Neutrophil gelatinase associated lipocalin (NGAL): Analytical issues (Review)

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1 Neutrophil gelatinase associated lipocalin (NGAL): Analytical issues (Review) Giuseppe Lippi, Rosalia Aloe Unità Operativa di Diagnostica Ematochimica, Azienda Ospedaliero-Universitaria di Parma, Parma, Italy ABSTRACT Neutrophil gelatinase-associated lipocalin (NGAL) is a component of the lipocalin family of proteins, which can be found in blood and urine in form of monomer, homodimer and heterodimer. Originally isolated from activated neutrophils, the protein is also found in tubular cells of kidney, cardiomyocytes, epithelia of prostate, uterus, salivary glands, lung, liver, trachea, stomach, bowel and colon, as well as some types of cancers including those of breast and urogenital tract. The monomeric form is principally produced by tubular cells, whereas the homodimer is prevalently synthesized by neutrophils. Although consolidated evidence has now been gathered in support of a diagnostic and prognostic role of NGAL in acute kidney injury and other renal disorders, there are some analytical issues that still impair its clinical usefulness, and that will be discussed in this article. These basically entail the lack of specificity of current immunoassays for detecting the monomer principally released by tubular cells, the poor standardization among different commercial methods, the identification of optimal sample matrix, the different approaches for reporting results, the role of additional demographical or clinical variables, as well as the calculation of appropriate reference ranges. Key Words: Neutrophil gelatinase-associated lipocalin; NGAL; Acute Kidney Injury; Renal Disease. INTRODUCTION Neutrophil gelatinase-associated lipocalin (NGAL) is a key component of the lipocalin family, which includes a number of small proteins capable to bind hydrophobic molecules and generate macromolecular complexes. The biological role of NGAL seems most likely attributable to this property, and entails effective binding to enterobactin and other siderophores, so reducing the concentration of Fe 3+ that is an important nutritional requirement for several microorganisms. An additional favorable activity of NGAL is attributable to its ability to bind to matrix metalloproteinase- 9 (MMP-9), thus preventing its endogenous degradation. 1,2 In humans, NGAL is constituted by a single 178 long, disulphide-bridged aminoacid chain, with an expected molecular weight of 22 kda, increasing to 25 kda after glycosylation. The protein can be found in three distinct molecular form, i.e., the 22 kda monomer, a 45 kda homodimer generated by dimerization of two identical NGAL monomers, and a larger 135 kda heterodimer, generated by association of the monomer with the 92 kda MMP-9, also known as gelatinase B. 1,2 The name of the protein is derived from the fact that NGAL was originally isolated from activated neutrophils, although other cellular sources could be later identified, including the tubular cell of the kidneys, cardiomyocytes, epithelia of prostate, uterus, salivary glands, lung, liver, trachea, stomach, bowel and colon, as well as some types of cancer cells including those of breast and urogenital tract. 3 Several lines of evidence now attest that the monomeric form is principally produced by tubular cells (even if little amounts can also be found in neutrophils), whereas the homodimer is prevalently synthesized by neutrophils (Fig. 1). A minor amount of heterodimer is also supposed to be generated in the kidney. 1,2 This complex pattern explains why NGAL can not be considered the troponin of the kidney, neither in its monomeric form. 4 The evidence that NGAL is physiologically produced by tubular cells, and that its generation is largely enhanced after renal injury, has paved the way to a large series of clinical studies that have assessed the diagnostic performance of this promising biomarker in the setting of acute kidney injury (AKI). The results of most of these studies, critically reviewed and meta-analyzed by Haase et al. in 2009, 5 showed that NGAL retains both diagnostic and prognostic value for AKI. In particular, data from 19 studies involving more than 2,500 patients, 487 (19%) of whom developed AKI, yielded to a cumulative area under the curve (AUC) for predicting AKI of (95% CI, ). The diagnostic accuracy of plasma or serum NGAL was similar but still lower than that of urine NGAL (i.e., AUC vs 0.837). Significant AUCs were also found for prediction of renal replacement therapy and in-hospital mortality (i.e., and 0.706, respectively). 