Medical care has been undergoing tremendous advances in the Middle East, not least in diagnostic testing. Roche Diagnostics Middle East (RDME) is a pioneer in leading this development in in vitro diagnostics (IVD), by supporting laboratories to achieve a higher level of performance, efficiency and sustainability. IVD testing directs over 60% of clinical decision-making and accounts for a small fraction of global healthcare spending. Whether it is in oncology, virology, blood screening, or research on infectious diseases, autoimmunity, inflammation, women’s health or metabolism, RDME has supported a large number of leading and prominent healthcare institutions to move from multiple analyzers and workflows to comprehensive, integrated laboratory solutions, meeting international standards and certifications.
Roche, with its unique privilege of having both pharmaceutical and diagnostics research under one roof, aims to improve healthcare and make a difference in patients’ lives. The medical solutions start from the stages of early detection and prevention of disease, to diagnosis, treatment selection and treatment monitoring. Roche Diagnostics is leading the industry by addressing unmet medical needs with new or medically enhanced diagnostic tests, supporting doctors and patients with an improved information basis for better medical decisions and treatment selection. Roche’s IVD test menu is one of the broadest in the industry and is continually being expanded based on the latest scientific advances.
Pioneering in Personalized Healthcare
Personalized Healthcare systematically uses patient characteristics, disease biology and diagnostic tests to tailor medicines to patients and improve disease management. Cooperating in the early development of new drugs is integral for the implementation of Personalized Medicine. Roche Diagnostics supports throughout the patient care chain, from screening, early detection, diagnosis and classification to therapy monitoring. Roche Diagnostic’s breakthrough HPV DNA test truly shows how Personalized Medicine works in practice, as it has offered clinicians the ability to detect the presence of specific HPV genotypes. Notably, having such a targeted test has enabled clinicians to choose the most appropriate treatment for their patients, rather than treating every HPV-infected individual with equal and aggressive therapy. This development has also given more confidence to patients, who are re-assured that the treatment they receive is tailored to their specific needs.
Leading in optimizing performance
Optimizing performance, automation and information technology are simplified with a common architecture that delivers tailor-made solutions for diverse workloads and testing requirements. Roche Diagnostics offers platforms that are designed to reduce the complexity of laboratory operation and provide efficient and compatible solutions for network cooperation. For example, Roche Diagnostics has developed medical diagnostic tests based on the Nobel prize-winning polymerase chain reaction (PCR; which exponentially amplifies small amounts of target DNA), that would otherwise be too time-consuming or impossible to perform.
Providing superior workflow solutions, including blood screening
Superior workflow solutions such as Task Targeted Automation (TTA) and Total Lab Automation (TLA) are designed to meet the needs of today’s fast developing healthcare systems. In RDME, a regional project management team is an added value to customers by providing consultancy and implementation support. TLA is customized to the specific needs of individual customers and, thanks to the modular system landscape, can be configured in 90 layouts, differing in size and shape. Roche Diagnostics is successfully delivering best in class Total Lab Solutions for Pathology and Cytology Laboratories to substantially improve the workflow with a unique and complete solution. Another example of superior workflow solutions and automation is with blood screening. Roche Diagnostics has been the preferred partner in the Blood Bank Industry by safeguarding patients through industry-leading assays and technologies. Besides offering Nucleic Acid Testing (NAT), Roche Diagnostics launched Roche Blood Safety Solutions (RBSS), which introduced serology testing of blood samples in an automated manner. As such, Roche Diagnostics is the only provider of a complete Blood Safety Solution to blood banks of any size. Fully integrated automation is offered; these standardized processes reduce manual steps, which guarantees the safety of the blood supply and offers state-of-the-art assay sensitivity and genotype coverage.
Improving therapeutic monitoring
Roche’s IVD offering can be used for treatment selection, response prediction and therapeutic monitoring once a condition has been identified. One of the best examples of this is in hepatitis, qualitative immunoassays (e.g. surface antigen; HBsAg II assay) screen for the presence of hepatitis B virus (HBV) skin, while other assays verify the existence of viral antigens or antibodies. The viral load, the amount of virus in the body, can be determined by quantitative tests. This test shows if therapy has effectively controlled the virus and whether it is replicating or not allowing doctors to monitor the stage and progression of the disease. The continual innovation in therapeutic monitoring is demonstrated in hepatitis C, where Roche Diagnostics has developed Elecsys anti-HCV II, a new state of the art diagnostic test that has an increased seroconversion sensitivity compared to other assays.
Enhancing centralization of data
Similarly, Roche offers centralization of data which is achieved with rapid and easy-to-operate systems that facilitate immediate healthcare decisions, thus placing an emphasis on patient-oriented diagnosis. One example of such a system is the Cobas IT 1000, a point-of-care IT solution that provides complete remote management of and access to all point-of-care diagnostic systems from just one hospital workstation. This automation and centralization of data management into just one workstation frees staff time and enhances the diagnostic service offered to patients.
