Antonio Fortunato

Neutrophil gelatinase-associated lipocalin (NGAL) as biomarker of acute kidney injury: a review of the laboratory characteristics and clinical evidences

Abstract Acute kidney injury (AKI) is a common and serious condition, currently diagnosed by functional biomarkers, such as serum creatinine measurements. Unfortunately, creatinine increase is a delayed and unreliable indicator of AKI. The lack of early biomarkers of structural kidney injury has hampered our ability to translate promising experimental therapies to human AKI. The recent discovery, translation and validation of neutrophil gelatinase-associated lipocalin (NGAL), possibly the most promising novel AKI biomarker, is reviewed here. NGAL may be measured by several methods both in plasma and urine for the early diagnosis of AKI and for the prediction of clinical outcomes, such as dialysis requirement and mortality, in several common clinical scenarios, including in the intensive care unit, cardiac surgery and renal damage due the exposition to toxic agent and drugs, and renal transplantation. Furthermore, the predictive properties of NGAL, may play a critical role in expediting the drug development process. A systematic review of literature data indicates that further studies are necessary to establish accurate reference population values according to age, gender and ethnicity, as well as reliable and specific decisional values concerning the more common clinical settings related to AKI. Furthermore, proper randomized clinical trials on renal and systemic outcomes comparing the use of NGAL vs. standard clinical practice are still lacking and accurate cost-benefit and/or cost-utility analyses for NGAL as biomarker of AKI are also needed. However, it is important to note that NGAL, in the absence of diagnostic increases in serum creatinine, is able to detect some patients affected by subclinical AKI who have an increased risk of adverse outcomes. These results also suggest that the concept and definition of AKI might need to be reassessed.

Distribution of plasma cardiac troponin I values in healthy subjects: pathophysiological considerations.

Abstract Background: The aim of this study was to evaluate the distribution of cardiac troponin I (cTnI) values, measured by the ADVIA TnI-Ultra method (Siemens Medical Solutions Diagnostics SrL) in healthy subjects and to characterize its relation to gender, age, as well as to N-terminal fragment of pro-brain natriuretic peptide (NT-proBNP). Methods: A Caucasian population of 692 healthy subjects (311 males and 381 females) with a mean (SD) age of 45.3 (17.3) years [range 11–89 years; females 46.5 (17.3) years, males 43.8 (17.1) years] was enrolled. The presence of cardiac or systemic acute or chronic diseases was excluded by history and accurate clinical evaluation. Results: A significant difference was found between the cTnI values in men and women (men: median 0.012 μg/L, range from undetectable values to 0.196 μg/L; women: median 0.008 μg/L, range from undetectable values to 0.130 μg/L; p<0.0001 by Mann-Whitney U-test). When a multiple regression analysis was performed, NT-proBNP, gender and age significantly contributed to the regression with cTnI (R=0.444, p<0.0001). Conclusions: Our data indicate that cut-off values, based on the 99th percentile of cTnI distribution in apparently healthy subjects, can significantly vary according to age and gender of the reference population. Clin Chem Lab Med 2008;46:804–8.

Efficacy of 7 day lansoprazole-based triple therapy for Helicobacter pylori infection in elderly patients.

Background: The prevalence of Helicobacter pylori increases with age. However, data regarding the effects of anti‐H. pylori treatments in the elderly are very scarce.

BETA 2-MICROGLOBULIN REMOVAL BY SYNTHETIC DIALYSIS MEMBRANES. MECHANISMS AND KINETICS OF THE MOLECULE

Beta 2-microglobulin (ß2-m) accumulation represents a possible complication of long term dialysis. It is therefore important to evaluate the capacity of removal of this molecule from the patient by different dialysis membranes. The present study is aimed at evaluating the mechanisms involved in ß2-m removal by three different synthetic membranes: a) highly asymmetric hydrophobic polysulfone (Biosulfane, NMC), b) moderately asymmetric and hydrophobic polysulfone (PS600, Fresenius), c) Polyacylonitrile (AN69HF, Hospal). The adsorption capacity and sieving coefficients of the three membranes for native and labeled ß2-m were studied in vitro utilizing human blood. The amount adsorbed by the membrane was measured by the elution of the molecule obtained with a detergent solution. Clearances, total removal and membrane adsorption were studied in six patients treated in a randomized sequence with the three membranes. For this purpose, plasma and dialysate measurements as well as total collection of spent dialysate and ß2-m elution from the used dialyzers were carried out. Ex novo generation of ß2-m did not take place during in vitro circulation. The molecule was removed by the studied membranes both by filtration and adsorption. The Biosulfane membrane removed ß2-m mostly by adsorption while the PS600 membrane removed ß2-m almost entirely by filtration. Intermediate behaviour was shown by AN69 membrane. Similar quantities of ß2-m were removed from the patients with the three membranes. Total removal could only be precisely measured by adding the quantity of ß2-m eluted from the membrane to the amount recovered in the spent dialysate. Out of total removal, adsorption was more than 90% with Biosulfane, while only 5% with the PS600. These findings contribute to the understanding of the discrepancy found between the clearance measured from the plasma side and that measured from the dialysate side. In conclusion, clearance and sieving measurements for ß2-m cannot be correctly performed unless the capacity of adsorption of the membrane is taken into account.