5 It is noteworthy that although the monomer is prevailing, the enhanced values of homodimer can also be found in patients with AKI, since the renal injury triggers migration and activation of leukocytes at the site of injury. The increased concentration of protein in blood in this setting is attributable to a mechanism of cellular release and following tubular reabsorption (Fig. 1). Although consolidated evidence has now been gathered in support of a diagnostic and prognostic role of NGAL in AKI and other renal disorders, there are however some analytical issues that are still impairing its clinical usefulness, and that will be discussed in the rest of this article. ANALYTICAL TECHNIQUES Before specifically addressing the leading analytical issues in the clinical assessment of NGAL, a brief overview about the current testing opportunities seems advisable. There are now several alternatives for routine measurement of NGAL in a variety of biological sample matrices, thus including whole blood, urine, serum, heparin or EDTA plasma. Regardless of general considerations about clinical advantages and limitations of assessing this challen- 332 LigandAssay 18 (4) 2013

2 Figure 1 Biology of neutrophil gelatinase-associated lipocalin (NGAL) and its relationship with tubular injury. ging molecule in different biological fluids (these aspects will be thoughtfully elucidated in another article of this journal), there are also several methods that are currently marketed. These basically entail point of care (POC) tests on whole blood (i.e. Triage NGAL, Biosite-Inverness Medical, Waltham, MA, USA, which is a rapid fluorescence immunoassay to be used with Triage Meters), manual enzyme linked immunosorbent assays (ELISAs) for urine, plasma and serum, and also some innovative fully-automated techniques for urine, plasma and serum, that would allow much wider implementation and routine use of this test, thus virtually replacing POC tests and manual ELISAs in most healthcare settings, including clinical laboratories. As such, the description of technology and analytical performance of POC and manual ELISAs is outside the scope of this article, and can be however found elsewhere. 6-9 It is also noteworthy that although promising techniques for NGAL quantification such as electrochemical determination are under development, their diffusion in clinical laboratories is still far from being realized. 10 ARCHITECT Urine NGAL The ARCHITECT Urine NGAL (Abbott Laboratories, Lake Forest, IL, USA) is a non-competitive, sandwich chemiluminescent immunoassay, employing a microparticle reagent where anti-ngal antibodyies are coated to paramagnetic particles and a conjugate reagent obtained by coupling acridinium to a second anti-ngal antibody. The test uses of sample, 50 μl of microparticle reagent and 50 μl of conjugate reagent. 11 A first 18 min incubation step of the sample (2.5 μl) with the microparticle reagent (50 μl) is then followed by a 4 min incubation step with the conjugate reagent (50 μl). The reagent mixture is then washed and the acridinium label is triggered with peroxide. The final concentration of NGAL in the sample is proportional to the optical signal that is produced. In a preliminary investigation, Grenier et al. assessed the analytical performance of ARCHITECT Urine NGAL and reported a total imprecision comprised between 2.1 and 5.3%, as well as optimal comparison with a manual ELISA technique (r=0.99). 11 The NGAL Test The NGAL Test (BioPorto Diagnostics A/S, Gentofte, Denmark, currently marketed in Italy by Sentinel Diagnostics, Milan, Italy) is a particle-enhanced immunoturbidimetric assay for quantitative assessment of the protein in human urine, as well as in heparin and EDTA plasma. The test can be implemented in a large number of clinical chemistry platforms, including Beckman Coulter AU series (640 and 5800), Roche Diagnostics (Modular and Cobas series), Siemens Dimension and Advia (1800 and 2400), Abbott Architect and Aeroset, Ortho Clinical Vitros. Basically, the sample is mixed with reaction buffer and the reaction is started after a short incubation by the addition of anti-ngal-coated immunoparticles (the assay typically requires 3 