Roche Diagnostics’ ongoing commitment to developing new analytical tools greatly benefits patients, and its technological innovations create a big impact on the healthcare development in the Middle East. As well as the analytical and technological advances described above, RDME has worked hard to establish the relevant infrastructure in the Middle East with a logistics hub, continual training for employees and a customer support center. These factors combined make RDME the leader in IVD and allow healthcare professionals to benefit from reliable, accurate and immediate results, which directly impact their diagnoses. RDME provides the deepest industry know-how and aims to become the region’s trusted IVD partner.
Fibroblast growth factor-23, a key regulator of phosphate and 1,25-dihydroxyvitamin D metabolism, appears to be an independent risk factor for mortality among chronic kidney disease patients. However, sample stability and poor analytical agreement between detection methods still need to be addressed for it to become a reliable biomarker.
by Dr A. Kumar, Dr W. Herrington, Dr S. Clark and Dr M. Hill
The function of fibroblast growth factor-23 and its role in chronic kidney disease
Fibroblast growth factor-23 (FGF-23) was identified as the key regulator of phosphate homeostasis from a study of renal phosphate-wasting condition autosomal dominant hypophosphatemic rickets [1]. It is secreted principally by bone-forming osteocytes and osteoblasts in response to increased dietary phosphate intake and abnormally elevated serum phosphate concentration (hyperphosphatemia). FGF-23 acts to correct raised phosphate levels by increasing urinary phosphate excretion, by direct inhibition of renal tubular phosphate reabsorption, and via reducing dietary phosphate absorption, by suppressing 1α-hydroxylation of 25-dihydroxyvitamin D to form active 1,25-dihydroxyvitamin D [1,25(OH)2D]. Low 1,25(OH)2D production also provides a negative feedback signal in phosphate homeostasis by inhibiting further FGF-23 secretion (Fig.1)[2, 3].
Chronic kidney disease (CKD) commonly causes a fall in glomerular filtration rate resulting in a reduced capacity for phosphate excretion [4]. FGF-23 levels increase early in CKD, often before any detectable rise in phosphate concentration [5] and those with the severest form of CKD, end-stage renal disease (ESRD), have FGF-23 levels that are 100 to 10,000-fold higher than healthy controls [4, 6]. Sustained suppression of renal 1,25(OH)2D synthesis by high FGF-23 levels contributes to lower serum calcium concentration, a stimulant of parathyroid hormone (PTH) secretion. PTH maintains normal serum calcium concentration by promoting reabsorption of the calcium from its reservoir in bone. The abnormal elevated levels of FGF-23 seen in CKD thus results in a disruption of the homeostatic balance of calcium and phosphate and this may impact on many normal processes including bone mineral metabolism and cardiovascular function. The consequences of prolonged derangement of bone mineral metabolism (known as CKD-mineral bone disease; CKD-MBD) is bone pain and increased fracture risk. CKD-MBD may also accelerate calcification of the vascular tree, a process that may explain some of the significantly increased cardiovascular risk in those with CKD [7]. Indeed, among CKD patients, several studies have shown FGF-23 to be an independent risk factor for mortality [8] and among those not on dialysis it appears to also predict CKD progression [4, 9]. Treatments that might positively impact on FGF-23 levels, for example, reducing dietary phosphate absorption with phosphate binders may therefore have beneficial effects on bone, renal and cardiovascular outcomes in those with CKD (both in those with hyperphosphatemia and those with high-normal serum phosphate concentration).
Methods for FGF-23 assessment
In vitro studies have shown that some of the FGF-23 synthesized by the osteocytes is cleaved between amino acid 179 and 180 by furin (a type I precursor convertase) releasing a C terminal fragment. Current immunometric methods detect either ‘intact’ FGF-23 (iFGF-23, ~32 KDa) or ‘C-terminal’ fragments (cFGF-23, ~14 KDa) in plasma or serum. The cFGF-23 assays recognize two epitopes in the C-terminus, thereby recognizing both iFGF-23 and cFGF-23 fragments. The intact assays recognize only the iFGF-23 because the epitopes flank the cleavage site [2]. At present, there is no reference method, or consensus to indicate which assay type is the most suitable for measuring circulating FGF-23. If all circulating FGF-23 is intact and biologically stable, concentrations detected by the intact and C-terminal assays should be comparable [10]. However, there is a paucity of data confirming this and so caution should be used when comparing studies using the different methods.