Vasopressin, atrial natriuretic factor and renal water homeostasis in premature newborn infants with respiratory distress syndrome

Arginine vasopressin (AVP), human atrial natriuretic peptide (hANP), and body fluid and electrolyte balance were examined during the first five days of life in eleven premature infants (birthweight 1610 +/- 240 g, gestation 30 +/- 1 weeks) receiving mechanical ventilation for respiratory distress syndrome (RDS). Plasma hANP and urine AVP concentrations were determined by radioimmunoassay on the first, third and fifth days. Arginine vasopressin urine levels remained constantly elevated during the study period (mean +/- SD 13.5 +/- 7.8 day 1, 12.0 +/- 9.9 day 3, 13.2 +/- 5.1 ng/l day 5, p = n.s.), while plasma hANP was significantly increased on the third day (626 +/- 495 vs. 298 +/- 240 pg/ml on day 1, p < .05). Urine sodium concentration, urine osmolality and osmolality and osmolar clearance were elevated significantly as well on day 3, p < .05, and correlated to hANP levels. Body weight decreased during the study by 8.2% on the third day and by 11.3% of birthweight on the fifth day. A significant increase in creatinine clearance occurred after the third day (p < .01), while free water clearance remained essentially the same during the first five days of life. We speculate that an increase in plasma hANP concentration on day 3 of life results in a natriuresis and osmolar diuresis without correlations or temporal relationships to hypervasopressinemia of the premature neonate with RDS.

Reference values for urinary neutrophil gelatinase-associated lipocalin (NGAL) in pediatric age measured with a fully automated chemiluminescent platform

Abstract Background: Neutrophil gelatinase-associated lipocalin (NGAL) has been suggested as the most promising biomarker of acute kidney injury. However, there are no reliable data on analytical sensitivity and reference limits of urinary NGAL (uNGAL) assay in pediatric age. The aim of the present study is to evaluate the analytical sensitivity and the reference range of uNGAL measured in urine specimens of pediatric age with the fully automated platform ARCHITECT® i1000. Methods: A total of 333 urine samples were collected from 25 healthy newborns (16 males and 9 females; age 1–4 days) and 308 children (150 males and 147 females; mean age 80.7 months, range 0.63–248 months) and assayed for uNGAL by two different Italian centers (Department of Laboratory Medicine of the Fondazione Toscana G. Monasterio of Pisa and Massa and the Clinical Pathology Laboratory Unit of Istituto Giannina Gaslini of Genova). Results: The calculated limits of blank (LOB) and detection (LOD) values were 0.5 ng/mL and 0.95 ng/mL, respectively. The distribution of uNGAL values approximated a log-normal distribution (median 5.2 ng/mL, interquartile range 2.5–12.8 ng/mL, 99th percentile 117.6 ng/mL). uNGAL values of the 25 neonates were significantly higher than those of 308 children (neonates: mean 44.2 ng/mL, median 30.3 ng/mL, range 5.2–137.4 ng/mL; children: mean 10.2 ng/mL, median 4.6 ng/mL, range 0.2–146.7 ng/mL; p<0.0001). Conclusions: uNGAL assay shows a good analytical sensitivity and imprecision, which allows the measurement of uNGAL values around the cut-off value (i.e., 117.6 ng/mL) with an imprecision <5 CV%. The distribution of uNGAL values in pediatric age approximates a log-normal distribution, with values which are higher in neonates compared to children.

Heart-Kidney Biomarkers in Patients Undergoing Cardiac Stress Testing

We examined association of inducible myocardial perfusion defects with cardiorenal biomarkers, and of diminished left ventricular ejection fraction (LVEF) with kidney injury marker plasma neutrophil gelatinase-associated lipocalin (NGAL). Patients undergoing nuclear myocardial perfusion stress imaging were divided into 2 groups. Biomarkers were analyzed pre- and poststress testing. Compared to the patients in the low ischemia group (n = 16), the patients in the high ischemia group (n = 18) demonstrated a significantly greater rise in cardiac biomarkers plasma BNP, NT-proBNP and cTnI. Subjects were also categorized based on pre- or poststress test detectable plasma NGAL. With stress, the group with no detectable NGAL had a segmental defect score 4.2 compared to 8.2 (P = .06) in the detectable NGAL group, and 0.9 vs. 3.8 (P = .03) at rest. BNP rose with stress to a greater degree in patients with detectable NGAL (10.2 vs. 3.5 pg/mL, P = .03). LVEF at rest and with stress was significantly lower in the detectable NGAL group; 55.8 versus 65.0 (P = .03) and 55.1 vs. 63.8 (P = .04), respectively. Myocardial perfusion defects associate with biomarkers of cardiac stress, and detectable plasma NGAL with significantly lower LVEF, suggesting a specific heart-kidney link.

Harmonization protocols for TSH immunoassays: a multicenter study in Italy.

