An accurate measure of glomerular filtration rate (GFR) is required in a variety of clinical situations. Plasma creatinine and creatinine-based equations have served as convenient estimates of GFR but they are subject to limitations. Formal assessment of GFR using clearance methods traditionally necessitates the use of radioactive traces such as 51Cr-EDTA. More recently, iohexol has become increasingly well recognized as an alternative exogenous marker which can now be used routinely, providing a safe, efficient and cost-effective measure.
by Zoe Maunsell and Prof. Tim James
Background
Glomerular filtration rate (GFR) describes the amount of fluid filtered by the glomerulus per unit time, and is therefore a useful indicator of renal function. Expressed in mL/min, it can be accurately measured from the rate of disappearance of an injected substance from the plasma or by collecting urine over a defined time period. The clearance formula, UV/P (where U represents urine concentration, V is urine volume per unit time and P is plasma concentration) can be used to calculate GFR when an ideal marker is used. Such a marker should be freely filtered at the glomerulus, and not secreted, absorbed or metabolized by the renal tubules. Since renal function is proportional to kidney size (which is proportional to body surface area), when estimating GFR, values are usually standardized to an average body surface area of 1.73 m2. Having an accurate yet convenient means to monitor renal function is extremely important in clinical practice. A compromise exists between highly accurate but time consuming, technically difficult reference methods and more accessible, readily available markers of renal function.
Gold standard methods
The ‘gold standard’ for the measurement of GFR is inulin clearance. Inulin is a polysaccharide derived from plants, which can be introduced into the body either by intravenous infusion or bolus dose and its rate of clearance measured. Since inulin is freely filtered by the glomerulus, is not absorbed, secreted or metabolized by the tubules, its elimination is proportional to GFR. Although it is the ‘gold standard’ for assessment of GFR, assay of inulin is technically demanding, and so this technique is not suitable for routine clinical practice.
Similarly, the radioactive tracer 51Cr-EDTA can be administered intravenously and its elimination rate monitored. This marker is widely used to measure GFR, but presents a risk to patients and healthcare workers since it involves exposure to ionizing radiation.
Iohexol clearance is an alternative to inulin clearance [1]. Iohexol is an iodine-containing, non-isotopic contrast agent, and studies have shown close agreement between GFR values obtained by iohexol and inulin clearance.
Creatinine and endogenous markers
Creatinine is a widely used clearance marker but suffers from well-described problems including the difficulty and inconvenience of 24-hour urine collection.
GFR has therefore been estimated without urine collection using endogenous markers, the most widely used of which is plasma/serum creatinine. Improvements have been made to the methodology used for creatinine measurement, including standardization of kinetic Jaffe creatinine assays, defining their traceability to isotope-dilution reference assays. However, the Jaffe method remains subject to numerous interferences and therefore laboratories may select the more specific enzymatic creatinine methods. Using creatinine as a marker of GFR has several limitations, including the relationship of creatinine concentration to muscle mass, meaning that estimation of GFR using creatinine alone is particularly a problem in children. It is for this reason that corrections for body surface area have been made. The major limitation of creatinine alone as a marker of GFR is that it is a relatively insensitive marker, and a large decline in renal function can occur before a change in plasma creatinine concentration is observed.
Cystatin C is another endogenous marker that can be used for the estimation of GFR. It is a cysteine protease inhibitor, is produced at a constant rate (independent of muscle mass, sex, age when older than 12 months and inflammatory conditions) and is freely filtered by the kidneys. It is almost completely reabsorbed by the proximal renal tubular cells so that little is normally excreted in the urine. The reciprocal of plasma cystatin C concentration has been shown to be correlated with GFR. Cystatin C has significant advantages over creatinine as a marker of renal function, since increases in serum concentration become apparent with mild renal impairment, such as GFR of 60–90 mL/min, and may be more useful than creatinine in detecting acute kidney injury. Like creatinine, the measurement of cystatin C suffers to some extent from problems with standardization, in particular with reported differences [2] in measured concentrations using turbidimetric versus nephelometric assays by different manufacturers, presumably due to the use of different antibody and detection systems. The availability of an international reference preparation (ERM–DA471/IFCC) is likely to lead to greater agreement between cystatin C methods.
Calculated estimates of GFR
In order to overcome the limitations of measuring creatinine and cystatin C concentrations alone, calculations have been devised. These involve corrections for body surface area, sex, age and ethnicity. Among the most well known are the 4- or 6-parameter MDRD (Modification of Diet in Renal Disease study), Cockroft–Gault (which estimates creatinine clearance) and CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) equations for adults and the Schwartz and the Counahan–Barratt prediction equations for children.
The Cockroft–Gault equation [3] provides an estimate of creatinine clearance. Devised in the 1970s, the equation was calculated by examining the mean of two 24-hour creatinine clearances from 249 adult men.
The 4-variable MDRD equation [4] (incorporating creatinine, age, sex and race) is the most widely used in the UK, being reported by laboratories in the form of eGFR, and forms the basis of the staging system for CKD. The formula was developed from the Modification of Diet in Renal Disease study, using data from 1628 patients. The patients had already been diagnosed with CKD, and the study aims were to slow the progression of CKD with a low protein diet and blood pressure control. However, significant limitations to the use of the equation exist, mainly connected with the use of creatinine as a marker of GFR. These include inaccuracies of the equation at extremes of body type such as malnourished or amputee patients and the unsuitability of the equation for use in pregnancy. The formula includes a correction factor for use in African American populations but its validity in other ethnic groups has not been established. The MDRD formula tends to underestimate function at normal GFR, therefore slight reductions in eGFR should not be over-interpreted and reporting eGFR >90mLs/min/1.73m2 is not recommended.
The CKD-EPI equation was developed in 2008 [5] and updated in 2012 [6]. The 2008 equation was developed using data from over 8000 patients from 10 studies. The 2012 versions used over 5000 patients from 13 studies. Importantly, both studies included patients with normal GFR, as well as those with CRF. It was found that the CKD-EPI equations performed better than the MDRD equation, particularly at higher levels of estimated GFR. The limitations of the studies include a limited number of elderly patients and those from ethnic groups other than black race.
In terms of estimation of GFR in children, the Schwartz [7] and Counahan–Barratt [8] equations were developed in the 1970s and have been widely used to estimate GFR from serum creatinine and height (length in infants). The Schwartz equation was modified in 2009 [ 9, 10]. Based on new studies of iohexol clearance, the original formula was found to overestimate GFR. It was postulated that this was in part due to the use of new, standardized creatinine assays. In 2009 the equation was updated, based on studies of 349 patients aged 1–16 years with mild to moderate chronic renal disease. The formula uses creatinine determined using an enzymatic method, urea, height and cystatin C.
Various modifications of these equations, using creatinine, cystatin C [11] or both [12] have been published and optimized in various patient cohorts. When using creatinine-based assays, it is important to know which creatinine assay is being used, since equations have been devised which include different coefficients depending on the methodology used.