Several studies have assessed the performance of commercially available enzyme-linked immunosorbent assays (ELISAs) for FGF-23. Heijboer and co-workers evaluated the performance of one cFGF-23 assay (Immutopics, USA) and two iFGF-23 assays (Immutopics and Kainos Laboratories Inc., Japan) using samples from healthy volunteers and patients with expected high levels of FGF-23 [11]. Intra- and inter-assay variations were assessed in approximately 100 samples with low, normal and high FGF-23 concentrations providing <20% CV for the cFGF-23 Immutopics and iFGF-23 Kainos assays. A high intra-assay variation (22–61%) was observed for the Immutopic intact assay which may be due to lot-to-lot variation [11]. A potential difficulty observed with the Kainos intact assay is poor assay performance when using an automated plate washer, as directed in their protocol. Heijboer and co-workers found acceptable results were only obtained when the wells were washed manually, which made this method impractical for measurement of large numbers of samples [11]. However, a later version of the Kainos assay protocol (from October 2010 onwards) includes an improved wash instruction. Using plasma samples from patients with renal impairment, Devaraj and co-workers also found good inter- and intra assay precision for cFGF-23 assay (CV between 4–10.5%), however, the CVs for iFGF-23 Immutopic assay were found to be poor (6–37.5%) [12].
Poor analytical agreement exists between the commercially available FGF-23 assays, due principally to the lack of a reference method. The performance of four commercially available methods [iFGF-23 assays from Immutopics, Kainos and Millipore (USA) and a cFGF-23 assay from Immutopics] were recently compared using plasma from 31 healthy adults and 36 patients undergoing hemodialysis [13]. A broad range of FGF-23 values were obtained: whereas the patient ranges fell between 154–2561 pg/mL and 447–2063 pg/mL for iFGF-23 and cFGF-23 assay respectively, the levels for healthy adults ranged from 9.9–62 pg/mL for the two assay types. Poor analytical agreement was observed between the assays particularly in the patient group. No agreement of test results was found between the iFGF-23 and cFGF-23 assays and this was more evident at physiological concentrations than in the haemodialysis group [13]. The lack of analytical agreement between these commercially available FGF-23 methods emphasizes that they cannot be used interchangeably and that a comparison of findings from different assays requires careful interpretation. The above evaluation study was performed with plasma samples [13]. A further consideration is some assays are restricted to the sample type that can be used. The iFGF-23 Kainos assay is suitable for both plasma and serum; however, the cFGF-23 Immutopics assay is established only for plasma [10, 13] providing lower or undetectable results in serum [10, 12]. Further method comparisons, ideally on larger numbers of samples and in different patient groups, would provide a valuable insight in this area and help identify which assay type is the most suitable for measuring circulating FGF-23. Nevertheless, studies measuring either intact or C-terminal FGF-23 have reported associations with mortality risk and decline in renal function [4, 14].
Stability of FGF-23: implication for large scale epidemiological studies
Limited evidence exists for the short-term stability of FGF-23 in collected blood samples (6 hours or less) and no information is available for its long-term stability in stored samples. Smith and co-workers investigated the short-term pre-analytical stability of FGF-23, measured using iFGF-23 and cFGF-23 ELISAs from Immutopics, by performing a number of timed experiments with blood taken from 15 patients with mild CKD [6]. The effect of aprotinin, a serine protease inhibitor, and a commercially available protease inhibitor cocktail to preserve FGF-23 after blood collection was also investigated [6]. In the absence of any preservative or inhibitor, iFGF-23 degraded by approximately 40% within 2 hours of collection even when the blood samples were separated into plasma. Conversely, 2 hours after blood collection the FGF-23 concentrations had increased by approximately 35% using the cFGF-23 assay. However, with the addition of the protease inhibitor cocktail the stability of both iFGF-23 and cFGF-23 in the samples extended up to 4 hours (less than 10% change). Based on this evidence, it appears that FGF-23 cannot be measured reliably in blood samples collected without the use of any preservative or inhibitors. This could be a serious limitation for large-scale epidemiological studies, particularly if samples have already been collected and are in storage and for blood collection methods that need to be simple in order to be cost-effective and feasible.
Preliminary findings from our laboratory using samples from 54 CKD patients suggest that FGF-23 (measured by both the iFGF-23 Kainos ELISA and the cFGF-23 Immutopics ELISA) remains stable in whole blood stored for up to 96 hours without the use of a preservative [15]. The apparent lack of agreement with the results from Smith et al. may be explained by differences in the methods of sample collection. Smith et al. used a single K2-EDTA blood tube for each participant which was re-sampled at each time-point whereas we collected separate blood tubes corresponding to each time-point.
Large-scale epidemiological studies often involve the long-term frozen storage of samples prior to biomarker analyses, particularly nested case-control designed studies where it may take several years for sufficient incident cases to materialize. Limited information is available to help understand the impact of long-term frozen storage on the stability of many biomarkers, including FGF-23. Studies investigating the stability of biomarkers in different sample types stored at various temperatures (for example, −40 °C, −80 °C and in liquid nitrogen vapour) will have immense value, particularly in support of long-term blood based prospective studies and biobanks.
Summary
FGF-23 is a key regulator of phosphate homeostasis and has emerged as an important biomarker in patients with CKD. Despite an increasing amount of literature, there are still unanswered questions related to FGF-23 sample stability and the availability of robust reliable methods for measuring FGF-23. Further studies into these areas will improve the quality of clinical research into the use of FGF-23 as a potential early biomarker in CKD.
