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Current approaches for the detection of acute kidney injury

, 26 August 2020/in Featured Articles /by 3wmedia

Acute kidney injury is a recognized complication in hospitalized patients and is associated with a high morbidity and high mortality. This brief article aims to summarize the need for early detection of acute kidney injury and the current approach within NHS England to identify such patients.

by Charlotte Fairclough

Background
Acute kidney injury (AKI) is a recognized complication in hospitalized patients. A report in 2009 from National Confidential Enquiry into Patient Outcome and Death (NCEPOD) suggested that AKI was frequently undetected in hospital patients thus contributing to patient morbidity and mortality [1]. Clinical guidelines for recognition and treatment for acute kidney injury were published by NICE (the National Institute for Health and Care Excellence) in 2013 and reported an associated mortality with AKI of more than 25–30% [2]. This guideline also recognized the prevalence of AKI in the primary care population in patients with or without acute illness. NICE also recognized the impact of AKI on healthcare resources, with costs (excluding those in the community) of £434–620 million per year, more than that associated with breast, lung and skin cancer combined [2].

AKI is characterized by an acute loss of the kidney’s excretory capacity leading to accumulation of waste products such as urea and creatinine, and decreased urine output. It is associated with rapid decline in glomerular filtration rate and increases in potassium, phosphate and hydrogen ions. It has varied causes and may be secondary to a non-renal event, thus may be common in hospitalized patients and critically ill patients. It may go undetected in primary care as it can occur without any symptoms. There are associations between co-morbidities, current medications, acute illness and AKI resulting in the high morbidity associated with the condition and the impact on healthcare resources [3].

One of the most common causes of AKI is pre-renal injury due to hypovolemia (a decreased volume of circulating blood). This is thought to be the cause of more than 70% of AKI in the community [4]. This may be exacerbated in patients prescribed certain medications and should be considered carefully by primary care clinicians when assessing patients for AKI [5]. Other causes of AKI are highlighted in Table 1.

Risk factors associated with development of AKI include age, ethnicity, co-morbidities and use of certain medications [3]. It is important to detect the injury as early as possible to prevent the long-term changes in renal function that have been noted to be associated with even less-severe AKI [6].

Defining acute kidney injury
Previous definitions of acute kidney injury had been published, such as RIFLE criteria (Risk Injury, Failure, Loss, End stage renal failure) and AKIN (acute kidney injury network) [7]. KDIGO (Kidney Disease Improving Global Outcomes) published clinical practice guidance in 2011 that categorized AKI based on changes in serum creatinine and/or urine output as defined in both of these previous publications [8]. This categorized AKI into stages 1, 2 and 3 dependent on severity. Evidence suggests that even small, reversible changes in creatinine are associated with worse outcomes, and indeed AKI and severity of AKI is associated with development of chronic kidney disease [6].

The KIDIGO criteria for AKI references changes in creatinine or changes in urine output as a marker for acute kidney injury [8]. Urine output may be the functional marker of kidney function, but can be difficult to monitor. Accurate fluid balance recordings are imperative in management and prevention of AKI in a hospitalized setting, but may be difficult to do accurately especially if the patient is mobile and able to use a toilet unaided. This is also difficult to assess in community patients who obviously will not have recorded urine output as specified in the guidelines. Thus serum creatinine measurements can be used as a marker of kidney function.

Detection of acute kidney injury
Creatinine is used a biomarker for renal function because it is easy and inexpensive to measure. It is also part of most common biochemical panels in blood tests ordered in both hospital and community patients. This means it is easy to monitor trends and to compare to historical data for the patient as required for the diagnosis of AKI. But it may be slow to respond to changes in renal function, and this may be important in the early detection of AKI. Creatinine concentration in the blood and urine is also influenced by other factors such as age, muscle mass, diet, tubular secretion, hydration status and is subject to analytical interferences. Two methods for measuring creatinine are in common use in biochemistry laboratories, the traditional Jaffe methodology and enzymatic methods. Enzymatic methodology for measurement of serum creatinine has been recommended by NICE in the AKI guidelines [2]. As noted above, it has been documented that changes in creatinine only occur when 50% of kidney function has been lost. Therefore, other markers of AKI such as neutrophil gelatinase- associated lipocalin (NGAL) and tissue inhibitors of metalloproteinases- 2 (TIMP-2) have been investigated as alternatives to serum creatinine.

NGAL is a 25-kDa protein in the lipocalin family and is associated with ischaemic kidney injury and may be measured in urine. NGAL is thought to increase in the early stages of AKI as it acts to limit and repair damage caused by the insult and is mediated by NF-κB which is rapidly increased after injury and promotes cell survival and proliferation. It has been found to be detectable in urine in the very early stages of AKI [9].

