Liquid chromatography-mass spectrometry (LC-MS/MS) is an analytical chemistry technique that combines the physio-chemical separation capabilities of liquid chromatography (via conventional chromatography within a column) with the analytic power of mass spectrometry. It allows the user to properly ascertain the individual mass/charge ratio of analytes present in a chromatographic peak. The high throughput capabilities of this technique will bring value to the clinical lab, where time taken to analyse samples is paramount. Bringing LC-MS/MS testing into the clinical setting has been a slow process, however, the medical device industry is on the verge of a fundamental breakthrough that could help drive the adoption of this technique.
LC-MS/MS is used primarily for the identification and quantification of particular molecules within a substance, and its application in diagnostics is a promising venture due to its potential ability to increase throughputs and streamline the processes needed. As such, patient data can be analysed quickly and accurately in order to provide improved patient care. Broadly speaking, the methodology can be divided into three parts. Initially, sample preparation is undertaken; be it whole blood, plasma, saliva or urine – the sample must be prepared to ensure large proteins and salts that may dirty the instrumentation are removed. Conventionally, this phase has been undertaken manually, which can be time-consuming and prone to human error. As such there is a need for the automation of this step to improve efficiency and reliability before LC-MS/MS is adopted by the clinical laboratory. Once sample preparation is complete, the liquid chromatography and mass spectrometry steps can take place, in which the sample is separated and analysed respectively.
LC-MS/MS and the clinical laboratory
Although adoption of LC-MS/MS in the clinical laboratory has been slow but steady, this technique has demonstrated vast improvements in analytical specificity when compared to conventional immunoassays. Mass spectrometry’s strength lies in its ability to be extremely specific to the target analyte, due to the absence of cross reactivity; the likes of which can be common in antibody-based immunoassay (IA) methods. However, the uptake of this technique by clinical labs has not been as rapid as expected, with many choosing to continue using immunoassay-based methods instead.
There are a number of factors causing clinical labs to be cautious about the mainstream use of LC-MS/MS systems. There are numerous LC and MS systems available to choose from, something which in itself can seem overwhelming to a clinical scientist who is not an LC-MS/MS expert. In addition, there is a range of options for calibrators and controls available, along with the internal expertise required to develop and validate methods, and set-up and run the instruments. The final factor to impact the decision is often cost, since investment in such systems is commonly high, especially when taking into account the automated components required to help reduce labour needs for sample preparation. As such, finance options are often limited. When combined, these factors can make immunoassay analysers seem like the simpler option.
The emergence of connected components
Although used in many clinical labs, immunoassay techniques are not always accurate. For example small molecule biomarkers, such as steroid hormones, prove challenging due to the lack of specificity in the binding sites on small molecules, a fact that many clinical scientists are all too aware of. Recent improvements to LC-MS/MS systems have focused on advancing both ease of use and efficacy, essentially to make them a viable alternative to IA methods. Laboratory managers can find ample published documentation that shows just how beneficial LC-MS/MS systems are when used in place of IAs. For instance, a study by Nigel W. Brown and colleagues published in Clinical Chemistry in 2005 demonstrated that LC-MS/MS was far more precise than a microparticle enzyme immunoassay (MEIA), which was ‘significantly affected by patient cohort’ (Brown, N et al. Clinical Chemistry 2005; 51(3): 586-593).
Clinical laboratories are faced with increasing complexities in their daily workflows, and there are pressures to provide detailed analyses of patient samples using streamlined and well-coordinated practices. The need to provide efficient turnaround on samples is also on the increase. There is, therefore, a trend where system manufacturers are looking to provide laboratories with the ability to advance efficiency through the implementation of compatible technologies, such as the combination of stand-alone elements (automated sample handlers, LC-MS/MS reagent kits, and software), which are supplied together to better manage workflows. These connected component-based systems, by which the different components of the LC-MS/MS system (sample preparation, liquid chromatography, and mass spectrometry) are placed in tandem with each other, is a big step in the right direction to increase productivity and efficiency, while simplifying the number of decisions that the lab needs to make. However, there are still improvements that can be made. The issue lies in the fact that connected components are not the same as a fully-integrated, automated system with dedicated assays and diagnostic kits that are regulatory compliant. The development of properly synergized components can truly simplify the decisions faced by clinical scientists and enable LC-MS/MS to become an integral part of the clinical laboratory.