References
1. ADHR Consortium. Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23. Nat Genet. 2000; 26: 345–348.
2. Wolf M. Forging forward with 10 burning questions on FGF23 in kidney disease. J Am Soc Nephrol. 2010; 21: 1427–1435.
3. Quarles LD. Role of FGF23 in vitamin D and phosphate metabolism: implications in chronic kidney disease. Exp Cell Res. 2012; 318: 1040–1048.
4. Isakova T, Xie H, Yang W, et al. Fibroblast growth factor 23 and risks of mortality and end-stage renal disease in patients with chronic kidney disease. JAMA 2011; 305: 2432–2439.
5. Russo D, Battaglia Y. Clinical significance of FGF-23 in patients with CKD. Int J Nephrol. 2011; 2011: 364890.
6. Smith ER, Ford ML, Tomlinson LA, Weaving G, Rocks BF, Rajkumar C, Holt SG. Instability of fibroblast growth factor-23 (FGF-23): implications for clinical studies. Clin Chim Acta 2011; 412: 1008–1011.
7. London GM, Guerin AP, Marchais SJ, Metivier F, Pannier B, Adda H. Arterial media calcification in end-stage renal disease: impact on all-cause and cardiovascular mortality. Nephrol Dial Transplant. 2003; 18: 1731–1740.
8. Jean G, Terrat JC, Vanel T, Hurot JM, Lorriaux C, Mayor B, Chazot C. High levels of serum fibroblast growth factor (FGF)-23 are associated with increased mortality in long haemodialysis patients. Nephrol Dial Transplant. 2009; 24: 2792–2796.
9. Titan SM, Zatz R, Graciolli FG, dos Reis LM, Barros RT, Jorgetti V, Moyses RM. FGF-23 as a predictor of renal outcome in diabetic nephropathy. Clin J Am Soc Nephrol. 2011; 6: 241–247.
10. Shimada T, Urakawa I, Isakova T, Yamazaki Y, Epstein M, Wesseling-Perry K, Wolf M, Salusky IB, Jüppner H. Circulating fibroblast growth factor 23 in patients with end-stage renal disease treated by peritoneal dialysis is intact and biologically active. J Clin Endocrinol Metab. 2010; 95: 578–585.
11. Heijboer AC, Levitus M, Vervloet MG, Lips P, ter Wee, PM, Dijstelbloem HM, Blankenstein MA. Determination of fibroblast growth factor 23. Ann Clin Biochem. 2009; 46: 338–340.
12. Devaraj S, Duncan-Staley C, Jialal I. Evaluation of a method for fibroblast growth factor-23: a novel biomarker of adverse outcomes in patients with renal disease. Met Syndr Relat Disord. 2010; 8: 477–482.
13. Smith ER, McMahon LP, Holt SG. Method-specific differences in plasma fibroblast growth factor 23 measurement using four commercial ELISAs. Clin Chem Lab Med. 2013; 51: 1971–1981.
14. Fliser D, Kollerits B, Neyer U, Ankerst DP, Lhotta K, Lingenhel A, Ritz E, Kronenberg F, Kuen E, Konig P, Kraatz G, Mann JF, Muller GA, Kohler H, Riegler P. Fibroblast growth factor 23 (FGF23) predicts progression of chronic kidney disease: the Mild to Moderate Kidney Disease (MMKD) Study. J Am Soc Nephrol. 2007; 18: 2600–2608.
15. Illingworth N, Edmans M, Clark S, Kumar A, Sutherland S, Herrington W, Hill M. Investigation of FGF-23 sample & assay suitability for large scale epidemiological studies. Ann Clin Biochem. 2013; 50: 73–74
The authors
Aishwarya Kumar1* PhD; Will Herrington2 MBBS, MRCP; Sarah Clark1 PhD; and Michael Hill1 PhD
1Clinical Trial Service Unit and Epidemiological Studies Unit (CTSU), University of Oxford, Oxford, UK
2Oxford Kidney Unit, Oxford University Hospitals, Oxford, UK
*Corresponding author
E-mail: Aishwarya.Kumar@ctsu.ox.ac.uk
Alzheimer’s disease (AD), a progressive and eventually fatal neurodegenerative condition, was first described over a century ago. The prevalence of the disease has greatly increased since then: indeed the World Health Organization estimates that around 36 million people are living with dementia, the majority of whom are suffering from AD. This number is expected to double by 2030 and triple by 2050, mostly due to increased human longevity: the incidence of AD increases exponentially after the age of 65, with nearly 50% of people over 85 affected. Very early diagnosis and timely and effective therapy are urgently needed if health and social services are not to be totally overwhelmed catering for the needs of both patients and their frequently elderly carers.