Tissue inhibitor of metallinoproteinases-2 (TIMP-2) and insulin-like growth factor binding protein 7 (IGFBP7) have been explored as biomarkers of AKI in critically ill patients in an intensive care setting in the Sapphire study [10]. Both of these proteins are inducers of the G1 cell cycle arrest thought to be critical in the development of AKI.

The management of AKI, especially in the community is often focused on removal of the risk factors and inducers of AKI. General Practice can play a role in reduction of the risk of developing AKI such as regular review of those patients on medication associated with increased risk of development of AKI and review of patients with chronic kidney disease who are inherently at increased risk of AKI [5].

NHS England AKI detection algorithm
It was recognized that detecting AKI based on identifying changes in serum creatinine as according to KDIGO guidelines was easily automatable using laboratory information management systems (LIMS). In 2014, NHS England published a patient safety alert to all NHS Trusts with pathology services, to standardize the reporting of AKI [11]. This recognized that some Trusts had already implemented an AKI alert system based on changes in creatinine and the KDIGO guidelines, but aimed to standardize the reporting and ensure reporting was done in real-time.

The alert system algorithm is based on comparison of a patient’s creatinine concentration with that of a baseline creatinine – either a result within the last 48 hours, 7 days or 12 months based on the KDIGO criteria [12]. The patient safety alert algorithm is mandatory for all pathology laboratories in the UK and was developed with the major LIMS providers, thus enabling standardization and a model that is compatible with all systems. The mode of alerting users is not described and thus subject to differing practices within the UK NHS Trusts. This allows for laboratory interaction with users to determine the required practice for each individual Trust. For example the alerts will be reported to the electronic patient record, but whether these results are to be telephoned, emailed, etc., to users is to be individually determined. Implementation into primary care is expected to occur by April 2016 [12].

Conclusion
In summary, AKI is an important issue in healthcare due to the high level of morbidity and mortality associated with it. It is also associated with increased demand on healthcare resources throughout the system including primary and secondary care. Early detection is vital in order to reduce the morbidity and mortality associated with the condition. Every part of the healthcare system, therefore, has a part to play, including GP identification of those patients at increased risk of development of AKI and reduction of that risk, laboratory detection of AKI from serum creatinine measurements or potentially other biomarkers, and to the clinician acting on those alerts and initiating treatment early to preserve renal function.

References
1. Stewart J, Findlay G, Smith N, Kelly K, Mason M. 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). National Confidential Enquiry into Patient Outcome and Death 2009. (http://www.ncepod.org.uk/2009report1/Downloads/AKI_summary.pdf)
2. NICE guidelines CG169. Acute kidney injury: prevention, detection and management. NICE 2013. (https://www.nice.org.uk/guidance/cg169)
3. Wang HE, Muntner P, Chertow GM, Warnock GE. Acute kidney injury and mortality in hospitalized patients. Am J Nephrol. 2012; 35: 349–355.
4. Kaufman J, Dhakal M, Patel B, Hamburger R. Community-acquired acute renal failure. Am J Kidney Dis. 1991; 17(2): 191–198.
5. Blakeman T, Harding S, O’Donoghue D. Acute kidney injury in the community: why primary care has an important role. Br J Gen Pract. 2013; 63(609): 173–174.
6. Chawler LS, Andur R L, Amodeo RL, Kimmel PL, Palant C. The severity of acute kidney injury predicts progression to chronic kidney disease. Kidney Int. 2011; 79 (12): 1361–1369.
7. Lopes JA, Jorge S. The RIFLE and AKIN classifications for acute kidney injury: a critical and comprehensive review. Clin Kidney J. 2013; 6: 8–14.
8. Kidney disease: improving global outcomes (KDIGO) Acute Kidney Injury Work Group. KDIGO Clinical practice guideline for acute kidney injury. Kidney Inter. Suppl. 2012; 2: 1–138.
9. Devarajan P. Neutrophil gelatinase associated lipocalin: a promising biomarker for human acute kidney injury. Biomark Med. 2010; 4(2): 265–280.
10. Pilarczyk K, Edayadiyil-Dudasova M, Wendt D, Demircioglu E, Benedik J, Dohle DS, Jakob H, Duss F. Urinary [TIMP-2]*[IGFBP7] for early prediction of acute kidney injury after coronary artery bypass surgery. Ann intensive care 2015; 5: 50
11. Standardising the early identification of acute kidney injury. NHS England 2014. (https://www.renalreg.org/wp-content/uploads/2014/08/Patient-Safety-Alert-AKI-algorithm-2014_06_04.pdf)
12. -Acute kidney injury warning algorithm best practice guidance. NHS England and UK Renal Registry 2014. (https://www.thinkkidneys.nhs.uk/aki/wp-content/uploads/sites/2/2014/12/AKI-Warning-Algorithm-Best-PracticeGuidance-10.03.16.pdf)

The author
Charlotte Fairclough, MSc
Department of Clinical Chemistry and Metabolic Medicine, Liverpool Clinical Laboratories, Royal Liverpool and Broadgreen University Hospitals NHS Trust,
Liverpool, UK