The needs of the lab
Clinical labs require a high level of automation with a number of its systems, owing to the high turnover rate demanded to meet the needs of patient care. In addition, easy to use technologies that include walk away operations are essential, and considered commonplace to clinical scientists, owing to the multitude of responsibilities placed on laboratory personnel. These busy labs require built-for-purpose, fully integrated analysers that are able to greatly reduce installation, validation, and training times, having the system ready to operate in a matter of weeks, rather than months. Streamlining the procedure without compromising the quality of the analysis via implementation of better integrated systems can be considered an essential next step in the medical devices industry. Furthermore, results obtained from these systems need not be in isolation: standardization between laboratories using the same system will be achievable owing to the inclusion of dedicated test kits that are fully validated and ready for use with the analyser. The ideal next-generation system for the clinical laboratory will encompass every step, including automated sample preparation, handling and LC-MS in one unit. Moreover, it will be labelled as a medical device, have dedicated assay kits, and be produced, serviced, and supported by a single manufacturer. Finally, such a device would ideally be able to connect bi-directionally with the laboratory information system (LIS) and furthermore to the laboratory automation system (LAS).
In the end, technologies that are able to advance the state of play for laboratory sample analysis are required in order to ensure laboratory personnel can be confident in the analyses they are making. Beyond connected components, the introduction of integrated LC-MS/MS systems into the laboratory could lead to a paradigm shift with regards to specificity in small molecule analysis that is expected by clinical scientists. Systems that can lead to better quality of care for patients and improved analysis for physicians will essentially help healthcare systems operate more efficiently.
The author
Sarah Robinson, Ph.D,
Market Development Specialist,
Thermo Fisher Scientific
& Expert Consultant to the EFLM
Working Group on Test Evaluation
Uromodulin kidney disease is a rare autosomal dominant kidney disease, characterized by hyperuricemia, gout and progressive kidney failure. Affected patients typically need renal replacement therapy in middle age. A considerable number of patients may reach end-stage kidney disease without a correct diagnosis, making improvements in diagnostic methods of vital importance.
by Dr Tamehito Onoe and Dr Mitsuhiro Kawano
Introduction
Uromodulin (UMOD), also known as Tamm–Horsfall protein, is the most abundant protein in healthy human urine. UMOD protein is a kidney-specific protein which is exclusively produced at the epithelial cells lining the thick ascending limb (TAL) of Henle’s loop.
The roles of urinary UMOD protein are assumed to be to protect against urinary tract infection, prevent urolithiasis formation and ensure water impermeability to create the countercurrent gradient. However, the accurate function and significance of UMOD protein are not yet fully elucidated [1].
Uromodulin kidney disease
Uromodulin kidney disease (UKD) is an inherited disease caused by UMOD gene mutations. So far, more than 100 mutations of the UMOD gene have been reported from all over the world [2]. Familial juvenile hyperuricemic nephropathy (FJHN), medullary cystic kidney disease type2 (MCKD2) and glomerulocystic kidney disease (GCKD), which are considered to be different diseases, have been proved to be caused by UMOD gene mutations [3]. Subsequently, because multiple names for one condition would be confusing and misleading, and also cysts are not pathognomonic for this disease, a new term, ‘Autosomal dominant tubulointerstitial kidney disease’ (ADTKD) was proposed in 2015 [4]. Mutations of renin (REN), hepatocyte nuclear factor 1β (HNF1β), and mucin-1 (MUC1) are also responsible for ADTKD besides UMOD. They all share common clinical characteristics, which are progressive kidney failure, tubulointerstitial nephritis and inheritance compatible with autosomal dominant trait with only trivial clinical differences. When UMOD mutation is identified in an ADTKD patient, the official diagnostic term is ADTKD-UMOD. However, UKD is also used to facilitate communication with patients, and so this term is used in the present article.