Changes in the brains of AD patients may commence up to two decades before clinical symptoms become apparent. The two major abnormalities, beta-amyloid plaques (Aβ) and neurofibrillary tangles (NFT), are very visible at autopsy and continued improvements in medical imaging technologies may allow eventual visualization in the brains of living patients. A definitive diagnosis of AD, though, is usually still based on neuropsychological testing and MRI and/or CT scans to rule out other causes of cognitive decline at a stage of the disease when the drugs currently available, which regulate neurotransmitters, are no longer very effective.
Ongoing research to allow earlier diagnosis has found that gradually increasing concentrations of both Aβ and NFT can be detected in the cerebrospinal fluid of AD patients. And two very recently published studies give additional cause for optimism. The first, published in Nature Genetics, was a large international study that scanned the DNA from more than 74,000 AD patients and healthy controls from 15 different countries to find novel genetic risk factors. As well as the genes already implicated in the disease, such as APOE4, which is strongly linked to late-onset AD, eleven new genes were discovered that had previously not been linked to the condition. This work could facilitate very early diagnosis in individuals at risk. And a smaller British Medical Research Council study discovered a compound that actually prevents further neurodegeneration in animal models.
It has been recognized, however, that an international approach would be most effective in reducing the impact of AD and other types of dementia. To this end health ministers from the G8 countries will be meeting in London in December to develop a coordinated plan of action. It is to be hoped that the result of their deliberations will be global cooperation between companies, researchers and clinicians, and ultimately timely diagnosis and therapy for this appalling condition.
by James D. Peele, PhD The HEp-2 immunofluorescence assay (IFA) for ANA screening is excellent for ruling out many connective tissue diseases, but a positive result seldom translates into a clinically meaningful diagnosis. A new automated, efficient, enzyme immunoassay for ANA screening provides reliable, objective information that can be applied clinically with confidence.
Acute kidney injury is a common and serious complication of many hospital admissions, yet there are often delays in recognizing its development. The laboratory can play a key role in ensuring large increases in serum creatinine do not go unnoticed so that deteriorating patients receive prompt medical attention.
by Nick Flynn
Introduction
Acute kidney injury (AKI) is a sudden decline in renal function, generally occurring over hours or days. AKI is increasingly recognized as a common healthcare problem associated with poor outcomes such as increased mortality and progression of chronic kidney disease [1], prolonged hospital stay and increased healthcare costs [2]. There is also evidence that management of patients with AKI is sometimes poor: in the UK, a National Confidential Enquiry into Patient Outcomes and Death (NCEPOD) report found severe deficiencies of care in a cohort of patients who died with a primary diagnosis of AKI [3]. For example, there was often a delay in recognizing post-admission AKI. This has prompted some hospitals to implement electronic alerts (e-alerts) to systematically detect and highlight cases of AKI. As current definitions of AKI are based mainly upon changes in serum creatinine, laboratories are well placed to implement these systems (Table 1) [4]. This review will briefly discuss options for e-alerts, some considerations for their implementation, and the evidence base for their use.
AKI e-alerts
The aim of AKI e-alert systems is to improve the outcomes of patients by facilitating earlier recognition and treatment of AKI. E-alerts may be triggered by a variety of different criteria, ranging from a single threshold creatinine value to full application of AKI diagnostic criteria. This may result in an automated comment being appended to the creatinine result, a phone call, email or text message to the requesting doctor, nephrologist or critical care outreach team, or a combination of the above. The intention is for the alert to prompt medical attention for these high-risk deteriorating patients, with a resulting improvement in patient outcomes (Fig. 1). The most successful e-alert systems are therefore likely to combine the alert with a clinical protocol for AKI management, and should be developed in collaboration with clinical colleagues.
Choosing alert criteria
Although a single threshold creatinine (for example, 300 µmol/L) is the simplest approach, this lacks both sensitivity and specificity for AKI. Creatinine may need to rise significantly before reaching the threshold, so the speed at which AKI is recognized may not be improved. In addition, depending on the population served by the laboratory, a large number of elevated creatinine results are likely to be from patients with stable chronic kidney disease, rather than AKI.
Accuracy can be improved by applying a ‘delta check’ to flag an absolute or percentage increase in creatinine, for example, a 75% increase in creatinine [5]. It is usually within the realms of most modern laboratory information management systems to offer one delta check for creatinine, and it is also sometimes possible to run multiple checks with different criteria. Finally, some systems aim to fully apply current definitions, such as those recommended by KDIGO (Table 1) [4].
Accurately estimating baseline creatinine is difficult
A problem faced both by simple delta checks and e-alerts based on AKI definitions is the difficulty in reliably estimating baseline creatinine. A system employing manual estimation of baseline by clinical biochemists at the Royal Derby Hospital has been shown to have good diagnostic accuracy for detection of AKI with a false negative rate of 0.2% and a false positive rate of 1.7% [6]. However, this approach is limited to normal working hours and many laboratories do not have the resources to replicate this labour intensive system. Instead, automatic surrogate estimation methods are used, such as the lowest, most recent or median creatinine value within a certain timeframe, such as the previous three months. Laboratories should be aware of the limitations of some of these estimation methods; for example, the lowest creatinine result has been shown to be a particularly poor estimate of baseline creatinine that can lead to high rates of potential AKI misclassification [7].
