (μg/L) and also normalized to urinary creatinine (μg/mmol). Fifty-two leukocyturic urine samples were also analysed for NGAL. Results The 95th centile for NGAL was determined to be 107 μg/L (13 μg/mmol). There were significant gender-related differences for NGAL, with women having higher concentrations. There were significant age-related differences for NGAL between the 40–59 and 60–88 y age categories. There were significant age-related differences between the <40 and 60–88 y categories when NGAL was normalized to creatinine. In addition, we found significantly higher concentrations of NGAL in leukocyturia (P < 0.0001). Conclusions We have established a 95th centile cut-off for urinary NGAL in a reference population. We have demonstrated the important potential interference of leukocyturia in confounding the interpretation of NGAL in the diagnosis of AKI.

Comparison of the prognostic accuracy of scoring systems, cardiopulmonary exercise testing, and plasma biomarkers: a single-centre observational pilot study

BACKGROUND Current approaches to risk assessment before major surgery have important limitations. The aim of this pilot study was to compare predictive accuracy of preoperative scoring systems, plasma biomarkers, and cardiopulmonary exercise testing (CPET) for complications after major non-cardiac surgery. METHODS Single-centre, observational study of patients aged ≥40 yr undergoing major elective non-cardiac surgery. Before surgery, risk scores were calculated and blood samples collected for measurement of plasma biomarkers. Patients underwent CPET for measurement of anaerobic threshold (AT) and peak oxygen consumption ( peak). After surgery, patients were followed for 28 days to evaluate complications and major adverse cardiac events (MACE). Data are presented as area under the receiver operating characteristic curve (AUROC) with 95% confidence intervals. RESULTS A total of 100 patients were recruited between April 2009 and October 2010; 17 of whom did not proceed to surgery. CPET variables suggested good predictive accuracy for MACE [AT: AUROC 0.83 (0.69-0.96); peak AUROC 0.81 (0.69-0.96)] and poor predictive accuracy for all complications [AT: AUROC 0.64 (0.52-0.77); peak AUROC 0.64 (0.52-0.77)]. There was a trend towards predictive accuracy of the plasma biomarkers B-type natriuretic peptide and estimated glomerular filtration rate (calculated from serum creatinine) for MACE but not all complications. C-reactive protein, ASA score, and revised cardiac risk index had little or no predictive value. CONCLUSIONS These pilot data suggest that CPET and plasma biomarkers may improve risk assessment before surgery. Only large clinical studies can confirm this observation and define the optimal use of these tests in clinical practice.

Reference Values for Urinary HMMA, HVA, Noradrenaline, Adrenaline, and Dopamine Excretion in Children Using Random Urine Samples and HPLC with Electrochemical Detection

Random urine samples were collected from 305 children aged from birth to 14 years and the values of hydroxymethoxymandelic acid, homovanillic acid, noradrenaline, adrenaline, and dopamine were measured by high-performance liquid chromatography with electrochemical detection. The results were reported relative to the urinary creatinine concentration and the values declined progressively with increasing age for each analyte with the exception of adrenaline. The results for each age group were not normally distributed and all values except outliers were retained in determining the upper reference limits.

Classical galactosaemia: novel insights in IgG N -glycosylation and N -glycan biosynthesis

Classical galactosaemia (OMIM #230400), a rare disorder of carbohydrate metabolism, is caused by a deficient activity of galactose-1-phosphate uridyltransferase (EC 2.7.7.12). The pathophysiology of the long-term complications, mainly cognitive, neurological and female fertility problems remains poorly understood. The lack of validated biomarkers to determine prognosis, monitor disease progression and responses to new therapies, pose a huge challenge. We report the detailed analysis of an automated robotic hydrophilic interaction ultra-performance liquid chromatography N-glycan analytical method of high glycan peak resolution applied to serum IgG. This has revealed specific N-glycan processing defects observed in 40 adult galactosaemia patients (adults and adolescents), in comparison with 81 matched healthy controls. We have identified a significant increase in core fucosylated neutral glycans (P<0.0001) and a significant decrease in core fucosylated (P<0.001), non-fucosylated (P<0.0001) bisected glycans and, of specific note, decreased N-linked mannose-5 glycans (P<0.0001), in galactosaemia patients. We also report the abnormal expression of a number of related relevant N-glycan biosynthesis genes in peripheral blood mononuclear cells from 32 adult galactosaemia patients. We have noted significant dysregulation of two key N-glycan biosynthesis genes: ALG9 upregulated (P<0.001) and MGAT1 downregulated (P<0.01) in galactosaemia patients, which may contribute to its ongoing pathophysiology. Our data suggest that the use of IgG N-glycosylation analysis with matched N-glycan biosynthesis gene profiles may provide useful biomarkers for monitoring response to therapy and interventions. They also indicate potential gene modifying steps in this N-glycan biosynthesis pathway, of relevance to galactosaemia and related N-glycan biosynthesis disorders.