Iohexol clearance: a gold standard method in routine use
Oxford University Hospitals NHS Trust has been routinely offering an iohexol clearance service for the measurement of GFR since June 2011. The service is widely used by the hospital’s pediatric service, and is used in a variety of clinical situations, including determining renal function in surgical patients and for chemotherapy dosing. Before the introduction of this service, GFR was assessed using 51Cr EDTA clearance. Although this provided an accurate measurement of GFR, a long waiting list and difficult patient preparation made this technique suboptimal for use in children. In addition, where GFR results are required before administration of chemotherapy agents, timely result availability is critical to prevent delayed treatment. With iohexol clearance we have been able to improve GFR turnaround time compared to 51Cr-EDTA clearance and audit of our first 2 years of service demonstrated that 89% of results were reported within 2 working days and 99% within 3 working days. Iohexol clearance also avoids the use of radioactive isotopes, reducing exposure for patients, carers and hospital staff. Therefore the service has several distinct advantages over conventional isotopic clearance methods (Table 1).
Iohexol clearance is measured according to a standardized protocol, as described previously [13]. In summary, the patient is cannulated, a baseline blood specimen is collected and a standard dose of Omnipaque containing 300 mg iodine/mL is administered through the cannula, (2 mL in patients weighing <40 kg, 5 mL in patients weighing >40 kg). Blood (1 mL) is collected into lithium heparin tubes at 120, 180 and 240 min after iohexol administration.
The iohexol concentration in each specimen is measured by a UPLC (ultra-high performance liquid chromatography) method involving precipitation of plasma samples with equal volumes of perchloric acid and injection of the supernatant onto a Waters 50 x 2.1 mm 1.8 μm reversed phase column with isocratic acetonitrile-based solvent elution. The assay uses a one-point (604 mg/L) calibration through zero. The assay is straighfoward, reliable and rapid; chromatography time is 6 min per specimen. The assay demonstrates excellent inter-assay CVs: 2.2%, 1.9% and 1.9% at 39, 163 and 322 mg/L respectively. The laboratory participates in external quality assessment through the Scandinavian EQUALIS scheme.
The two-point model is used to calculate iohexol clearance, according to the Brochner–Mortensen method [14]. The model assumes a one-compartment model where iohexol is cleared by first-order kinetics. The estimated clearance (Cl ) of the GFR marker is expressed as:
Cl= Q• b/c1 (ml/min)
Where Q = amount of injected marker, b = disappearance rate of marker (min−1), c1= intercept on the y-axis.
The clearance correction for non-immediate mixing of the tracer substance is expressed as:
Cl = 0.990778 x estimated Cl−0.001218• estimated Cl2
Alternatively, single point estimates of GFR at appropriate time points can be calculated using the Jacobsson model. This method can be used in cases where the two-point method cannot be used, for example when contamination has occurred due to poor flushing of lines between sample collection. Here:
GFR = ln(D/VCt) / (t/V + 0.0016)
Where V = volume of distribution in mL (V = 187 x weight in kg + 732), t = time of sampling in min, D = dose of iohexol administered, Ct = concentration of iohexol at time t.
Iohexol clearance is corrected for actual body surface area, using the Dubois and Dubois method, calculated as 0.007184 x (weight in kg)0.425 x (height in cm)0.72, to yield a measure of GFR in mL/min/1.73 m2.
Since initiating the iohexol GFR service in 2011, we have analysed over 400 sets of patient samples. The use of UPLC over HPLC allows for smaller sample volumes to be collected, making the technique particularly suited to pediatrics. Ongoing close liaison with clinicians and nursing staff has revealed that patients and their families have responded favourably to the new procedure and in particular prefer being able to remain on the Daycare Ward throughout the investigation, which is a child-friendly environment.
Iohexol clearance has the added benefit of being significantly less costly than 51Cr-EDTA clearance. The approximate cost is £80 compared to £250 for 51Cr-EDTA.
Conclusions
The measurement of glomerular filtration rate, GFR, is important in a number of clinical scenarios. Much work has been undertaken to develop methods of estimating GFR in order to avoid the time-consuming and relatively invasive formal measurement of renal function using clearance methods. These include the development of eGFR equations based on factors such as plasma creatinine, cystatin C, age, race and body surface area. Equations such as the MDRD and CKD-EPI formulae for adults, and Schwartz and Counahan–Barratt equations for children have been widely used in clinical practice.
However, despite their widespread use, limitations of these equations have been described, including the problems of creatinine-based equations tending to underestimate GFR at normal GFR levels and the issue of creatinine-based formulae being unsuitable for use in patients with non-normal muscle mass.
For the formal measurement of GFR, clearance studies must be performed which have traditionally used radioactive tracers such as 51Chromium-EDTA. However, in recent years the use of iohexol has increased. Thanks to its stability in vitro and relative ease of measurement, the assay of iohexol is rapid, reliable and can be performed on small specimens. This fits in particularly well with the clinical needs of a pediatric service for GFR measurement and is cheaper, safer and more convenient than traditional methods.
References
1. Ng DK, Schwartz GJ, Jacobson LP, Palella FJ, Margolick JB, Warady BA, Furth SL, Muñoz A. Universal GFR determination based on two time points during plasma iohexol disappearance. Kidney Int. 2011; 80: 423–430.
2. Herget-Rosenthal S, Bökenkamp A, Hofmann W. How to estimate GFR-serum creatinine, serum cystatin C or equations? Clin Biochem. 2007; 40: 153–161.
3. Cockcroft DW, Gault MH. Prediction of creatinine clearance from serum creatinine. Nephron 1976; 16: 31–41.
4. Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D. A more accurate method to estimate glomerular filtration rate from serum creatinine: A new prediction equation. Modification of Diet in Renal Disease Study Group. Ann Intern Med. 1999; 130: 461–470.
5. Levey AS, Stevens LA, Schmid CH, Zhang YL, Castro AF III, Feldman HI, Kusek JW, Eggers P, Van Lente F, Greene T, Coresh J, for the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI). A new equation to estimate glomerular filtration rate. Ann Intern Med. 2009; 150: 604–612.
6. Inker LA, Schmid CH, Tighiouart H, Eckfeldt JH, Feldman HI, Greene T, Kusek JW, Manzi J, Van Lente F, Zhang YL, Coresh J, Levey AS, for the CKD-EPI Investigators. Estimating glomerular filtration rate from serum creatinine and cystatin C. N Engl J Med 2012; 367: 20–29.
7. Schwartz GJ, Haycock GB, Edelmann CM Jr, Spitzer A. A simple estimate of glomerular filtration rate in children derived from body length and plasma creatinine. Pediatrics 1976; 58: 259–263.
8. Counahan R, Chantler C, Ghazali S, Kirkwood B, Rose F, Baratt TM. Estimation of glomerular filtration rate from plasma creatinine concentration in children. Arch Dis Child 1976; 51: 875–878.
9. Schwartz GJ, Muñoz A, Schneider MF, Mak RH, Kaskel F, Warady BA, Furth SL. New equations to estimate GFR in children with CKD. J Am Soc Nephrol. 2009; 20: 629–637.