Should every case fulfilling AKI criteria be highlighted?
When choosing criteria for an e-alert system, it may seem sensible to use current definitions for AKI. However, there are arguments against this approach. The KDIGO definition of AKI relies on small changes in serum creatinine based on epidemiological studies which show that even these small increases are associated with an increase in mortality risk in large populations [2]. However, in many cases an increase of 0.3 mg/dl (≥26.5 µmol/L) is within the realms of normal biological variation, particularly amongst patients with chronic kidney disease. As an illustrative example, creatinine increased by between 69% and 129% after the consumption of 300 g of animal protein in healthy volunteers, even with creatinine measurement using a specific enzymatic method [8]. The limitations of the more widely used Jaffe method for serum creatinine are well known amongst laboratory professionals, and any of a wide range of non-creatinine chromogens may cause an increased result in the absence of renal disease. When KDIGO criteria are combined with a poor method of baseline estimation (lowest previous creatinine), the proportion of creatinine results causing an AKI e-alert can approach 10%; this is unlikely to be helpful. Strict application of current AKI definitions could therefore lead to annoyance and unresponsiveness amongst clinicians alerted to minor creatinine elevations, unnecessary interventions, anxiety for patients and families, and diversion of limited healthcare resources to a large and relatively low risk group. It is therefore important for laboratories to consider both local IT and resource capabilities and the relative benefit and harm of different criteria for e-alerts before implementation.
Evidence base
A small number of studies have investigated the effect of AKI e-alerts on clinician behaviour or patient outcomes. For example, a real-time alert of worsening AKI stage through a text message sent to the clinician’s telephone was found to increase the number of early therapeutic interventions in an ICU in Belgium [9]. There was also an increase in the proportion of patients who recovered their renal function within 8 hours after an alert indicating less severe AKI, but not amongst those with more severe AKI. There was no significant effect on renal replacement therapy, ICU length of stay, mortality, maximum creatinine or maximum AKI stage. Importantly, 9 out of 10 AKI alerts were based on urine volume criteria, so the applicability of these findings to creatinine based e-alerts is questionable.
Hospitals that have already implemented AKI e-alerts have noted improved outcomes following their introduction. For example, a hospital-wide e-alert system based on changes in serum creatinine at the Royal Derby Hospital, led to a progressive reduction in 30 day mortality over consecutive 6 month periods (23.7%, 20.8%, 20.8%, 19.5%, chi-square for trend P=0.006) [10]. This improvement in survival was maintained after adjustment for age, co-morbid conditions, severity of AKI, elective/non-elective admission and baseline renal function. However, the e-alert was introduced as part of a range of educational interventions so it is difficult to determine the contribution made by the e-alert component.
The evidence base for AKI e-alerts is therefore not strong, and would benefit from further studies to demonstrate that this approach can lead to measurable improvements in patient outcomes.
Conclusions
E-alerts represent an opportunity for the laboratory to assist in the early detection of acute kidney injury. This could improve the outcomes of patients with this life threatening condition. Aside from AKI, there are undoubtedly many other opportunities for the laboratory to optimize existing resources by helping clinicians to digest the large amount of laboratory data produced on a daily basis, to highlight trends and to ensure that important changes are recognized and acted upon. The laboratory can play a key role to ensure that these systems are implemented, that they are effective in selectively capturing a high risk population, and that evidence is gathered to justify their continued use.
References
1. Coca SG, et al. Long-term risk of mortality and other adverse outcomes after acute kidney injury: a systematic review and meta-analysis. Am J Kidney Dis. 2009; 53(6): 961–973.
2. Chertow GM, et al. Acute kidney injury, mortality, length of stay, and costs in hospitalized patients. J Am Soc Nephrol. 2005; 16: 3365–3370.
3. Stewart J, et al. Adding Insult to Injury: a review of the care of patients who died in hospital with a primary diagnosis of acute kidney injury (acute renal failure). A report by the National Confidential Enquiry into Patient Outcome and Death. London: NCEPOD, 2009. www.ncepod.org.uk/2009report1/Downloads/AKI_report.pdf
4. Kidney Disease: Improving Global Outcomes (KDIGO) Acute Kidney Injury Work Group. KDIGO Clinical Practice Guideline for Acute Kidney Injury. Kidney Int. Suppl. 2012; 2: 1–138.
5. Thomas M, et al. The initial development and assessment of an automatic alert warning of acute kidney injury. Nephrol Dial Transplant 2011; 26: 2161–2168.
6. Selby N, et al. Use of electronic results reporting to diagnose and monitor aki in hospitalized patients. Clin J Am Soc Nephrol. 2012; 7: 533–540.