μl of sample, 50 μl antibody reagent and 150 μl of reaction buffer). The presence of NGAL in the sample causes aggregation of immunoparticles, which is quantified by amount of light scattering measured as absorption of light at 570 nm for 5 min. 12 The test is completed in 10 min and the final concentration is determined by interpolation on an established calibration curve. A recent evaluation of this test on the Beckman Coulter AU5800 (Beckman Coulter Inc., Brea, CA, USA) reported a intra- and interassay imprecision comprised between % and %, respectively. The linearity was excellent between 18 and 790 ng/ml (r = 1.000; p<0.001) and a highly significant correlation was found between this test and Abbott ARCHI- TECT NGAL (r=0.925; p<0.001). 12 LigandAssay 18 (4)

3 Antibodies specificity As previously described, NGAL can be found in blood and urine in three molecular forms, i.e., the monomer, the homodimer and the heterodimer (Fig. 1). The two principal forms, the monomer and the homodimer, are virtually indistinguishable one from the other except from the protein domain where the two monomers are linked. According to this premise, it is hence predictable that the specific configuration of antibodies in the diagnostic kits strongly influences the analytical and clinical performance of the assay. In particular, all the methods currently available in the market are based on cocktails of antibodies that do not distinguish the monomer from the homodimer and thereby fail to discriminate the fraction of NGAL released from the kidney (i.e., principally the monomer) from that released from neuthrophils (i.e., the homodimer). 13 As such, any kind of pathological or even physiological increase of the protein that is sustained by release of extra-renal sources inevitably decreases the diagnostic specificity of the test for detecting renal injury. 14,15 Rather understandably, this drawback is only evident in patients with important causes of enhanced release of NGAL outside the kidney, thus being mainly limited to subjects with important leukocytosis and/or cancer. It is hence advisable that future research may be strongly focused to develop commercial methods based on cocktail of antibodies capable to specifically target the monomer and more clinically specific for diagnosing renal injury. Some promising studies are underway, and preliminary results have been produced by Mårtensson et al., who showed that the combination of two ELISAs is effective to distinguish monomeric /NGAL produced by the kidney epithelium, from the dimeric isoform released by neuthrophils. 16 The potential commercialization of this test would represent indeed a breakthrough in the effective diagnosis of AKI. Standardization issues In a recent evaluation, Kift et al. compared the analytical performance of two clinical methods (i.e., NGAL Test, BioPorto on Siemens ADVIA 1800 and ARCHITECT Urine NGAL on ARCHITECT i2000sr), with that of three research-use-only ELISAs from R&D Systems, Hycult and BioPorto, and reported that a broad variability exists between these urinary NGAL methods in terms of performance, and also emphasized that this aspect should be carefully considered when interpreting results from different studies. 8 In particular, some drawbacks were found for the limit of quantification of BioPorto NGAL Test on ADVIA, for parallelism for BioPorto NGAL ELISA, and for several analytical aspects for the Hycult test. The inter-method disagreement was wide, especially for values obtained with the Hycult assay, thus globally highlighting a poor standardization among the methods tested. Influence of the sample matrix In an earlier study, Cavalier et al. compared the analytical performance of whole blood NGAL measured using Biosite Triage with that of ARCHITECT Urine NGAL and concluded that the former approach did not meet their expectations, showing important analytical limitations and - namely - unsuitable imprecision around the suggested diagnostic threshold (i.e., 130 ng/ml). 17 These important findings are in support of the evidence that the urinary assessment may provide better results than whole blood. As regards plasma, Bakker and Syperda recently investigated the correlation between paired specimens collected in heparin and EDTA blood tubes and found an excellent correlation and a very limited bias of approx 10 ng/ml, which would only require a modest adjustment when shifting from one sample matrix to another. 