10. Schwartz GJ, Work DF. Measurement and estimation of GFR in children and adolescents. Clin J Am Soc Nephrol 2009; 4: 1832–1843.
11. Grubb A, Nyman Un, Björk J, Lindström V, Rippe BG, Christensson A. Simple cystatin C–based prediction equations for glomerular filtration rate compared with the modification of diet in renal disease prediction equation for adults and the Schwartz and the Counahan–Barratt prediction equations for children. Clinical Chemistry 2005; 51: 1420–1431.
12. The Swedish Council on Health Technology Assessment (SBU). Methods to estimate and measure renal function (glomerular filtration rate). Stockholm: The Swedish Council on Health Technology Assessment (SBU). 2012; Yellow 214.
13. James TJ, Lewis AV, Tan GD, Altmann P, Taylor RP, Levy JC. Validity of simplified protocols to estimate glomerular filtration rate using iohexol clearance Ann Clin Biochem. 2007: 44; 369–376.
14. Bröchner-Mortensen J. A simple method for the determination of glomerular filtration rate. Scand J Clin Lab Invest. 1972; 30(3): 271–274.
The authors
Zoe Maunsell* MBiochem MSc DipRCPath and Tim James PhD
Department of Clinical Biochemistry, Oxford University Hospitals NHS Trust, Oxford, UK
*Corresponding author
E-mail: zoe.maunsell@ouh.nhs.uk
Teicoplanin in therapeutic drug monitoring
, /in Featured Articles /by 3wmediaRecent years have seen the emergence of teicoplanin usage for staphylococcal infections, in particular endocarditis, osteomyelitis, septic arthritis, and methicillin-resistant Staphylococcus aureus (MRSA). Teicoplanin is now routinely used as an alternative to vancomycin, due to its safety profile and ease of administration. This article discusses the advantages for teicoplanin, the need for routine monitoring, and the various associated methodologies.
by Dr Francis H. Y. Fung
Background
Teicoplanin was first isolated and identified from antibiotic-producing strains of Actinoplanes teichomyceticus in 1978, and was shown to be highly active against both aerobic and anaerobic Gram-positive pathogenic bacteria [1]. This anti-bacterial agent was initially thought to belong in the class of glycopeptide antibiotics that act as cell wall inhibitors such as vancomycin, ristomycin, and mannopeptins. Barna et al. [2] first described the molecular structure of teicoplanin A2 in 1984, as five similar glycopeptides characterized by different fatty acid chains of 10 to 11 carbon atoms (which make up the majority of teicoplanins found in vivo) (Fig. 1), and four minor related compounds that may also be present in very minute quantities. The five major side chains (A2-1 to A2-5) have terminal groups of CH–NH2, whereas in the four minor compounds (R2-1 to R2-4) this is substituted by a C=O group.
The letter ‘R’ denotes where each fatty acid chain attaches to the side group on the teicoplanin molecule. Its structure contains the identical ABCD ring system that is also found in vancomycin, another antibiotic within the same family. In addition to a small modification in the DE ring system, teicoplanin lacks the β-hydroxyl group, a site of sensitivity in vancomycin, and it also has an additional FG ring system that is not present in vancomycin. It is these subtle yet crucial differences that will prove to be a major differentiating factor between the two drugs.
Teicoplanin versus vancomycin
In vitro binding studies revealed that teicoplanin interferes with the final stage of peptidoglycan synthesis (glycan polymerization and cross-linking) by binding to the terminal amino N-acyl-D-alanyl–D-alanine of the growing peptidoglycan or its precursors [2]. This N-acyl-D-alanyl–D-alanine group binds and forms a stoichiometric complex via the formation of five hydrogen bonds between the peptide backbone of the glycopeptide and the D-Ala–D-Ala dipeptide. The formation of this complex prevents any further peptidoglycan synthesis by sterically hindering the correct alignment of transglycosylase and its substrate. The antibiotic activity and potency of teicoplanin, particularly against Streptococcus and Enterococcus species, are also well documented [3]. Despite the increasing frequency of glycopeptide resistance, teicoplanin has always shown clinical worth, especially in treatment of life-threatening sepsis.
Vancomycin, a similar antibiotic in the same glycopeptide family, has historically been the drug of choice for the treatment of diseases caused by methicillin-resistant Staphylococcus aureus (MRSA). The emergence of MRSA strains in the community setting can cause infections ranging from cellulitis with skin abscesses to pneumonia in otherwise healthy individuals. Although vancomycin hydrochloride has been the accepted standard therapy for MRSA infections, its potential nephrotoxicity is one of the major limitations for its routine use. Some studies have found an increased risk of renal failure following vancomycin treatment [4], because of its effects on proximal tubular cells where the antibiotic can accumulate inside lysosomes.
Teicoplanin has been shown to have essentially the same efficacy as vancomycin but with some advantages, such as once-daily bolus administration for dose management. In addition, the pharmacokinetic profile of teicoplanin also offers the patient a choice of administration routes of either intramuscular or intravascular delivery. Several side effects of vancomycin treatment are known, such as ‘red man’ syndrome, a combination of erythema, pruritus, and hypotension, which can be an immediate adverse event of vancomycin infusion. The likelihood of this reaction is greatly reduced with teicoplanin [5]. Perhaps the most widely recognized clinical problem with the vancomycin regime is nephrotoxicity in the patient. Meta-analysis of comparative trials have demonstrated that adverse events were less likely to occur with teicoplanin (13.9%) compared with vancomycin (21.9%) (P=0.0003). This was particularly significant when nephrotoxicity was considered: 4.8% versus 10.7% (P=0.0005) for teicoplanin and vancomycin, respectively [6]. This is of added relevance for patients with renal failure, as evidence suggests dosage adjustment is of paramount importance because of their impaired renal clearance.
A number of studies have been carried out comparing the efficacy and safety of these two drugs, showing the potential for teicoplanin as a suitable alternative to vancomycin [7]. This systematic review and meta-analysis of 24 randomized controlled trials in 2009 highlighted the advantages of teicoplanin over vancomycin. More importantly, total adverse events were found to be less frequent, and nephrotoxicity to be lower for teicoplanin usage, especially when administered in combination with aminoglycosides. The safety profile of teicoplanin has also been shown to be favourable, where severe skin reactions can result due to vancomycin-related infusion and the subsequent release of histamine [8]. Comparison studies with other drugs also used for bacterial infection treatments such as clindamycin, rifampicin, netilmicin, and enoxacin provide further support for the pharmacological profile of teicoplanin [9].
There is evidence of disadvantages in teicoplanin administration, such as hypersensitivity manifesting as fever and chills. Some patients with a positive reaction to teicoplanin can also tolerate vancomycin, where no major advantage lies with either drug. Thrombocytopenia can also develop, although this occurred almost exclusively in patients receiving much larger teicoplanin doses than are now recommended. The majority of the available evidence favours the bacteriolytic effect of teicoplanin [10], where base concentrations of just 1 µg/mL can cause rapid lysis of streptococcal cells in exponential phase. Due to increased bacterial resistance to vancomycin through the substitution of the D-Ala–D-Ala terminus of the peptidoglycan precursor by D-Ala–D-Lac, teicoplanin does seem to hold several major advantages. In addition to the similar and competitive cost of treatment, teicoplanin is emerging as a suitable and appropriate alternative to vancomycin for Staphylococcus infections.