Paediatric Reference Ranges for Urinary Catecholamines/Metabolites and Their Relevance in Neuroblastoma Diagnosis

catecholamines are adrenaline (epinephrine), noradrenaline (norepinephrine) and dopamine, the latter being most frequently elevated in neuroblastoma. Dopamine (the precursor of noradrenaline) is found in the adrenal medulla, in noradrenergic neurons, in the brain, in specialized interneurons in the sympathetic ganglia and in the carotid body where it acts as a neurotransmitter. The catecholamines are synthesized from tyrosine, which may be derived from ingested food or synthesized from phenylalanine in the liver. Tyrosine enters neurons and chromaffin cells by an active transport system and is converted to dihydroxyphenylalanine (dopa). This reaction is catalysed by tyrosine hydroxylase. Dopa is converted to dopamine by the enzyme dopa decarboxylase, which is present in highest concentration in the liver, kidney and brain. Hydroxylation of the dopamine side chain by dopamine {3 hydroxylase produces noradrenaline. Dopamine {3 hydroxylase only occurs in the neuron and is a mixed function oxidase requiring oxygen and an external electron donor. Noradrenaline is methylated to adrenaline only in the adrenal medulla by the enzyme phenylethanolamine N-methyl-transferase (PNMT) using S-adenosyl-methionine as a methyl donor. Catecholamines are stored together with adenosine triphosphate (ATP), calcium, magnesium Additional key phrases: 4-hydroxy-3-methoxy mandelic acid; homovanillic acid; adrenaline; noradrenaline

Establishment of reference intervals for aldosterone and renin in a Caucasian population using the newly developed Immunodiagnostic Systems specialty immunoassay automated system

Background Measurement of aldosterone and/or renin is essential to aid the differential diagnosis of secondary hypertension, guide strategy for therapeutic management of hypertension and assess adequacy of mineralocorticoid replacement. Aim The objective was to establish normative data for aldosterone and renin using the Immunodiagnostic Systems specialty immunoassay system platform in a Caucasian population. Methods Following informed consent, 365 subjects were recruited to this study. Subjects were ambulatory and attended clinic for blood pressure measurement and phlebotomy between the hours of 7:00 and 11:00. Blood pressure was measured according to the 2013 European Society of Hypertension/Cardiology guidelines. The inclusion criteria: age ≥18 years, BMI <30 kg/m2, non-pregnant, blood pressure <140/90, normal electrolytes and kidney function and not taking prescribed/over the counter medications. Ninety-four subjects were excluded based on these criteria. A total of 271 volunteers (females n = 145), aged 18–65 years formed the reference cohort. Blood for aldosterone/renin was collected into ethylenediaminetetraacetic acid specimen tubes. Samples were kept at room temperature and transported within 30 min of blood draw to the laboratory for immediate processing (centrifugation, separation and freezing of plasma). Plasma was stored at −20℃ prior to analysis on the Immunodiagnostic Systems specialty immunoassay system instrument. Results The established reference intervals in an Irish Caucasian population for renin: females: 6.1–62.7 mIU/L, males: 9.0–103 mIU/L, for aldosterone: females: <138–1179 pmol/L, males: <138–670 pmol/L, respectively. Conclusion This study demonstrates that reference intervals for aldosterone and renin should be gender specific. These automated immunoassays offer rapid stratification of patients with refractory hypertension and will better facilitate the optimization of therapeutic management.

Peri-operative troponin monitoring using a prototype high-sensitivity cardiac troponin I (hs-cTnI) assay: comparisons with hs-cTnT and contemporary cTnI assays

Background Non-cardiac surgery is associated with major vascular complications and higher incidences of elevated plasma troponin (cTn) concentration. Goal-directed therapy (GDT) is a stroke volume (SV)-guided approach to intravenous (IV) fluid therapy that improves tissue perfusion, oxygenation and reduces post-operative complications. In patients undergoing major gastro-intestinal surgery, we compared high sensitive and contemporary troponin assays and correlated results with patient outcome. Methods Patients (n = 135) were randomized to receive IV fluid, guided by either the central venous pressure (CVP group, n = 45) or SV (± dopexamine inotrope, n = 45 per group). Serum was obtained pre- and post-operatively (0, 8 and 24 h) for troponin analysis by a prototype hs-cTnI assay (Abbott Laboratories), hs-cTnT (Roche Diagnostics) and contemporary cTnI (Beckman Coulter) assays. Results All troponin measurements were increased (P ≤ 0.05) post-operatively but there was no difference (P > 0.05) amongst treatments. Post-operative increases were reported more frequently (P ≤ 0.05) and earlier with hs-cTnI. Temporal increases (P ≤ 0.05) were reported in patients with and without complications for hs-cTnI/T assays but only in the complications group for cTnI measurements. Elevations ≥99th centile occurred most often (P ≤ 0.05) for hs-cTnT measurements but with similar frequency for both outcome groups (all assays). Only the hs-cTnI assay showed an increased relative risk of mortality (P ≤ 0.05) for elevations ≥99th centile Conclusions Our study may suggest a possible preference for the hs-cTnI assay in the peri-operative setting; however, our findings should be verified for larger cohort studies where emerging reference range data is incorporated for improving risk prediction with hs-cTn assays.