18 In general, plasma should be preferred over serum, since NGAL may be released from neutrophils during coagulation of the specimen. Adjustment of urine NGAL for urinary creatinine Helmersson et al. evaluated the biological variability of urine NGAL and its ratio with urine creatine in healthy subjects. Interestingly, the absolute value of urine NGAL was positively associated with urine creatinine concentration, thus providing a reasonable support to the recommendation of adjusting urinary values for creatinine and thereby reduce the bias due to variations in urine output. The biological variability of urine NGAL was however nearly fourtime higher that of its ratio with urine creatinine (i.e., 191 vs 27%). 19 An additional and reasonable approach seems that suggested by Chen et al., who recently proposed to use the NGAL changing ratio, calculated by dividing the baseline value with that of a further measurement during serial sampling. 20 Influence of biological variables and extra-renal disorders In a recent article we have measured serum and urine NGAL in trained male athletes who completed a 60-km ultramarathon. 21 Interestingly, serum NGAL, urine NGAL and its ratio with urine creatinine increased by 1.6, 7.7 and 2.9-fold after the run, respectively. Diagnostic values of the biomarker (i.e., >200 ng/ml) were found in one athlete for serum NGAL and in none for urine NGAL before the run, whereas they were observed in one for serum NGAL and two for urine NGAL after the run. A significant association was observed between pre- and post-exercise variation of serum creatinine and serum NGAL (r=0.81; p<0.001), but not with variation of either urine NGAL (r=0.25; p=0.34) or its ratio with urine creatinine ratio (r=0.25; p=0.33). Even more interestingly, a significant correlation was found between post-exercise increase of serum NGAL and variation of both leukocytes (r=0.77; p<0.001) and polymorphonuclear neutrophils (r=0.69; p=0.002), whereas no significant correlation was found between variations of urine NGAL and leukocytes (r=0.26; p=0.30) or polymorphonuclear neutrophils (r=0.19; p=0.46), as well as between urine NGAL/creatinine ratio and leukocytes (r=0.26; p=0.31) or polymorphonuclear neutrophils (r=0.15; p=0.56). 22 These findings clearly attest that the concentration of NGAL, especially when measured in serum, may be strongly influenced by strenuous physical exercise, which is likely attributed to 334 LigandAssay 18 (4) 2013

4 the increased of leukocytes number that typically characterizes this condition. On the other hand, leukocytes involvement when bacteriuria is present is another potential cause of decreased specificity of urine testing. 23 Although urine centrifugation is always advisable, serum or plasma may be preferable in this setting. The potential confounding role of additional conditions that may lead to hyper-expression of ectopic or leukocyte NGAL have been poorly investigated so far, but they should be carefully considered when interpreting test results in patients with suspected renal injury. The reference range The establishment of a reliable reference range is another important issue, wherein recent evidence suggests that the use of a single cut-of may be inappropriate under some circumstances. Stejskal et al. originally showed that serum NGAL is significantly associated with alanine aminotransferase (ALT) (r=-0.3, p<0.01) aspartate aminotransferase (AST) (r=-0.3, p<0.01), cholesterol (r=-0.21, p=0.047), creatinine (r=0.19, p=0.05) and high-sensitivity C-reactive protein (hs-crp) (r=0.22, p=0.036), whereas no significant correlation was found with body mass index (BMI), waist circumference and arterial blood pressure. 24 Cullen et al. also showed a significant gender-related differences for NGAL (i.e., values are higher in women). A meaningful agerelated difference was also observed, especially among some categories of ages (i.e., vs y for urine NGAL, and between <40 and y for its ratio with urine creatinine). 