Therapeutic drug monitoring
Therapeutic drug monitoring (TDM) plays an important role in the optimization of drug therapy, especially for drugs with narrow therapeutic ranges. Since the introduction of home therapy and the use of teicoplanin in the community, clinicians have paid close attention to serum concentrations of teicoplanin in their administration regimen, as multifactorial education programmes with active TDM have been shown to be efficacious in maintaining the appropriate use of antibiotics in various healthcare settings [11].
Predominately (>90%) bound to plasma proteins, teicoplanin is mostly cleared renally and only 2–3% of an intravenously administered dose is metabolized. Total teicoplanin clearance rate has been reported to be 11 mL/h/kg, and steady state is achieved slowly – 93% after 14 days of repeated administration [12]. It is believed an optimal loading dose followed by appropriate maintenance doses should achieve the teicoplanin trough serum concentration of 25 µg/mL rapidly and steadily, increasing the chances of full recovery for the patient. Care should be taken when teicoplanin is substituted for vancomycin, as it has a longer half-life: about 40 hours compared to that of 3–5 hours for vancomycin [13]. Because of its longer half-life, teicoplanin only needs to be administered once daily. Administration should be tailored to the individual, as each patient has varying degrees of hepatic and renal function: age, infection, and concomitant use of certain drugs are a few factors that can alter the rate of clearance. Nevertheless, an 800 mg initial dose followed by 400 mg maintenance doses for the following 48 hours has been accepted for safely achieving an optimal teicoplanin trough level. Re-assessment would then take place to determine if additional maintenance doses are needed, and, if so, what the course of action would be. It is important to ascertain the ideal pre-dose serum concentrations, as it can be a predictor of outcome for the patient. However, the rate of success with the treatment declines with age, again highlighting the need to consider other factors pertaining to the individual patient.
Measurement of teicoplanin: a brief history
The solid-phase enzyme receptor assay (SPERA) was most widely used for TDM initially, capable of measuring glycopeptides of the vancomycin family. The assay was based around the interaction of teicoplanin and acyl-D-alanyl–D-alanine, where the antibiotic of interest and enzyme-labelled teicoplanin compete for a synthetic analogue of the biological receptor, albumin-E-aminocaproyl-D-alanyl–D-alanine. Using this method for teicoplanin determination in human serum the intra-assay CV (coefficient of variation) was reported to be 7.2%, inter-assay CV to be 11.2%, and the analytical recovery to be 94% [14]. SPERA correlated well with other available microbiological assays at the time, and it was accepted as a good tool for identification and quantitative detection of teicoplanin.
The disk diffusion (or Kirby Bauer) test used Petri dishes containing the antimicrobial agent covering the surface of the agar. The size of the area free of the microbe being tested was proportional to the effectiveness of the antibiotic. Similarly, the agar incorporation method used viable colonies from overnight cultures to inoculate Sensitivity Test Agar plates containing doubling teicoplanin concentrations. The lowest concentration that inhibited growth would then be assigned the MIC (minimum inhibitory concentration) value. These were more of a qualitative rather than a quantitative assay, and its clinical usage was fairly limited. There were also commercially available kits, such as the high-inoculum Etest method (AB Biodisk, Solna, Sweden) that detects intermediate glycopeptide susceptibility. Etest strips covered with teicoplanin are exposed to infusion agar and incubated for 48 hours at 37°C, and results read at the point of complete growth inhibition. A VITEK assay from bioMerieux (Marcy l’Etoile, France) also used overnight agar plate cultures to measure teicoplanin effect and determined its MIC. This latter method could be automated for the clinical laboratory; however, it still lacked the precision one expects from an assay used for TDM in MRSA resistance.
Increasing popularity of immunoassays paved the way for the implementation of fluorescence polarization immunoassay (FPIA). Based on the principle of competitive binding and the reagent limited concept, the TEICOPLANIN Assay System (Abbot TDx) uses fluorescein-labelled antigen to compete with sample antigen for a fixed number of antibody binding sites. In recent years there has been an emergence of high-performance liquid chromatography (HPLC) as an alternative to FPIA, a method that has several advantages over the traditional immunoassay. The improvements lie within the various components that constitute an HPLC system, since the characteristics of various compounds in a sample matrix can be very different (Fig. 2). Tandem mass spectrometry assays have also recently been developed to measure teicoplanin in patient serum [15], providing the most sensitive method yet for monitoring this therapeutic drug.
Conclusion
As teicoplanin administration gains popularity with clinicians dealing with TDM, there lies a growing need for close assessment of its serum levels. In addition to clinical outcomes, accurate measurement of teicoplanin allows the administration of a definitive loading dose and appropriate maintenance doses to ensure the rapid recovery of patients and improved quality of life. A large number of assays are now available in clinical biochemistry laboratories, and end users are encouraged to adopt these methodologies.
References
1. Parenti F, Beretta G, Berti M, Arioli V. Teichomycins, new antibiotics from Actinoplanes teichomyceticus Nov. Sp. I. Description of the producer strain, fermentation studies and biological properties. J Antibiot. (Tokyo) 1978; 31: 276–283.
2. Barna JC, Williams DH. The structure and mode of action of glycopeptide antibiotics of the vancomycin group. Annu Rev Microbiol. 1984; 38: 339–357.
3. Parenti F, Schito GC, Courvalin P. Teicoplanin Chemistry and Microbiology. J Chemother. 2000; 12(Suppl 5): 5–14.
4. Hidayat LK, Hsu DI, Quist R, Shriner KA, Wong-Beringer A. High-dose vancomycin therapy for methicillin-resistant Staphylococcus aureus infections: efficacy and toxicity. Arch Intern Med. 2006; 166: 2138–2144.
5. Wilson AP. Comparative safety of teicoplanin and vancomycin. Int J Antimicrob Agents 1998; 10: 143–152.
6. Wood MJ. Comparative safety of teicoplanin and vancomycin. J Chemother. 2000; 12(Suppl 5): 21–25.
7. Svetitsky S, Leibovici L, Paul M. Comparative efficacy and safety of vancomycin versus teicoplanin: systematic review and meta-analysis. Antimicrob Agents Chemother. 2009; 53: 4069–4079.
8. Davey PG, Williams AH. A review of the safety profile of teicoplanin. J Antimicrob Chemother. 1991; 27(Suppl B): 69–73.
9. Covelli I, Nani E. Microbiological profile of teicoplanin. J Chemother. 1991; 3(Suppl 1): 39–42.
10. Chmara H, Ripa S, Mignini F, Borowski E. Bacteriolytic effect of teicoplanin. J Gen Microbiol. 1991; 137: 913–919.
11. Pea F, Viale P, Pavan F, Tavio M, et al. The effect of multifactorial, multidisciplinary educational interventions on appropriate use of teicoplanin. Int J Antimicrob Agents 2006; 27: 344–350.