Patients with UKD have urinary concentration defect, hyperuricemia and gout from a young age. Their kidney function gradually deteriorates, and reaches end-stage kidney disease (ESKD) from 25 to 75 years of age. Their kidneys are usually of normal size or small and there are sometimes cysts, although the frequency of cysts does not differ from that of ‘non-cystic’ kidney diseases. Their urine tests usually show no or only very mild proteinuria or hematuria. The most prominent characteristic of UKD is a marked abundance of chronic kidney disease (CKD) patients in their pedigree, compatible with an autosomal dominant trait. Our group detected a novel A247P UMOD mutation in a UKD family (Fig. 1), many of whose members have hyperuricemia, CKD and ESKD, and are on hemodialysis (HD) therapy [5].
UKD is reported to be a rare disease, with a frequency of about 1.5 cases per million population. However, because hyperuricemia is a frequent complication in all CKD patients, when their family history is absent or unknown, it is difficult to suspect UKD, and so the frequency of UKD may be underestimated. This means that a certain proportion of UKD patients may reach ESKD without a correct diagnosis.
Diagnosis of uromodulin kidney disease
Clinically UKD should be suspected when a CKD patient has an abundant family history compatible with autosomal dominant trait, hyperuricemia, gout and bland urine findings. The final diagnosis of UKD is made by genetic test, which is, however, not commercially available, and only a limited number of laboratories are capable of performing it. So easier laboratory tests supportive of genetic tests would be helpful for the diagnosis of UKD and are awaited.
The renal histology of UKD patients shows nonspecific interstitial fibrosis, tubular atrophy and normal glomeruli. So it is difficult to make a diagnosis of UKD by ordinary histological methods. Moreover, not many UKD patients seem to undergo renal biopsy because their urine sediment shows no or only slight abnormalities and so clinicians may hesitate to undertake this invasive test. However, we believe that renal pathological examination is very informative not only for ruling out other kidney diseases but also for the diagnosis of UKD. UMOD proteins synthesized from mutated UMOD gene have protein folding disability and cannot escape from the endoplasmic reticulum (ER) of the epithelial cells. Immunostaining using anti-UMOD antibody in kidney sections of UKD patients shows massive UMOD accumulation in their epithelial cells (Fig. 2). Because of the question of whether there are any UKD patients among those who received kidney biopsy and were diagnosed as having nephrosclerosis or interstitial nephritis, we performed the following investigation.
In a 3787-sample kidney biopsy database of Kanazawa University, patients meeting all of the following criteria were selected for UMOD immunostaining. (1) Renal insufficiency (serum creatinine >1.0 mg/dL) below 50 years of age; (2) hyperuricemia: serum uric acid higher than 7mg/dl or under treatment for hyperuricemia; (3) no or only very mild abnormalities in urinalysis; and (4) no other apparent renal disease present clinically or histopathologically. Finally, 15 patients were selected and abnormal UMOD accumulations were detected in three independent patients by UMOD immunostaining. A247P UMOD gene mutations were detected in the proband of the family in Figure 1 and the other independent patient, indicating that they may share the same ancestor. The other patient had no family history of CKD. These results show that there may be more UKD patients than expected before, and also indicate that when kidney biopsy shows only nonspecific interstitial fibrosis in patients with renal insufficiency, UMOD immunostaining may be considered to detect UKD with or without a family history of CKD, especially with hyperuricemia and bland urinary findings.
Most of the synthesized UMOD protein is carried to the apical membrane of epithelial cells and excreted in the urine. However, a low but considerable amount of UMOD protein goes to the basolateral membrane and is secreted into the serum [6]. Serum UMOD protein concentrations are reported to be 45–490 ng/mL, while urine UMOD protein concentrations are 1000–80 000 ng/mL. The functions and significance of serum UMOD proteins are unknown.
Some results of animal experiments indicate that UMOD protein has a renoprotective effect against various types of injury. A renal ischemia-reperfusion experiment in UMOD knockout mice showed significantly worse results than in wild-type animals [7]. It is well known that urinary UMOD concentrations in UKD patients are decreased. The authors recently reported that serum UMOD protein concentrations are also significantly decreased in UKD patients besides urinary UMOD (Fig. 3). Serum and urinary UMOD concentrations decline in parallel with the decrease of estimated glomerular filtration rate (eGFR) due to the diminishment of UMOD producing epithelial cells in CKD patients. In UKD patients, the serum and urine UMOD concentrations were significantly lower compared with CKD patients beyond their eGFRs. Decreased serum and urinary UMOD concentrations may be good clues to suspect and diagnose UKD; however, verification in more UKD patients with various mutations will be indispensable.