25 In another study, Cangemi et al. recently found that the urine NGAL values of neonates were significantly higher than those of children (i.e., 44 vs 10 ng/ml, p<0.0001), 26 thus reinforcing the concept that the reference range should be defined according to the different demographical variables, especially age and gender. CONCLUSIONS Routine NGAL assessment is a valuable approach for investigating a variety of acute and chronic renal injuries, and is also emerging as an appealing perspective in the diagnostics of extra-renal disorders, such as bacterial peritonitis, 27 and different forms of malignant cancers Nevertheless, some important analytical issues still impair the otherwise promising diagnostic performance of this novel biomarker. It is hence advisable that further efforts should be placed for refinement of current analytical techniques, mainly aimed to develop more robust and clinically effective assays to support physicians in the challenging diagnosis of AKI. A remarkable perspective is represented by the ongoing development and future commercialization of tests specifically designed for targeting the NGAL monomer, 31 which are indeed expected to increase the refine the current armamentarium for efficient diagnosis of acute and chronic renal disorders. REFERENCES 1. Cai L, Rubin J, Han W, et al. The origin of multiple molecular forms in urine of HNL/NGAL. Clin J Am Soc Nephrol 2010;5: Lippi G, Plebani M. Neutrophil gelatinase-associated lipocalin (NGAL): the laboratory perspective. Clin Chem Lab Med 2012;50: Makris K, Rizos D, Kafkas N, Haliassos A. Neurophil gelatinase-associated lipocalin as a new biomarker in laboratory medicine. Clin Chem Lab Med. 2012;50: Devarajan P. 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5 from kidney epithelial cells and neutrophils. Clin Chim Acta 2012;413: Cavalier E, Bekaert AC, Carlisi A, et al. Neutrophil gelatinase-associated lipocalin (NGAL) determined in urine with the Abbott Architect or in plasma with the Biosite Triage? The laboratory's point of view. Clin Chem Lab Med 2011;49: Bakker AJ, Syperda H. Neutrophil gelatinase-associated lipocalin: comparison of the use of EDTA and heparin plasma. Clin Chem Lab Med 2012;50: Helmersson-Karlqvist J, Arnlöv J, Larsson A. Day-today variation of urinary NGAL and rational for creatinine correction. Clin Biochem 2013;46: Cheng CW, Chen YC, Chang CH, et al. The ratio of plasma neutrophil gelatinase-associated lipocalin predicts acute kidney injury in patients undergoing liver transplantation. Transplant Proc 2012;44: Lippi G, Sanchis-Gomar F, Salvagno GL, et al. Variation of serum and urinary neutrophil gelatinase associated lipocalin (NGAL) after strenuous physical exercise. Clin Chem Lab Med 2012;50: Lippi G, Salvagno GL, Banfi G.. Serum but not urine concentration of neutrophil gelatinase-associated lipocalin is influenced by acute leukocyte variations. Leuk Lymphoma 2012;53: Decavele AS, Dhondt L, De Buyzere ML, Delanghe JR. Increased urinary neutrophil gelatinase associated lipocalin in urinary tract infections and leukocyturia. Clin Chem Lab Med 2011;49: Stejskal D, Karpísek M, Humenanska V, et al. Lipocalin-2: development, analytical characterization, and clinical testing of a new ELISA. Horm Metab Res 2008;40: Cullen MR, Murray PT, Fitzgibbon MC. Establishment of a reference interval for urinary neutrophil gelatinase-associated lipocalin. Ann Clin Biochem 2012;49(Pt 2): Cangemi G, Storti S, Cantinotti M, et al. Reference values for urinary neutrophil gelatinase-associated lipocalin (NGAL) in pediatric age measured with a fully automated chemiluminescent platform. Clin Chem Lab Med 2013;51: Lippi G, Caleffi A, Pipitone S, et al. Assessment of neutrophil gelatinase-associated lipocalin and lactate dehydrogenase in peritoneal fluids for the screening of bacterial peritonitis. Clin Chim Acta 2013;418: Budzynska A, Nowakowska-Dulawa E, Marek T, et al. Differentiation of pancreatobiliary cancer from benign biliary strictures using neutrophil gelatinase-associated lipocalin. J Physiol Pharmacol 2013;64: Martí J, Fuster J, Solà AM, et al. Prognostic value of serum neutrophil gelatinase-associated lipocalin in metastatic and nonmetastatic colorectal cancer. World J Surg 2013;37: Kaur S, Chakraborty S, Baine MJ, et al. Potentials of plasma NGAL and MIC-1 as biomarker(s) in the diagnosis of lethal pancreatic cancer. PLoS One 2013;8:e Bangert K, Rosenkilde N, Ploug Jørgensen J, et al. Immunochemical determination of monomer, homodimer and total Neutrophil Gelatinase-Associated Lipocalin [Abstract]. J Am Soc Nephrol 2012;23:830A. Per corrispondenza: Prof. Giuseppe Lippi U.O. Diagnostica Ematochimica Azienda Ospedaliero-Universitaria di Parma Via Gramsci 14, Parma, Italy Tel.: Fax: glippi@ao.pr.it, ulippi@tin.it 336 LigandAssay 18 (4) 2013