12. Wilson AP. Clinical pharmacokinetics of teicoplanin. Clin Pharmacokinet. 2000; 39: 167–183.
13. Boger DL. Vancomycin, teicoplanin, and ramoplanin: synthetic and mechanistic studies. Med Res Rev. 2001; 21: 356–381.
14. Corti A, Rurali C, Borghi A, Cassani G. Solid-phase enzyme-receptor assay (SPERA): a competitive-binding assay for glycopeptide antibiotics of the vancomycin class. Clin Chem. 1985; 31: 1606–1610.
15. Fung FH, Tang JC, Hopkins JP, Dutton JJ, et al. Measurement of teicoplanin by liquid chromatography-tandem mass spectrometry: development of a novel method. Ann Clin Biochem. 2012; 49(Pt 5): 475–481.
The author
Francis Fung PhD
Department of Clinical Biochemistry, Royal Liverpool University Hospitals, Liverpool, UK
E-mail: francis.fung@nhs.net
Testing for gastrointestinal cytomegalovirus infection
, /in Featured Articles, Gastrointestinal Disorders /by 3wmediaby Dr Jingmei Lin and Dr Rong Fan Cytomegalovirus infection in the gastrointestinal tract likely occurs in periods of immunosuppression. The diagnosis of cytomegalovirus infection in the gastrointestinal tract can be made in several ways including serology, testing for viremia, viral culture from biopsy material, histopathologic examination coupled with immunohistochemistry, and molecular method […]
Improving the diagnosis of pulmonary tuberculosis in hard-to-reach patients in London, UK
, /in Featured Articles /by 3wmediaDeveloped countries have not escaped the rise in cases of multidrug-resistant tuberculosis. In the UK a mobile X-ray unit has been operating in London since 2005 and this service has been augmented with point-of-care testing (POCT) since 2011. POCT has been well received by patients and has greatly reduced the number of unnecessary hospital visits.
by Dr R. J. Shorten and Dr A. Story
Background
Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis. TB is primarily a respiratory disease, although it can affect any part of the body and it is spread from person to person via expectorated droplets by individuals with active pulmonary disease. TB is a significant international problem and in 1993 the World Health Organization declared tuberculosis a global emergency [1]. In 2012 there were 8.6 million new cases and 1.3 million deaths, the vast majority of these occurring in Asia and sub-Saharan Africa [2].
Multidrug-resistant TB
This global epidemic is further complicated by the increase in drug resistant M. tuberculosis. Multidrug-resistant TB (MDR-TB) is a form of TB caused by bacteria that do not respond to, at least, isoniazid and rifampicin, the two most effective, first-line anti-TB drugs. There were approximately 450 000 cases of MDR-TB globally in 2012. More than half of these cases were in India, China and the Russian Federation [2].
This epidemic has not been avoided in developed countries and following a century of decline, the incidence of TB in the UK has been rising since 1988. Data from Public Health England [3] shows that the majority of these cases are concentrated in urban areas, with almost 40% being in London (3426 cases in 2012). Additionally, one or more of the following risk factors were present in 7.3% of cases; history of problem drug use, alcohol misuse/abuse, homelessness or imprisonment. Furthermore, some of these individual risk factors are associated with smear positivity (those patients who are more likely to be contagious), drug resistance, poor adherence to treatment and loss to follow up [4]. Indeed, the growing problem of TB in these hard-to-reach groups has led to specific UK guidance [5].
Searching for patients
Find & Treat was established in 2005 to strengthen TB control in the socially excluded communities of London by working with over 200 partner organizations. The service has been evaluated as an innovative and cost-effective health and social care model [6]. The mobile X-ray unit (MXU) has been operating in London since this time and screens approximately 8000 patients per year (Fig. 1). It is staffed by TB nurse specialists, reporting radiographers, social workers and outreach workers and former TB patients with a lived experience of homelessness who work are Peer Educators. Its role is to identify hard-to-reach patients with suspected TB using digital chest radiographs. These patients are then linked into local healthcare provision via Accident & Emergency (A&E) departments or community TB programmes. In December 2011 the service was augmented with the Xpert MTB/RIF assay (Cepheid, Sunnyvale, California, USA), which was used as a point-of-care test (POCT) for the molecular detection of M. tuberculosis in sputum [7].
Point-of-care testing
Prior to the implementation of the Xpert assay, if a digital chest radiograph indicated active pulmonary TB (seen in 1–2% of patients screened), then the patient underwent immediate referral to the nearest hospital with a TB service. This involved a TB nurse specialist or outreach worker accompanying the patient to an A&E department for assessment, conventional microbiological investigation and possible admission. Approximately 20% of referred patients are subsequently confirmed to have TB and are initiated on a complex course of multiple antibiotics over a period of at least 6 months [8].
The Xpert MTB/RIF assay is a nucleic acid amplification test (NAAT). The easy to use analyser extracts, amplifies and detects DNA of M. tuberculosis in the patients’ sputum. The assay also detects approximately 95% of the common rifampicin resistance-conferring mutations in the rpoB gene (a surrogate marker of multidrug resistant TB) [9].
The hands-on time of the assay is minimal and a specimen container with a rubber septum in the lid allows the staff on the MXU to safely process sputum samples without the need for containment facilities or a microbiological safety cabinet (Fig. 2). MXUs exist in other European cities, but we believe that this is the first example of a NAAT POCT being used in this way anywhere in Europe.
Patients with abnormal X-rays that indicate active pulmonary TB are asked whether they can produce sputum. Those who can, expectorate a sample into the specific sample container. They seal the container and return it to the team on the MXU who assess the sputum for quality (sputum, rather than watery saliva is required). The sample reagent is carefully added through the rubber septum in the lid and swirled gently to allow homogenization of the sample. This process ensures that minimal aerosols are generated and that they are not released to cause exposure to the operator. The assay takes approximately 2 hours in total and the result will determine whether the patient requires immediate referral to a TB service. Patients with negative NAAT are followed up by two further sputum samples, including one early morning specimen, collected in the community and processed for routing smear microscopy and culture in hospital laboratories. Due to the high negative predictive value of the assay (94%) and the increased sensitivity compared to sputum microscopy [10], it is highly unlikely that any infectious cases will go undetected using this algorithm.
Considerations and advantages of POCT for TB
As the POCT is performed by non-laboratory staff, it is imperative that the MXU staff are comprehensively trained and assessed for competence. None of the staff had any laboratory or analytical experience prior to the implementation of this assay. Registered clinical laboratory staff are involved in all stages of implementation and training. A close working relationship between the MXU team and the clinical laboratory is required to ensure appropriate refresher training and review of standard operating procedures and risk assessments.