7. Siew ED, et al. Estimating baseline kidney function in hospitalized patients with impaired kidney function. Clin J Am Soc Nephrol. 2012; 7: 712-719.
8. Butani L, et al. Dietary protein significantly affects the serum creatinine concentration. Kidney Int. 2002; 61: 1907.
9. Colpaert K, et al. Impact of real-time electronic alerting of acute kidney injury on therapeutic intervention and progression of RIFLE class. Crit Care Med. 2012; 40: 1164–1170.
10. Kohle N, et al. Impact of a combined, hospital-wide improvement strategy on the outcomes of patients with acute kidney injury (AKI) [abstract]. Joint Congress of the British Transplantation Society & Renal Association, 2013. Bournemouth. Abstract O30. www.btsra2013.com/
The author
Nick Flynn, Pre-registration clinical scientist
Department of Clinical Biochemistry, University College London Hospitals, London, UK
E-mail: nick.flynn@nhs.net
The diagnosis of acute kidney injury (AKI) is often hindered by the reliance on serum creatinine as a marker of kidney function, which can delay detection. Neutrophil gelatinase-associated lipocalin is a promising biomarker which increases within hours of kidney damage and could therefore improve the early diagnosis of AKI.
by Dr Ashley Garner
Clinical background
Acute kidney injury (AKI) is a common condition associated with significant morbidity and mortality. It is currently diagnosed using serum creatinine and urinary output as markers of kidney function, as defined in the recent KDIGO criteria [Fig. 1][1]. However, these are relatively late markers of AKI since they mainly reflect a decrease in glomerular filtration rate and the time required for serum creatinine to accumulate can delay diagnosis. Biomarkers that can detect structural injury to the kidney rather than a loss of function may allow better and earlier detection of AKI. Earlier diagnosis of AKI could facilitate earlier intervention, potentially reduce the risk of irreversible kidney damage and improve patient outcomes. Much research in recent years has therefore focused on the discovery of improved biomarkers for AKI and neutrophil gelatinase-associated lipocalin (NGAL) is one of the most promising candidates [2].
Pathophysiology of NGAL
NGAL is a small 25kDa protein which belongs to the superfamily of lipocalins. It is expressed in many cells including neutrophils, hepatocytes and renal tubular cells and is induced in response to pathological stimuli including infection, inflammation, ischemia and malignancy. NGAL has a functional role in the innate immune system as a bacteriostatic agent, depleting iron-binding siderophores and thereby preventing bacterial iron acquisition. The iron-binding properties of NGAL are also proposed to provide protection from oxidative stress. There is growing evidence that NGAL also acts as a growth factor in some tissues including renal epithelial cells where it modulates cell proliferation, differentiation and apoptosis and may provide protection against renal tubular damage in AKI [3].
Animal studies of AKI induced by ischemia or nephrotoxicity have shown that NGAL is one of the most upregulated proteins in the kidney and is detectable in the urine within 2–3 h. It has been reported that urine NGAL concentrations increase 25–100 fold and plasma NGAL increases 7–16 fold following AKI. Unlike serum creatinine, NGAL is not increased when there is impaired glomerular filtration without renal tubular damage, often termed ‘pre-renal’ uraemia.
Low plasma concentrations of NGAL are found in health as it is expressed at a low constant rate from various tissues. NGAL is then freely filtered at the kidney and the majority is reabsorbed in the proximal tubule, resulting in low NGAL concentrations in the urine. Following AKI, NGAL is greatly upregulated in the cells lining the ascending loop of Henle and collecting ducts of the kidney and is then excreted in the urine. The origin of the increase in plasma NGAL following AKI is less clear and there is evidence to suggest that NGAL expression is increased in other organs such as the lungs and liver following kidney injury.
Since NGAL can be produced by different tissues in response to various stimuli, it is not specific to AKI. Other common conditions that can cause elevated NGAL, and therefore complicate the interpretation of results, include sepsis, heart failure, chronic kidney disease (CKD), malignancy and urinary tract infections.
NGAL assays
Commercial CE marked assays are available for measuring NGAL in plasma, whole blood and urine. It is not clear from the literature whether any of these sample types are preferred or have better diagnostic performance but there are limiting factors for each that may require consideration. Plasma and whole blood samples are invasive and may be contaminated by haemolysis releasing NGAL from neutrophils. Urine NGAL may theoretically be more sensitive for AKI due to greater induction in renal tubular cells but can be falsely elevated in urinary tract infections due to leukocyturia and it is still unclear whether the NGAL should be corrected for urine concentration effects or whether this is unnecessary or even misleading in AKI. Although non-invasive, urine samples may be more difficult to obtain especially at specific time points or if the patient has reduced urine output.