*Corresponding author
E-mail: charlotte.fairclough@nhs.net

Zinc oxide nanorod-based acute kidney injury biomarker detection technology and potential clinical implications

, 26 August 2020/in Featured Articles /by 3wmedia

We developed an ultrasensitive bioassay using micropatterned zinc oxide nanorods (ZnO NRs) for the multiplexed detection and quantification of trace levels of cytokines implicated in acute kidney injury (AKI) directly from patient samples. The remarkable limits of detection of the novel ZnO NR-based assay are compared directly with conventional methods.

by Manpreet Singh, Anginelle Alabanza, Lorelis E. Gonzalez, Weiwei Wang, Prof. W. Brian Reeves, and Prof. Jong-in Hahm

Introduction
Cytokines and chemokines are important immunoregulatory molecules produced by many cells such as neutrophils, monocytes, macrophages and T-cells that can serve as biomarkers of inflammatory diseases to predict and track disease pathogenesis [1–4]. Various cytokines and chemokines like interleukins (ILs) and tumour necrosis factors (TNFs) can serve as valuable clinical biomarkers of acute kidney injury (AKI), a rapidly acquired disorder associated with high morbidity and mortality that is commonly seen in hospitalized patients. As elevated levels of cytokines may reveal the activation of signalling pathways leading to inflammation and disease progression, methods enabling prompt and sensitive detection and quantification of multiple cytokines/chemokines simultaneously in a clinical setting are highly sought. Although conventional techniques such as enzyme-linked immunosorbent assays (ELISAs) are widely available and reliable, their applications may not be suitable for the rapid, multiplexed detection of weakly expressed cytokines due to their detection limits (DLs) of typically greater than tens of pg/mL, long assay times of several hours, and extensive serial workflows for detecting multiple protein analytes.

As the typical levels of important AKI-implicated cytokines and chemokines in healthy populations can often be well below the customary DLs of standard cytokine assays, which are generally around tens of pg/mL, there is great clinical interest in reducing the lower limits of detection down to the fg/mL range. In particular, the increasing need for early diagnosis and treatment in AKI and other cytokine-implicated diseases has driven the development of innovative detection schemes capable of reaching even lower DLs than have conventionally been offered. In this context, we have shown that zinc oxide nanorods (ZnO NRs) permit enhanced detection of fluorescence signals emitted by fluorophore-coupled biomolecules in the forms of custom-prepared oligonucleotide constructs and highly purified single-composition proteins in simple media [5–8]. In our most recent work, which is highlighted here, we have developed and validated an ultrasensitive fluorescence-based bioassay using micropatterned ZnO NRs as a novel optical platform for the multiplexed detection and quantification of two urinary biomarkers of AKI, tumour necrosis factor-α (TNF-α) and interleukin-8 (IL-8), in samples of patients at risk for and diagnosed with AKI [9].

In addition to the biomedical relevance of TNF-α and IL-8 in the pathophysiology of AKI, the biomarkers are ideal model cytokines and chemokines for this study due to the differences in their typical concentration levels found in human urine. The baseline expression of IL-8 in healthy populations generally ranges from tens of pg/mL to ng/mL and can be ascertained using conventional approaches, whereas TNF-α levels are typically below the DLs of traditional cytokine detection platforms. Accordingly, we performed ELISA- and ZnO NR-based assays on the same set of patient samples to first examine whether the highly expressed IL-8 levels agree between both detection methods and further demonstrated the detection capability of the ZnO NRs to reveal the ultralow protein levels of TNF-α that cannot otherwise be ascertained via ELISA.

Results and discussion
Overall approach of ZnO NRs-based fluorescence assay
Using a micropatterned array of densely grown, vertically oriented ZnO NRs synthesized using a facile, low-cost, chemical vapour-phase method, we employed a sandwich assay scheme for the multiplexed detection of both AKI-relevant biomarkers. The sequential assay steps included incubation of the ZnO NR platform with primary TNF-α and IL-8 antibodies, bovine serum albumin for surface blocking, standards for both proteins for generating calibration curves or patient urine samples for determining biomarker levels in subject individuals, and fluorophore-conjugated secondary antibodies. In Figure 1(A), representative emission data from the multiplexed assay are qualitatively presented for Alexa 488-labelled TNF-α (left) and Alexa 546-labelled IL-8 (right). As a direct comparison, the panels in Figure 1(B) display scanning electron microscope (SEM) images of the ZnO NR array platform to show the morphology and dimensions of the individual square patches of ZnO NRs. The highly crystalline ZnO NRs do not exhibit any background fluorescence, as evidenced in the inset (F) of image 1(B), and, hence, all optical signals detected for protein quantification are derived only from the surface-adsorbed fluorophore-tagged biomolecules.