Abstract Background: Systematic difference between thyroid-stimulating hormone (TSH) immunoassays may produce misleading interpretation when samples of the same patients are measured with different methods. The study aims were to evaluate whether systematic differences are present among TSH immunoassays, and whether it is possible to obtain a better harmonization among TSH methods using results obtained in external quality assessment (EQA) schemes. Methods: Seven Italian clinical laboratories measured TSH in 745 serum samples of healthy subjects and patients with thyroid disorders. These samples were also re-measured by two reference laboratories of the study with the six TSH immunoassays most popular in Italy after 2 months of storage at −80 °C. Moreover, these data were compared to 53,823 TSH measurements, obtained by laboratories participant to 2012–2015 EQA annual cycles in 72 quality control samples (TSH concentrations from about 0.1 mIU/L to 18.0 mIU/L). TSH concentrations were recalibrated using a mathematical approach based on the principal component analysis (PCA). Results: Systematic differences were found between the most popular commercially available TSH immunoassays. TSH concentrations measured by the clinical laboratories were very closely correlated to those measured with the same method by reference laboratories after 2 months of storage at −80 °C. After recalibration using the PCA approach the variation of TSH values significantly decreased from a median pre-calibration value of 13.53% (10.79%–16.53%) to 9.63% (6.90%–13.21%) after recalibration. Conclusions: Our data suggest that EQA schemes are useful to improve harmonization among TSH immunoassays and also to produce some mathematical formulas, which can be used by clinicians to better compare TSH values measured with different methods.

Multicenter evaluation of the new immunoassay method for TSH measurement using the automated DxI platform

AIM OF THE STUDY Recently, Beckman Coulter Diagnostics set up a new TSH immunoassay for the automated DxI platform. The aim of this study was to evaluate and compare the analytical performance and clinical results of this method with those of previous method. MATERIAL AND METHODS A multicenter study (named TSH ELAS Study) was organized using 593 serum samples, collected from healthy subjects and patients with thyroid disorders, and 13 control samples, circulated in an External Quality Assessment (EQA) scheme. RESULTS The values of LoB and LoD, and LoQ at 20% CV were 0.0004mIU/L, 0.001mIU/L and 0.0023mIU/L, respectively. Moreover, TSH concentrations >0.01mIU/L actually show imprecision values lower than 5% CV. This new TSH assay showed a systematic underestimation (on average of 6.25%) compared to old method, which is mainly due to larger differences between methods for samples with low TSH concentrations, related to the better analytical sensitivity of new compared to old method. In a reference population, including 279 apparently healthy adult subjects, Caucasian volunteers (mean age 43.6years, age 20-63years, 138 women and 141 males) the distribution of TSH concentrations was: mean (CI 95%) 1.694mIU/L (1.588-1.779), median 1.495mIU/L (1.412-1.588mIU/L), 97.5th percentile 3.707mIU/L. CONCLUSIONS The new TSH immunoassay for DxI platform shows some relevant improvements compared to the previous one: use of the most recent WHO 3rd IRP 81/563 standard and monoclonal antibodies (instead of polyclonal antibodies of the old method), and better analytical sensitivities and reproducibility.

The Effect of Omeprazole on Serum Concentrations of Theophylline, Pepsinogens A and C, and Gastrin in Elderly Duodenal Ulcer Patients.

With the aim of verifying the effect of omeprazole treatment on theophylline serum concentration in elderly peptic ulcer patients, we studied 10 male subjects aged > 65 years (mean age = 75.2, range = 67–86) with chronic obstructive bronchopneumonia and endoscopically diagnosed duodenal ulcer in acute phase. All subjects were treated with a slow-release formulation of theophylline 200 mg b.i.d. plus omeprazole 20 mg daily for 4 weeks. In all subjects serum concentrations of azote, creatinine, theophylline were determined at the beginning and after 1 and 4 weeks; at the beginning and end of the study, pepsinogen group A (PGA), pepsinogen group C(PGC) and gastrin were measured. Statistical analysis was performed with the Students f-test for paired data.The results showed no statistically significant differences after 1 and 4 weeks of omeprazole treatment in serum concentration of theophylline (T0 = 7.4, T1 week = 7.5, T4 weeks = 6.0, p = ns), azote (T0 = 45.2, T1 week = 30.5, T4 weeks = 36.1, p = ns), creatinine (T0 = 1.27, T1, week = 1.02, T4 weeks = 1.16, p = ns), PGA (T0 = 99.5, T4 weeks, = 126.2, p = ns), and PGC (T0 = 10.6, T4 weeks = 12.1, p = ns); however serum gastrin increased from T0 = 70.2 ± 13.2 to T4 weeks = 130.3 ± 18.3 (p < 0.0001). It was concluded that (1) serum concentration of theophylline is not affected by the concomitant omeprazole treatment lasting 1 month in elderly patients suffering from chronic obstructive bronchopneumonia and peptic ulcer, (2) modifications of dosages of theophylline and/or omeprazole are not necessary in the elderly with normal renal function, (3) the increase in fasting serum gastrin after 4 weeks of treatment may indicate that omeprazole 20 mg daily is efficacious in inhibiting gastric acid secretion in the elderly people.