The assay has been well received by patients, particularly as a negative result prevents a referral to hospital. This allows MXU staff to focus resources on screening more patients, rather than accompanying patients to hospital who subsequently are confirmed to be clear of TB. The MXU regularly screens in custodial settings where capacity to effectively isolate suspected cases is limited and significant resources are required accompanying patients with suspected TB to hospital appointments for further investigations. The value of the assay in determining which patients are potentially infectious is especially useful in these settings. The ability of the Xpert MTB/RIF to detect most cases of rifampicin resistant (and therefore likely to be MDR) TB, allows the MXU team to initiate appropriate second-line therapy more rapidly. Additionally, the simplicity and safe use of the assay has been well adopted by the MXU staff. We have demonstrated the potential of this technology in focussing resources on the most appropriate individuals and therefore improving the quality of care in this vulnerable group of patients. A randomized controlled trial to assess the benefit of using this assay on patients with an abnormal chest X-ray against them being referred to secondary care is underway to accurately quantify the benefits of this assay in tackling TB in this group of patients.
Summary
The epidemiology of TB in 21st century big cities is characterized by a concentration of disease in hard-to-reach medically underserved populations. Capacity to outreach effective diagnostic platforms directly to high risk populations is likely to become an increasingly important feature of TB control [11].
References
1. World Health Organization (WHO). Tuberculosis. Fact sheet 104, 2012. 2. WHO. Tuberculosis. Media centre; Fact sheet 104, 2014.
3. Tuberculosis in the UK: Annual report on tuberculosis surveillance in the UK, 2013. London: Public Health England 2013.
4. Story A, Murad S, Roberts W, et al. Tuberculosis in London: the importance of homelessness, problem drug use and prison Thorax 2007; 62(8): 667–671.
5. National Institute for Health and Clinical Excellence (NICE): Public health guidance. Tuberculosis – hard-to-reach groups. PH37 2012.
6. Jit M, Stagg HR, Aldridge RW, et al. Dedicated outreach service for hard to reach patients with tuberculosis in London: observational study and economic evaluation. BMJ 2011; 343: d5376.
7. Boehme CC, Nabeta P, Hillemann D, et al. Rapid molecular detection of tuberculosis and rifampin resistance. N Engl J Med. 2010; 363: 1005–1015
8. NICE: Clinical guidelines. Tuberculosis: Clinical diagnosis and management of tuberculosis, and measures for its prevention and control. CG117 2011. (http://guidance.nice.org.uk/CG117)
9. Zhang Y, Yew WW. Mechanisms of drug resistance in Mycobacterium tuberculosis. Int Tuberc Lung Dis. 2009; 13(11): 1320–1330.
10. O’Grady et al. Evaluation of the Xpert MTB/RIF assay at a tertiary care referral hospital in a setting where tuberculosis and HIV infection are highly endemic. J Clin Infect Dis. 2012; 55(9): 1171–1178.
11. van Hest NA, et al. Tuberculosis control in big cities and urban risk groups in the European Union: a consensus statement. Euro Surveill. 2014;19(9): Article 7.
The authors
Rob J. Shorten1* BSc, MSc, PhD and A Story2 RGN MPH PhD
1Clinical Scientist, Department of Medical Microbiology, Central Manchester Foundation Trust and Honorary Research Associate, University College London Centre for Clinical Microbiology, UK
2Clinical Lead Find & Treat, University College Hospitals NHS Foundation Trust, London, UK
*Corresponding author
E-mail: rob.shorten@nhs.net
QC considerations for point-of-care testing
, /in Featured Articles /by 3wmediaPatient testing is increasingly being carried out at the bedside. Indeed the availability of point-of-care testing (POCT) instruments and devices has grown significantly in recent years. Undoubtedly POCT helps speed up the availability of results, but when such testing is moved outside of the laboratory setting how can accuracy be ensured? `
by Sarah Kee
In all patient testing, Quality Control (QC) exists to ensure accuracy and reliability. For many of the health care workers using POCT instruments, QC will be unfamiliar territory. Many of the standard QC procedures applied in laboratories cannot be applied to POCT devices. However, it is essential that both primary and community care settings apply well-structured QC procedures to ensure the accuracy and reliability of results, minimizing risk to patients and improving patient outcomes.
Designing a QC strategy for POCT
When designing a QC strategy for POCT devices there are three main issues that need to be considered:
1. Design of the devices
POCT devices can be broadly split into three categories with procedures varying according to instrument design:
For laboratory type instruments the design mirrors that of analysers found within the laboratory environment. Therefore, QC procedures should mirror those found within the laboratory: if patient tests are performed every day, then multi-level QC samples should also be run every day. The accuracy and reliability of these results should be monitored over time to provide a true reflection of performance. External Quality Assessment should be run in conjunction with Internal Quality Control (IQC).
For cartridge-based instruments, the technology differs from that found in standard laboratory type analysers and therefore to reflect this, QC should be performed differently.
Cartridge-based devices usually consist of a cartridge-based component and an electronic reader based component. The cartridge-based component contains all the necessary “ingredients” for the analysis of the patient sample while the electronic reader component is responsible for converting the result from the cartridge component into a numerical value. QC can be problematic as the QC technician is only ever testing the one particular disposable cartridge and the electronic function of the analyser that is in use at that specific time. Nevertheless, performing QC for these devices is still essential to ensuring the cartridge is performing correctly as damage may have occurred during transport, or the on-board reagents may have deteriorated. As a minimum, QC should be run when changing cartridge lot and periodically throughout the lot’s lifespan to ensure the stability of the on-board components. To fully ensure the instrument’s accuracy over time, technicians should also participate in an EQA scheme.
Strip-based instruments are very similar in design to cartridge-based instruments. Like cartridges, the strips are responsible for analysis of the sample, however, as the electronic component has no QC self-check feature, a faulty analyser could be producing erroneous results which remain undetected for some time. Because of this, QC processes should be more stringent for strip-based devices than cartridge-based devices. Strips should be checked on delivery using multi-level QC to ensure they have not been damaged during transit, as well as every day of patient testing. It is also important to participate in a frequent EQA scheme.
2. Possible risks to the patient
When implementing QC for POCT devices the risk of harm to the patient should be the foundation of the QC strategy: where and why do errors occur and what are the consequences of an erroneous result to the patient?
The QC strategy should balance the risk of harm to the patient with the stringency of the QC procedure applied. Studies have shown that the most common phase for errors in POCT is analytical, with 65.3% of errors occurring during this phase. In contrast, the analytical phase in laboratory testing is the least common source for errors. This highlights the need for QC procedures for POCT devices as the potential risk of harm to the patient is greater for POCT than laboratory-based tests.
As many POCT are used by non-laboratory professionals it is vital that users are trained to undertake QC – without QC results may not be accurate. Inaccurate results could have serious implications for the treatment the patient receives.
3. Who is responsible for QC in POCT?
According to ISO 22870:2006, a POCT management group should be set up with responsibility for managing and training staff using the equipment. This group should be responsible for the quality management strategy and implementing a programme of staff training, to include quality control, for all personnel performing POCT and interpreting results.
The running of QC samples on POCT devices should be performed by those who are using the devices regularly, as QC samples should be run as a patient sample would be and therefore must be performed by personnel who are responsible for patient testing.