Reproducibility and calibration
In Figure 1(C), exemplar fluorescence intensity plots show the different amounts of TNF-α and IL-8 simultaneously detected from selected patient urine samples as obtained by averaging the optical signal from about 550 NR square patches on different areas of the same ZnO NR detection platform. The reproducibility of the fluorescence signal on the ZnO NRs-based platform is shown in Figure 1(D) in which the same patient sample was assayed five times on the same ZnO NR platform for intra-assay variability (**) as well as on three different ZnO NR plates for inter-assay variations (*) that may arise from assay or array-to-array differences. This scheme was conducted for two patient samples, and the coefficients of variation for the intra-assay (16.5% for TNF-α and 2.5% for IL-8) and inter-assay (12% for TNF-α and 2.8% for IL-8) results were found to be below the generally accepted value of 10–20%.

In order to quantitatively compare the levels of TNF-α and IL-8 obtained via the ELISA- and ZnO NRs-based assays, calibration curves were generated using standard solutions of each cytokine. The DLs of the ELISA-based method, defined as 2 standard deviations above the mean of 20 zero concentration replicates, were determined as 5.5 and 7.5 pg/mL for TNF-α and IL-8, respectively. On the other hand, the DLs of the ZnO NR platform, assessed using the upper boundary of blank samples with a 95% accuracy goal, were found to be 4.2 and 5.5 fg/mL for TNF-α and IL-8, respectively. The unparalleled sensitivity down to the several fg/mL range enabled by the ZnO NR platform can reveal the levels of weakly expressed, disease-implicated cytokines such as TNF-α to promote early clinical diagnostics.

IL-8 testing and statistical analysis
Following calibration, the same patient samples were assayed on both the ELISA and ZnO NR platforms for quantitative comparison between both assays. When comparing the highly expressed IL-8 levels in the urine of 38 patients that ranged between several tens of pg/mL to a few ng/mL, the ZnO NRs-based assay had strong statistical agreement with the ELISA-based results allowing for direct validation of the novel bioassay. In Figure 2(A), a correlative plot displays the IL-8 readings from the same patients determined by the ELISA and ZnO NR assays on the x and y axes, respectively. The linear fit of the data points, shown in the dashed red line, lies very close to the superimposed line of y = x, shown in black, indicating excellent agreement between the two assay methods. In Figure 2(B), a histogram distribution chart reporting the differences in IL-8 readings between the two methods shows the majority of IL-8 readings from both assays fell within the range of ±2.5 pg/mL of each other. The IL-8 levels were further evaluated using the Bland-Altman analysis in Figure 2(C & D) in which the differences between the ELISA and ZnO NR readings for each patient were plotted against the mean concentration values. As shown, the data analysed over a large range of concentrations centred near the black lines, which represent the case of equivalent IL-8 readings obtained from the two different assays. The results of these comparative analyses validate the ZnO NR platform as a reliable technique to accurately quantify urinary biomarker proteins directly from patient samples.

TNF-α testing
To substantiate the applicability of the ZnO NR platforms in ultrasensitive cytokine detection using the weakly expressed biomarker of TNF-α, the protein levels for 46 patients were determined using both assay platforms. As seen in Figure 3(A), many of the patient samples exhibited values too close or below the DL of the ELISA assay (5.5 pg/mL) and are marked accordingly as grey blocks in the ELISA row. By contrast, the TNF-α values of all the patients were successfully quantified on the ZnO NR platform, well below several tens of pg/mL and into the low fg/mLrange. For the ZnO NR row in Figure 3(A), those samples that could not be measured via ELISA are shown with a magnifier sign, and their TNF-α concentrations, as determined by the ZnO NRs-based assay, are then shown in Figure 3(B & C) on two different scales for clarity. As demonstrated, the optical signal enhancement provided by the ZnO NR array platform enables the ultrasensitive detection of trace levels of proteins directly from patient samples.

Advantages of the ZnO NR-based approach and future outlook
Within the realm of biodetection, the ZnO NR-based approach can provide many direct advantages including facile platform fabrication, desirable optical properties, biocompatibility, and promising multiplexed/high-throughput integration capacities. The ZnO NR arrays are easily fabricated using a gas-phase method through well-established synthesis procedures and can be used directly after growth without any post-synthetic modifications. Further, the highly crystalline NRs exhibit many desirable optical properties including no intrinsic fluorescence (i.e. absence of autofluorescence) as well as enhancement of the optical intensity and photostability of nearby signal emitters. Since the ZnO NRs do not display any photoluminescence in the visible and near-infrared range, they do not interfere with the spectroscopic profiles of fluorophores commonly used in biology and biomedical detection. At the same time, the reduced dimensions and high shape anisotropy of the ZnO NRs enable optical enhancement and prolonged stability of the signal from fluorophore-tagged biomolecules adsorbed on their surface allowing for the ultrasensitive detection of trace levels of bioconstituents.