NGAL exists in monomeric, dimeric and heterodimeric or complexed forms. It has been reported that the monomer is the predominant form produced by renal tubular cells and the homodimer is predominantly released by neutrophils. The relevance of these different forms of NGAL will depend on the extent to which NGAL assays detect them and the sample type used. Even though the monomer form may be most relevant for urine NGAL the origin of plasma NGAL in AKI is less clear and may therefore include the other forms. This variation in NGAL assays and sample types makes it difficult to directly compare study results and derive clinically relevant cut-off values. Standardization of NGAL assays using an internationally approved reference material would greatly improve this variation but this is not currently available and would require agreement on what forms of NGAL should be measured.
Research from large heterogeneous populations suggests that urine NGAL concentrations are dependent on gender, age and ethnicity. Biological variation for urine NGAL has also been reported to be as high as 84%. These factors will need to be taken into account when establishing reference intervals for NGAL although they may not be clinically significant if a cut-off value is used to diagnose AKI, especially if it greatly exceeds the expected reference intervals in health.
Clinical utility of NGAL in AKI
There is evidence that NGAL could be useful as an early diagnostic and prognostic biomarker for AKI. Many studies have demonstrated that NGAL rises 24–72 h before creatinine in patients with AKI and is associated with poorer outcomes. It is difficult to determine the diagnostic performance of NGAL for AKI in terms of clinical sensitivity and specificity however, due to the limitations of using serum creatinine as the gold standard comparator. For example, the rise in creatinine caused by pre-renal uraemia will not be associated with a raised NGAL. In addition, a multicentre pooled analysis of prospective studies has shown that patients who have raised NGAL without increases in serum creatinine are at increased risk of adverse outcomes and suggests these patients have a condition termed ‘subclinical AKI’ where there may be tubular damage without glomerular impairment [Fig. 2][4].
The majority of studies assessing NGAL testing in AKI have focused on specific patient populations at high risk of AKI: namely post-cardiac surgery, post contrast infusion, intensive care and emergency admissions.
The advantage of using NGAL in post-surgery or post-contrast patients is that the time of insult is known and therefore NGAL can be measured at set time points for the early detection of AKI and timely intervention. It is more difficult to determine the best application of NGAL in ICU patients in regard to the timing and frequency of tests and it is less clear whether earlier detection can improve outcomes in these patients frequently complicated by multi-organ failure. In the emergency admissions setting NGAL has fewer advantages over serum creatinine since early detection (within hours) is less likely to be applicable. Also NGAL, like creatinine, can be raised in CKD so may require multiple measurements to detect AKI but more patients are likely to have had a previous creatinine result than an NGAL result.
Although there is an abundance of observational studies showing that AKI can be detected earlier using NGAL compared to serum creatinine there is an absence of randomized clinical trials to demonstrate that using NGAL instead of current practice will improve patient outcomes or provide cost benefits. This is probably the biggest barrier to the adoption of NGAL testing in routine practice and better treatments and interventions may be required to overcome it. This would suggest that one of the most important roles for NGAL and earlier biomarkers of AKI is in the discovery and development of effective interventions and therapeutics.
Another consideration regarding interventions for AKI is that NGAL only detects renal tubular damage, it does not distinguish between different causes. However, effective treatments may require the underlying cause to be determined and therefore further biomarkers may be needed to differentiate between causative factors and indicate the most appropriate intervention.
Conclusion
A large number of clinical studies suggest that NGAL may provide an early diagnostic and prognostic biomarker for AKI. However, further randomized clinical trials comparing the use of NGAL to standard practice are required to show cost benefits or improvements in patient outcomes. It seems that even if biomarkers like NGAL enable us to detect AKI earlier, this alone may not be sufficient to improve patient care but hopefully they will facilitate the development of better interventions that will eventually lead to improved outcomes for patients with AKI.
References
1. Kidney Disease: Improving Global Outcomes (KDIGO) Acute Kidney Injury Work Group. KDIGO Clinical Practice Guideline for Acute Kidney Injury. Kidney Int. Suppl. 2012; 2: 1–138.
2. ADQI Consensus on AKI Biomarkers and Cardiorenal Syndromes. Contrib Nephrol. Basel: Karger, 2013; 182: 13–29.
3. Schmidt-Ott KM, Mori K, Li JY, Kalandadze A, Cohen DJ, Devarajan P, Barasch J. Dual action of neutrophil gelatinase-associated lipocalin. J Am Soc Nephrol. 2007; 18: 407–413.
4. Haase M, Devarajan P, Haase-Fielitz A, Bellomo R, Cruz DN, Wagener G, Krawczeski CD, Koyner JL, Murray P, Zappitelli M, Goldstein SL, Makris K, Ronco C, Martensson J, Martling CR, Venge P, Siew E, Ware LB, Ikizler TA, Mertens PR. The outcome of neutrophil gelatinase-associated lipocalin-positive subclinical acute kidney injury: a multicenter pooled analysis of prospective studies. J Am Coll Cardiol. 2011; 57(17): 1752–1761.
The author
Ashley Garner PhD
Department of Blood Sciences,
Leeds Teaching Hospitals Trust, Leeds, UK
E-mail: Ashley.Garner@leedsth.nhs.uk
November 2024
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