IQC and EQA/PT – making appropriate choices for POCT Devices
Internal Quality Control (IQC) involves running samples containing analytes of known concentration, to monitor the accuracy and precision of the analytical process over time. Depending on the design of the device and risk of harm to the patient the IQC strategy will differ accordingly. When choosing appropriate IQC material for performing QC on POCT devices, it is important to look for material that offers the following benefits:
Interlaboratory data management software is available that will allow a laboratory to manage and interpret their QC data. This software is an extremely cost effective and efficient way to ensure that a laboratory’s POCT devices are performing at a high standard, allowing accuracy and precision to be monitored over time, thus providing a true reflection of performance of both the device and the personnel using the device.
This software is usually available with a peer group comparison functionality that will allow a laboratory to directly compare the results of their POCT devices to other laboratories using the same devices worldwide.
Proficiency Testing (external quality assessment) is strongly recommended for all point of care devices. ISO 22870:2006 states, “There shall be participation in external quality assessment schemes”. An EQA scheme assesses the accuracy of the POCT devices through direct results comparison of one device to identical devices worldwide. This peer comparison allows a laboratory to assess the accuracy of a device over time and provides confidence that the patient results being reported are accurate.
There are many PT schemes available for POCT devices; when choosing a scheme it is important that a laboratory considers the following:
The benefits of POCT are undisputed but only if we are assured of accurate results. Getting the right QC strategy in place now will ensure the contribution POCT makes to patient testing is a wholly positive one.
The author
Sarah Kee, BSc PGCE
QC Scientific Consultant
Randox Laboratories Ltd
E-mail: sarah.kee@randox.com
www.randox.com
Chronic kidney disease: an ‘invisible epidemic’ ?
, /in Featured Articles /by 3wmediaA growing challenge for nephrologists is to quickly identify patients with chronic kidney disease (CKD). Experts believe there is now “coherent, undisputable evidence” that timely treatment of CKD can delay the onset of end-stage renal failure (ESRF).
The challenge of timely diagnosis
Several studies suggest that early identification and management of CKD can reduce the risk of kidney failure progression and halve co-morbidities such as cardiovascular disease. The latter is a significant achievement on its own, since cardiovascular complications are estimated to increase mortality in CKD patients by 2-4 times over that of the general population.
Nevertheless, early diagnosis of CKD has not proven to be easy. Chronic kidney disease tends to develop slowly, with few evident symptoms. As a result, most patients seek clinical expertise only after the disease has advanced to a stage where dialysis becomes unavoidable.
Epidemiological data limited
Confounding the problem is a lack of epidemiological data. In the US, the Centers for Disease Control (CDC) can only estimate that “more than 20 million people may have CKD.”
The picture is even less encouraging in Europe. In a first-of-its-kind effort in 2010 to explore what they labelled a “silent epidemic,” Dutch and Italian researchers were “struck by the fact that epidemiological data for CKD existed only in a tiny minority of European countries.” More puzzling was official data from the European Commission or the bulk of EU Member States, which “did not even mention CKD as an issue of public health concern.”
Attempts to establish aggregated CKD data in Europe have led to mixed results. The ambitious EUGLOREH (Status of Health in the European Union) survey established prevalence rates of (higher risk) Stages 3-5 CKD ranging from 3.6% in Norway to 7.2% in Germany, but little about earlier Stages (1 and 2) where treatment best delays progression.
Prevalence of Stages 3-5 CKD was similar for males and females in Germany and Italy, but higher for females in other countries – Belgium, England, Iceland and Norway. In countries for which data was available, prevalence also rose with age.
However, EUGLOREH underlined the total lack of time-trend epidemiological data in Europe, unlike the US where the so-called NHANES III and IV surveys showed prevalence of Stages 1-5 CKD rising from 14.5% in 1988-94 to 16.8% in 1999-2004, while that of Stages 3-5 remained near unchanged, at about 6%.
Delaying dialysis
Recent clinical practices, which advise delaying the onset of dialysis as far as possible, are driving the need for early identification of CKD. In 2003, ‘Kidney International’, the Official Journal of the International Society of Nephrology, stated that “dialysis delayed is death prevented.”
The authors noted that hundreds of thousands of renal failure patients lived in developing countries with inadequate resources or infrastructure; however, many Western patients too were unsuitable for dialysis, “because of age, frailty, or a co-morbid illness with poor prognostic outcome.”
Costs of CKD
The financial impact of CKD is high. In the US, the cost of CKD and its co-morbidities in the pre-ESRF phase are estimated to be as much as $26,000 per patient each year.
Delaying the onset of ESRF provides significant, additional benefits. For example, Italy spends 1.8% of its total health care budget on ESRF patients, who represent only 0.083% of the general population. In the UK, ESRF accounts for 2% of health spending.
The situation in the US is dramatic, with kidney treatment accounting for 24% of Medicare spending, higher than congestive heart failure.
Testing for CKD
Typically, detection and monitoring of CKD requires both urine and blood tests in the laboratory. The former typically focus on kidney function and the latter on damage, according to the US National Institutes of Health (NIH). Key markers for CKD are abnormal urinary levels of albumin (albuminuria) and other proteins (proteinuria), along with a persistent reduction in the glomerular filtration rate (GFR). Variations in urine concentration are corrected by urine albumin-to-creatinine ratio (ACR) or protein-to-creatinine ratio (PCR) on a spot specimen. However, there is considerable debate about the clinical and cost-effectiveness evidence of ACR versus PCR.
So far, CKD testing has largely been driven by diabetes patients, with best practices based on care standards from professional bodies such as the US National Kidney Foundation, the American Diabetes Association and the National Institute for Health and Care Excellence (NICE) in the UK. At-risk groups considered to be typical targets for screening consist of patients with hypertension, cardiovascular disease or a family history of kidney failure. In the US, the CDC estimates that about “1 of 3 adults with diabetes and 1 of 5 adults with high blood pressure has CKD.”
Limitations to screening
With some exceptions, testing urine is not recommended in the general population. In the US, the NIH explains that the “benefit of CKD screening in the general population is unclear.”
The reasons for this restraint are also practical. Albumin levels fluctuate over the day due to metabolism and diet. As a result, urine samples are collected on a random, 4-hourly, or overnight basis. If abnormalities are indicated, re-confirmation is required via a timed, 24-hour collection cycle, accompanied by the labelling and refrigerated storage of the samples. All this clearly poses logistical challenges for large scale CKD screening.
Secondly, any mass screening would clearly use colour-changing dipsticks, the first point of call for urine tests. Though the National Kidney Foundation considers dipsticks satisfactory for a first screening, it warns clinicians to be especially cognizant of false negative results, and specifies the need for lab analyses to quantify urine PCR and ACR ratios. Indeed, while ACR of over 30 mg/g is considered to indicate CKD, the sensitivity level of dipsticks begins at 300 mg/g.
The only alternative to urine is a blood sample, but this too has evident limitations as a mass screening tool for CKD.