In addition to the demonstrated sensitivity permitted by the ZnO NR-based platform, the cytokine bioassay also has the direct advantages of rapid analysis, minimal volume requirements, and reusability. The multiplexed detection was achieved with 90 min of total assay time and only 60 μL of total bioreagent/sample volume using commonly employed fluorescence microscopy instrumentation. Further, the highly biocompatible ZnO NRs platform was found to withstand at least 25 repeated assays in complex biological and chemical reaction environments that include urine samples.

As modern automation strategies in high-throughput screening have seen great advancements in the sophistication of robotic sample delivery strategies and the detection of many analytes simultaneously via multichannel optical sensors, the ZnO NR-based platform may be able to provide much-sought detection sensitivity when integrated into these breakthrough technologies. In the microarray, each square patch of densely grown ZnO NRs with a typical dimension of 3~50 μm in side length can be configured to serve as a discrete detection element for different patient samples when coupled with appropriate sample delivery and multiplexed optical sensing/readout platforms. The demonstrated detection capabilities combined with this integration potential suggests that the ZnO NR-based approach serves as more than just an alternative or tandem detection platform to existing methods, but rather provides an advanced approach which allows the much needed, ultrasensitive detection of biomarker proteins in samples that exhibit concentration levels much lower than those which standard techniques can ascertain.

Conclusion
We successfully demonstrated a ZnO NRs-based fluorescence bioassay for the rapid, ultrasensitive, quantitative and multiplexed detection of AKI-related biomarkers in patient urine samples. We first statistically validated the ZnO NR-based approach against a conventional ELISA-based method by comparing the measurements of highly expressed levels of IL-8 that were above the DLs of ELISA. We further revealed the full detection capabilities of the ZnO NRs platform by quantifying ultratrace amounts of a weakly expressed cytokine, TNF-α, whose levels in urine are often below the DLs of conventional cytokine assays. The unparalleled detection sensitivity and other discussed advantages of the ZnO NR-based bioassay can be readily extended to advance other optical-sensing applications in biological research and clinical diagnostics.

Acknowledgement
This article is a summary of the work first presented in Singh M, Alabanza A, Gonzalez LE, Wang W, Reeves WB, Hahm J. Ultratrace level determination and quantitative analysis of kidney injury biomarkers in patient samples attained by zinc oxide nanorods. Nanoscale 2016; 8: 4613–4622 [9].

References
1. Feldmann MJ. Many cytokines are very useful therapeutic targets in disease. Clin Invest. 2008; 118: 3533–3536.
2. Fichorova RN, Richardson-Harman N, Alfano M, et al. Biological and technical variables affecting immunoassay recovery of cytokines from human serum and simulated vaginal fluid: a multicenter study. Anal Chem. 2008; 80: 4741–4751.
3. Borish LC, Steinke JWJ. Cytokines and chemokines. Allergy Clin Immunol. 2003; 111: S460–S475.
4. Nathan C, Sporn M. Cytokines in context. J Cell Biol. 1991; 113: 981–986.
5. Adalsteinsson V, Parajuli O, Kepics S, et al. Ultrasensitive detection of cytokines enabled by nanoscale ZnO arrays. Anal Chem. 2008; 80: 6594–6601.
6. Dorfman A, Kumar N, Hahm J. Nanoscale ZnO-enhanced fluorescence detection of protein interactions. Adv. Mater. 2006; 18: 2685–2690.
7. Singh M, Song S, Hahm J. Unique temporal and spatial biomolecular emission profile on individual zinc oxide nanorods. Nanoscale 2014; 6: 308–315.
8. Singh M, Jiang R, Coia H, et al. Insight into factors affecting the presence, degree, and temporal stability of fluorescence intensification on ZnO nanorod ends. Nanoscale 2015; 7: 1424–1436.
9. Singh M, Alabanza A, Gonzalez LE, et al. Ultratrace level determination and quantitative analysis of kidney injury biomarkers in patient samples attained by zinc oxide nanorods. Nanoscale 2016; 8: 4613–4622.

The authors
Manpreet Singh¹ BS, Anginelle Alabanza¹ BS, Lorelis E. Gonzalez¹ BS, Weiwei Wang² BS, W. Brian Reeves³ MD, and Jong-in Hahm*¹ PhD
¹Department of Chemistry, Georgetown University, Washington, DC 20057, USA
²Division of Nephrology, The Penn State College of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033, USA
³Department of Medicine, University of Texas Health Sciences Center at San Antonio, San Antonio, TX 78229, USA

*Corresponding author
E-mail: jh583@georgetown.edu

Novel nephrological markers: anti-PLA2R, anti-THSD7A and uromodulin

, 26 August 2020/in Featured Articles /by 3wmedia

Autoantibody diagnostics have in recent years transformed the diagnosis of the rare kidney disease primary membranous nephropathy (MN). The identification of the target antigens M-type phospholipase A2 receptor (PLA2R) and thrombospondin type 1-domain-containing 7A (THSD7A) paved the way for the development of specific immunological assays to detect the corresponding antibodies. Determination of both anti-PLA2R and anti-THSD7A antibodies allows serological diagnosis in 75% to 80% of cases of primary MN. Anti-PLA2R tests are, moreover, an indispensable tool for patient monitoring. A further new biomarker, uromodulin, acts as an indicator of impaired renal function, especially in chronic kidney disease, supplementing established markers such as creatine and cystatin C.