Clinical utility of C-terminal telopeptide of type 1 collagen in multiple myeloma.

Myeloma bone disease (MBD) is a major cause of morbidity in multiple myeloma (MM). We investigated bone turnover markers (BTM) as relapse predictors and biomarkers for monitoring MBD. We measured C‐terminal telopeptide of type I collagen (CTX‐1), and Procollagen type 1 N Propeptide (P1NP) in 86 MM patients and 26 controls. CTX‐1 was higher in newly diagnosed patients compared to control, remission and relapse (P < 0·05), and decreased following treatment. In the setting of relapse, a CTX‐1 rise greater than the calculated least significant change (LSC) was observed in 26% of patients 3–6 months prior to relapse (P = 0·007), and in 60·8% up to 3 months before relapse (P = 0·015). Statistically significant changes in CTX‐1 levels were also observed in patients who were with and without bisphosphonate therapy at the time of relapse. In patients with normal renal function, mean CTX‐1 level was highest in the newly diagnosed group (0·771 ± 0·400 μg/l), and lowest in the remission group (0·099 ± 0·070 μg/l) (P < 0·0001). P1NP levels were not statistically different across the patient groups. We conclude that CTX‐1, measured on an automated hospital laboratory platform, has a role in routine treatment monitoring and predicting relapse of MBD, even in patients on bisphosphonates.

Use of a highly-sensitive cardiac troponin I assay in a screening population for hypertrophic cardiomyopathy: a case-referent study

BackgroundHypertrophic cardiomyopathy (HCM) is a genetic condition, and relatives of affected persons may be at risk. Cardiac troponin biomarkers have previously been shown to be elevated in HCM. This study examines the new highly-sensitive cardiac troponin I (hsTnI) assay in a HCM screening population.MethodsNested case–control study of consecutive HCM sufferers and their relatives recruited from May 2010 to September 2011. After informed consent, participants provided venous blood samples and clinical and echocardiographic features were recorded. Associations between the natural log (ln) of the contemporary troponin I (cTnI) and hsTnI assays and markers of cardiac hypertrophy were examined. Multiple regression models were fitted to examine the predictive ability of hsTnI for borderline or definite HCM.ResultsOf 107 patients, 24 had borderline and 19 had definite changes of HCM. Both TnI assays showed significant, positive correlations with measures of cardiac muscle mass. After age and sex adjustment, the area under the receiver operator characteristic (AUROC) curve for the outcome of HCM was 0.78, 95% CI [0.65, 0.90], for ln(hsTnI), and 0.66, 95% CI [0.51, 0.82], for ln(cTnI) (p=0.11). Including the hsTnI assay in a multiple-adjusted “screening” model for HCM resulted in a non-significant improvement in both the AUROC and integrated discrimination index.ConclusionsBoth cTnI and hsTnI show a graded, positive association with measures of cardiac muscle mass in persons at risk of HCM. Further studies will be required to evaluate the utility of these assays in ECG- and symptom-based identification of HCM in at-risk families.

Transitioning high sensitivity cardiac troponin I (hs-cTnI) into routine diagnostic use: More than just a sensitivity issue

Objectives High sensitivity cardiac troponin T and I (hs-cTnT and hs-cTnI) assays show analytical, diagnostic and prognostic improvement over contemporary sensitive cTn assays. However, given the importance of troponin in the diagnosis of myocardial infarction, implementing this test requires rigorous analytical and clinical verification across the total testing pathway. This was the aim of this study. Design and methods Analytical verification included assessment of critical outlier frequency, for hs-cTnI and cTnI assays. Concordance for paired cTnI and hs-cTnI measurements (n=1096) was verified using 99th percentiles for both genders (cTnI: 30 ng/L, hs-cTnI: 25 ng/L) and for men and women separately (hs-cTnI: M: 34;F: 16 ng/L). Discordant data was correlated with clinical and laboratory information. Diagnosis of Acute Coronary Syndrome (ACS) or Non-ACS was adjudicated by two cardiologists independently. Results The hs-cTnI assay showed a lower (10-fold) critical outlier rate (0.091%) and more detectable results above the limit of detection (LOD) (23.4%) and 99th percentile (2.4%), compared to cTnI. Analytical concordance between the two assays was high (94.5%) but decreased (91.7%) when gender-specific hs-cTnI cut-offs were used. The hs-cTnI assay gave fewer false negatives (up to 1.0%) but disproportionately more false positives (up to 6.7%) overall, which improved (3.9%) for serial measurements. Conclusions Laboratories should analytically and clinically verify hs-cTn assays before use, with attention to performance and the clinical and diagnostic algorithms that support appropriate testing and result interpretation. Work in the pre- and post-analytical phases is necessary to augment the analytical improvement in the new era of troponin testing.