Conclusions
So far no treatment has been devised that slows the rate of renal functional deterioration of UKD. At present, management for UKD patients is not different from that for other CKD patients. Anti-hyperuricemia drugs or anti-hypertensive therapy is used when necessary and the appropriate renal replacement therapy or renal transplantation should be considered when ESKD is reached. To clarify the pathogenesis and achieve effective treatment for UKD, establishment of more efficient diagnostic methods for UKD is expected. UMOD immunostaining for renal sections and measurement of serum and urinary UMOD concentrations are considered to be good modalities for the diagnosis of UKD. It is expected that through these tests, more UKD patients will be diagnosed at earlier stages and will be able to benefit from starting appropriate therapy before ESKD.
Recently some particular SNPs of UMOD promoter areas have been proved to be associated with hypertension or renal insufficiency from the genome-wide association study [8]. UMOD will likely attract greater attention as a renal-prognostic marker for not only UKD patients but also the general population.
References
1. Lhotta K, Piret SE, Kramar R, Thakker RV, Sunder-Plassmann G, Kotanko P. Epidemiology of uromodulin-associated kidney disease – results from a nation-wide survey. Nephron Extra 2012; 2: 147–158.
2. Scolari F, Izzi C, Ghiggeri GM. Uromodulin: from monogenic to multifactorial diseases. Nephrol Dial Transplant. 2015; 30: 1250–1256.
3. Hart TC, Gorry MC, Hart PS, Woodard AS, Shihabi Z, Sandhu J, Shirts B, Xu L, Zhu H, Barmada MM, Bleyer AJ. Mutations of the UMOD gene are responsible for medullary cystic kidney disease 2 and familial juvenile hyperuricaemic nephropathy. J Med Genet. 2002; 39: 882–892.
4. Eckardt KU, Alper SL, Antignac C, Bleyer AJ, Chauveau D, Dahan K, Deltas C, Hosking A, Kmoch S, Rampoldi L, Wiesener M, Wolf MT, Devuyst O. Autosomal dominant tubulointerstitial kidney disease: diagnosis, classification, and management-A KDIGO consensus report. Kidney Int. 2015; 88(4): 676–683.
5. Onoe T, Yamada K, Mizushima I, Ito K, Kawakami T, Daimon S, Muramoto H, Konoshita T, Yamagishi M, Kawano M. Hints to the diagnosis of uromodulin kidney disease. Clin Kidney J. 2016; 9: 69–75.
6. Bachmann S, Koeppen-Hagemann I, Kriz W. Ultrastructural localization of Tamm-Horsfall glycoprotein (THP) in rat kidney as revealed by protein A-gold immunocytochemistry. Histochemistry 1985; 83: 531–538.
7. El-Achkar TM, Wu XR, Rauchman M, McCracken R, Kiefer S, Dagher PC. Tamm-Horsfall protein protects the kidney from ischemic injury by decreasing inflammation and altering TLR4 expression. Am J Physiol Renal Physiol. 2008; 295: F534–544.
8. Trudu M, Janas S, Lanzani C, Debaix H, Schaeffer C, Ikehata M, Citterio L, Demaretz S, Trevisani F, Ristagno G, Glaudemans B, Laghmani K, Dell’Antonio G, Loffing J, Rastaldi MP, Manunta P, Devuyst O, Rampoldi L. Common noncoding UMOD gene variants induce salt-sensitive hypertension and kidney damage by increasing uromodulin expression. Nat Med. 2013; 19: 1655–1660.
The authors
Tamehito Onoe MD, PhD and Mitsuhiro Kawano* MD, PhD
Division of Rheumatology,
Department of Internal Medicine,
Kanazawa University Hospital,
Kanazawa, 920-8641,
Japan
*Corresponding author
E-mail: sk33166@gmail.com
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
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