Membranous nephropathy
Membranous nephropathy is an organ-specific autoimmune disease and a major cause of nephrotic syndrome in adults. The disease is characterized by formation of immune complexes in the glomerular basement membrane, resulting in complement-mediated proteinuria and progressive loss of kidney function. 70-80% of cases are of the primary or idiopathic form. The remaining 20-30% of cases are secondary, arising from underlying causes such as malignancy, infection, drug intoxication or another autoimmune disease such as systemic lupus erythematosus. Diagnostic differentiation of primary and secondary forms is crucial due to different treatment regimes. Primary MN is treated with immunosuppressants, while therapy for the secondary form is targeted at the underlying disease. Treatment decisions for primary MN are further complicated by the extreme variability in clinical outcome. Patients can experience spontaneous remission or persistent proteinuria without renal failure, or progress to end-stage renal disease.

Anti-PLA2R antibodies
Autoantibodies against PLA2R are a highly specific marker for primary MN. They occur in around 70% to 75% of patients at time of diagnosis, while they are only very rarely found in patients with secondary MN or in healthy individuals. Their titer, moreover reflects the disease activity and severity. The target antigen, which was identified in 2009, is a type 1 transmembrane glycoprotein which is expressed on the surface of podocytes.

Following the discovery of the target antigen, standardized assays for the determination of anti-PLA2R antibodies in a routine setting were rapidly developed. The recombinant-cell indirect immunofluorescence test (RC-IIFT, Figure 1) utilizes transfected cells expressing full-length PLA2R on the cell surface as the antigenic substrate. The RC-IIFT is a reliable screening test for qualitative detection of anti-PLA2R autoantibodies. Using this assay, anti-PLA2R antibodies were detected with maximum specificity (100%) and a sensitivity of 77% in a cohort of 275 biopsy-proven primary MN patients. In the Anti-PLA2R ELISA, purified recombinant receptor is used as a solid-phase coating of microtitre plates. This assay provides accurate quantification of autoantibody concentrations and is particularly useful for disease monitoring. In a large cohort of clinically well characterized patients, the assay revealed very high sensitivity with respect to the RC-IIFT (96.5%) at a set specificity of 99.9%. The quantitative results of ELISA and RC-IIFT show a good correlation.

Anti-PLA2R is now an established parameter for diagnosing primary MN, differentiating it from secondary MN, assessing the disease status and monitoring responses to therapy [1, 2]. The antibody titre reflects the immunological as opposed to the clinical disease activity, and a change in the antibody titer, either spontaneous or treatment-induced, precedes the corresponding change in proteinuria by weeks or months (Figure 2) [3]. Thus, anti-PLA2R measurements provide a much earlier indicator than proteinuria of patient improvement or deterioration, helping to guide therapy decisions. Complete remission is always preceded by complete antibody depletion.

Anti-PLA2R titres also allow predictions regarding clinical outcome. High antibody titres are associated with a lower chance of spontaneous remission, a longer therapy period to achieve remission, and progression to kidney failure (Table 1) [4]. A low anti-PLA2R antibody titre at baseline, on the other hand, is the most pronounced independent predictor of spontaneous remission [5]. Patients with low anti-PLA2R titres are less likely to require immunosuppressive therapy than those with high titres. Overall, anti-PLA2R assessment is recommended every two months before starting immunosuppressive therapy to avoid unnecessary treatment in patients entering remission, and every month for the first six months of immunosuppression [2].

Anti-PLA2R analysis is also useful for predicting primary MN recurrence after kidney transplantation. Up to 40% of patients relapse after transplantation, and anti-PLA2R positivity is associated with a higher risk of recurrence. In a recent study, pre-transplant anti-PLA2R determination demonstrated a positive predictive value of 100% and a negative predictive value of 91% for a diagnosis of recurrent MN [6]. Further, if anti-PLA2R antibodies are persistently found during the first six months after transplantation, the risk of relapse is particularly high. Antibody determination may therefore be helpful for assessing the necessary and intensity of immunosuppressive therapy following transplantation.

Anti-THSD7A antibodies
Autoantibodies against THSD7A have been recently identified as a further marker in primary MN [7]. Similarly to PLA2R, THSD7A is an N-glycosylated, high-molecular-mass protein expressed on the podocyte membrane. Antibodies against THSD7A occur in around 2.5% to 5% of patients with idiopathic MN. Significantly, they are found predominantly in patients who are negative for anti-PLA2R, suggesting a distinct disease subgroup. Nevertheless, some rare cases with dual positivity for anti-PLA2R and anti-THSD7A have recently been described [8]. No reactivity to THSD7A has been observed in healthy controls or patients with other proteinuric or renal autoimmune diseases.

Anti-THSD7A serves as an additional, complementary marker in primary MN, reducing the diagnostic gap of anti-PLA2R analysis. Moreover, like anti-PLA2R, anti-THSD7A antibody levels also appear to be associated with disease activity. Further studies are currently underway to investigate this link.

Circulating anti-THSD7A antibodies can be determined by RC-IIFT using transfected cells expressing recombinant antigen (Figure 3). Combined testing for anti-PLA2R and anti-THSD7A provides a comprehensive screening for primary MN.

Uromodulin
Uromodulin, also known as Tamm-Horsfall protein, is a glycoprotein which is synthesized exclusively in the kidneys in the ascending limb of the loop of Henle, and subsequently secreted. When renal function is impaired, the uromodulin concentration in the serum or plasma decreases [9]. The concentration exhibits a linear correlation to the estimated glomerular filtration rate (eGFR) (Figure 4). Thus, uromodulin shows inverse kinetics to conventional markers like creatine and cystatin C, which increase with declining kidney function. Moreover, uromodulin concentrations change already in the early stages of chronic kidney disease, when there are few symptoms. Thus, uromodulin measurements enable detection of renal insufficiency in the creatine-blind area in the initial stages of kidney disease. Measurement of uromodulin is also suitable for monitoring kidney vitality during therapy and as a predictive marker after kidney transplantation.

Uromodulin can be measured in the serum or plasma by ELISA based on microplates coated with anti-uromodulin antibodies. The patient uromodulin concentrations are established using a simple cut-off-based interpretation, with a normal value being above 100 ng/ml. External factors such as body weight, nutrition or muscle mass do not need to be factored into the results by additional calculations, as is the case with classic markers. Further, since the uromodulin concentration is measured in serum or plasma, the laborious and error-prone collection of 24-hour urine is not required. This makes it a fast, easy and sensitive supplementary test for the early identification of nephropathies and loss in renal function.

Perspectives
Anti-PLA2R and anti-THSD7A assays are now a mainstay for the diagnosis of primary MN. Due to the high specificity, anti-PLA2R detection may even enable biopsy to be postponed or omitted in elderly patients, persons with poor clinical condition, or patients with life-threatening complications of nephrotic syndrome such as lung emboli. Nevertheless, a proportion of primary MN patients (around 20%) shows negative results for both anti-PLA2R and anti-THSD7A antibodies. This may reflect the disease activity at time of blood sampling (e.g. spontaneous remission) or a misclassification of patients who actually have secondary MN. It is also supposed that some primary MN patients react to other, as yet unidentified antigens. Anti-PLA2R measurements are also playing an increasingly central role in therapy decisions and prognosis, as the relationship between the anti-PLA2R titre and clinical outcome becomes better understood. Current research is directed at further elucidating the complex pathogenesis of primary MN and applying this knowledge to improve therapeutic care.

References
1. Mastroianni-Kirsztajn et al. Frontiers in Immunol. 2015: 6:221
2. Ronco et al. Lancet 2016: 385 (9981): 1983-92
3. Beck et al. Kidney Int. 2010: 77: 765-70
4. Hofstra et al. J. Am. Soc. Nephrol. 2012: 23(10): 1735-43
5. Timmermans et al. Am. J. Nephrol. 2015: 42(1): 70-7
6. Gupta et al. Clin. Transplant. 2016: 30: 461-9
7. Tomas et al. N. Engl. J. Med. 2014: 371(24): 2277-87
8. Larsen et al. Modern Pathol. 2016: 29: 421-6
9. Steubl et al. Medicine 2016: 95(10): e3011

The author
Jacqueline Gosink, PhD
EUROIMMUN AG
Seekamp 31,
23560 Luebeck, Germany
E-mail:j.gosink@euroimmun.de

Faster viral load results for an improved clinical service

, 26 August 2020/in Featured Articles /by 3wmedia

With 1,300 beds and over 6,000 employees, the Hospital Universitario 12 de Octubre in Madrid is one of the largest hospitals in Spain, serving a population of more than 500,000 people in and around the capital. It is an important teaching and research centre with a number of areas of expertise, including organ transplantation and the diagnosis and treatment of cancer.

The hospital’s Clinical Microbiology Department has a significant serology workload, processing more than 250 serology samples every day. This includes viral load testing for targets such as cytomegalovirus (CMV), hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1).

The serology laboratory faces a number of challenges that need to be addressed in order to meet future workload and service user requirements. Not least, the available space in the laboratory is limited due to the instrumentation that is required. Our existing viral load method requires separate sample preparation and amplification/detection platforms, and involves considerable manual intervention. Furthermore, as samples are processed in batches, this requires additional space for pre-analytical sample storage.

On reviewing our processes, we identified the need for increased automation within the laboratory, to reduce the number of manual steps and improve workflow efficiencies, and improve laboratory response times.

Evaluating new technology
In 2014-2015, we had the opportunity to evaluate a new, fully automated, random access platform for viral load analyses. The DxN VERIS Molecular Diagnostics System (Beckman Coulter) consolidates DNA extraction, nucleic acid amplification, quantification and detection onto a single automated instrument for a number of molecular targets. We evaluated the performance of the VERIS assays for CMV, HBV, HCV and HIV-1 using standard and control samples, as well as clinical samples, comparing them to our existing viral load method (COBAS Ampliprep^®/COBAS TaqMan^® assays, Roche).

All four assays were found to have comparable performance to our existing method, demonstrating excellent sensitivity, specificity and precision [1,2]. The correlation between both methods for HBV viral load quantification, for example, is shown in figure 1. A precision analysis for the VERIS HBV assay, which was calculated for five levels tested in duplicate over 20 days, gave a ‘within run’ standard deviation of ≤0.09 Log IU/mL and a ‘between run’ standard deviation of ≤0.09 Log IU/mL (table 1). Moreover, repeated analysis of negative samples alongside high positive samples at different rack positions showed no cross contamination, giving confidence in results. This random access technology provided the first result in just 75 minutes for HBV and CMV DNA, and in 90 minutes for HCV and HIV-1 RNA, with subsequent results every 2.5 minutes.

Our experiences in evaluating the DxN VERIS system led us to appreciate its potential as an enabler for an improved molecular biology clinical service. The increased automation and random access offer workflow improvements that simplify laboratory tasks and reduce the potential for human error. Furthermore, its overall performance and ease of use facilitated the smooth introduction of the technology in our laboratory.

Rapid results inform prompt treatment decisions
Early in 2016, we began to use the DxN VERIS System routinely for HBV and HCV viral load quantifications. Our annual volume of HBV and HCV samples is around 7,000 and, as a clinical laboratory working closely alongside medical staff, our viral load results support timely clinical decision making and subsequent patient management. In this respect the DxN VERIS system is ideal for our needs, providing same day results to our outpatient clinics.

One of the most important aspects of the system for our laboratory is the ability to process samples as they are received in the laboratory. With our previous method, we had to work in batches of 24 or 48, collecting and storing samples throughout the day (or overnight) until we had a sufficient number of samples for a single run. Then results were not available until the entire run was completed. This had a huge impact on response times.

Now, with the random access capabilities of the DxN VERIS system, this has changed. We receive samples around the clock and we are able to run them straight away. This has improved our response times significantly, from 24-28 hours to just 4-5 hours from sample receipt, and with comparable quality of results compared to our previous method.

At the moment we enter results into the patient record manually, but soon we will be moving to a barcode system that will transfer all details and results into the electronic patient record automatically, saving time and further reducing opportunities for human error.

Expanding laboratory capabilities
The DxN VERIS system has been well received by laboratory staff and has expanded our service capabilities. Fully automated from the loading of samples to obtaining results, it is easy to operate by laboratory technicians of all abilities. In addition, since it involves minimal manual intervention and fewer steps than our previous method, there is less opportunity for error and staff have more time to perform other important tasks in the laboratory.

One of our objectives as a clinical microbiology department is to offer a more complete panel of assays on a 24-hour basis. Previously, this was not possible for molecular diagnostic investigations such as HBV and HCV viral loads, because it was not practical to run one or two samples at a time on our previous system. The random access and ease of use of the DxN VERIS system has enabled us to operate our HBV/HCV viral load service 24 hours per day, making it ideal to meet the variable needs of our laboratory in terms of workload volume and response times.

For further information about the DxN VERIS Molecular Diagnostic System and the VERIS assays currently available, please contact: Tiffany Page, Senior Pan European Marketing Manager Molecular Diagnostics, Email: info@beckmanmolecular.com or visit www.beckmancoulter.com/moleculardiagnostics

References
1. Rafael Delgado (2015) Evaluation in a Clinical Setting of the General Performance of DxN VERIS CMV and HBV Viral Load Assay. Oral presentation, ECCMID, Copenhagen.
2. Gutiérrez, F, Zurita, S, Pérez-Rivilla, A and Delgado, R (2015) Evaluation of the Automated DxN VERIS System for Human Immunodeficiency Virus Type-1 (HIV-1) and Hepatitis C Virus (HCV) Viral Load (VL) Monitoring. Poster presentation ESCV, Edinburgh.

The author
Rafael Delgado, Head of Clinical Microbiology Hospital
Universitario 12 de Octubre, Madrid, Spain.