Expanding screening: recent developments
Nevertheless, there is growing evidence that larger-scale screening for CKD is likely in the future.
In 2005, Dutch researchers sought to address the challenge of a 24-hour urine collection cycle. They reported that a level of about 11 mg/L urinary albumin concentration (UAC) in a single spot morning urine sample could identify microalbuminuria, and do this as effectively as ACR. They suggested the above as a cut-off point for requiring subsequent urine sample collections, to avoid “huge numbers of individuals” having to undergo “the cumbersome procedure of a 24-hour urine collection” in any mass screening program.
In 2008, the UK’s NICE formally enlarged the scope of testing. It recommended that apart from diabetes, screening for CKD be undertaken in patients with hypertension, cardiovascular diseases, structural renal tract disease, renal calculi or prostatic hypertrophy and multisystem diseases such as systemic lupus erythematosus. The list extended to people with a family history of Stage 5 CKD or hereditary kidney disease.
In its guidelines, NICE also attempted to come to grips with vexing questions on CKD markers such as albuminuria and proteinuria, and the selection and sequencing of tests. The Institute however left open room for further interpretation. Its consensus recommendation was that “ACR should be the test of choice” , but also said there would “often be good clinical reasons for subsequently using PCR to quantify and monitor” proteinuria.
In 2012, the Australasian Proteinuria Consensus Working Group took what may be one of the biggest steps so far to scale up screening. Based on a retrospective longitudinal cohort study of 5,586 CKD patients, it extended the UK NICE screening envelope to include obesity and smoking, and “strongly” advocated targeted opportunistic testing for CKD risk in “all adults attending an appointment with their health care practitioner”. The Working Group also concluded that ACR from a morning (or random) spot urine sample was as good as PCR and 24-hour urinary albumin and protein measurements, and recommended testing for albuminuria rather than proteinuria in individuals at risk of CKD.
The group advised “all pathology laboratories in Australia” to implement its recommendations as part of an “integrated national approach to CKD detection.”
The UK: A ‘World Leader’ in early detection of CKD
Although the Australian recommendations appear to be much more explicit than the NICE guidelines, the UK has ensured that CKD is incorporated into its wider Quality and Outcomes Framework (QOF). As a result, GPs are now rewarded based on how well they identify and manage patients with CKD. CKD is given 27 points in QOF, alongside 9 points for diabetes related to kidney disease. This is considered to provide significant support for the Renal National Service Framework (NSF), which aims to minimize the impact of kidney disease in its early stages.
Indeed, the EUGLOREH report mentioned previously considers the CKD-directed steps in the Quality and Outcomes Framework to have made the UK “a world leader in this field.”
Telediagnosis may offer breakthrough in mass CKD screening
Meanwhile, a breakthrough in the US could remove the final barriers to a true mass screening program for CKD.
In August 2013, researchers from the University of California at Los Angeles (UCLA) announced they had developed a compact, field-portable device to conduct albumin tests and transmit data via smartphone, thus reducing the need for “frequent office visits by people with diabetes and others with chronic kidney ailments” or the use of “bulky and costly benchtop urine analysers” which limit testing and diagnosis to laboratory settings. The new system projects beams of visible light through two attached fluorescent tubes, one of which contains a control liquid and the other a urine sample mixed with fluorescent dyes. The smartphone camera captures the fluorescent light after it passes through an additional lens.
The device measures albumin concentration to less than 10 micrograms per millilitre, which its inventor states is “more than 3 times lower than the clinically accepted normal range.” It weighs less than 1 kg and is estimated to cost $50 to $100, with tests taking about 5 minutes.
Measuring renal function: traditional methods and iohexol clearance
, /in Featured Articles /by 3wmediaAn accurate measure of glomerular filtration rate (GFR) is required in a variety of clinical situations. Plasma creatinine and creatinine-based equations have served as convenient estimates of GFR but they are subject to limitations. Formal assessment of GFR using clearance methods traditionally necessitates the use of radioactive traces such as 51Cr-EDTA. More recently, iohexol has become increasingly well recognized as an alternative exogenous marker which can now be used routinely, providing a safe, efficient and cost-effective measure.
by Zoe Maunsell and Prof. Tim James
Background
Glomerular filtration rate (GFR) describes the amount of fluid filtered by the glomerulus per unit time, and is therefore a useful indicator of renal function. Expressed in mL/min, it can be accurately measured from the rate of disappearance of an injected substance from the plasma or by collecting urine over a defined time period. The clearance formula, UV/P (where U represents urine concentration, V is urine volume per unit time and P is plasma concentration) can be used to calculate GFR when an ideal marker is used. Such a marker should be freely filtered at the glomerulus, and not secreted, absorbed or metabolized by the renal tubules. Since renal function is proportional to kidney size (which is proportional to body surface area), when estimating GFR, values are usually standardized to an average body surface area of 1.73 m2. Having an accurate yet convenient means to monitor renal function is extremely important in clinical practice. A compromise exists between highly accurate but time consuming, technically difficult reference methods and more accessible, readily available markers of renal function.
Gold standard methods
The ‘gold standard’ for the measurement of GFR is inulin clearance. Inulin is a polysaccharide derived from plants, which can be introduced into the body either by intravenous infusion or bolus dose and its rate of clearance measured. Since inulin is freely filtered by the glomerulus, is not absorbed, secreted or metabolized by the tubules, its elimination is proportional to GFR. Although it is the ‘gold standard’ for assessment of GFR, assay of inulin is technically demanding, and so this technique is not suitable for routine clinical practice.
Similarly, the radioactive tracer 51Cr-EDTA can be administered intravenously and its elimination rate monitored. This marker is widely used to measure GFR, but presents a risk to patients and healthcare workers since it involves exposure to ionizing radiation.
Iohexol clearance is an alternative to inulin clearance [1]. Iohexol is an iodine-containing, non-isotopic contrast agent, and studies have shown close agreement between GFR values obtained by iohexol and inulin clearance.
Creatinine and endogenous markers
Creatinine is a widely used clearance marker but suffers from well-described problems including the difficulty and inconvenience of 24-hour urine collection.
GFR has therefore been estimated without urine collection using endogenous markers, the most widely used of which is plasma/serum creatinine. Improvements have been made to the methodology used for creatinine measurement, including standardization of kinetic Jaffe creatinine assays, defining their traceability to isotope-dilution reference assays. However, the Jaffe method remains subject to numerous interferences and therefore laboratories may select the more specific enzymatic creatinine methods. Using creatinine as a marker of GFR has several limitations, including the relationship of creatinine concentration to muscle mass, meaning that estimation of GFR using creatinine alone is particularly a problem in children. It is for this reason that corrections for body surface area have been made. The major limitation of creatinine alone as a marker of GFR is that it is a relatively insensitive marker, and a large decline in renal function can occur before a change in plasma creatinine concentration is observed.
Cystatin C is another endogenous marker that can be used for the estimation of GFR. It is a cysteine protease inhibitor, is produced at a constant rate (independent of muscle mass, sex, age when older than 12 months and inflammatory conditions) and is freely filtered by the kidneys. It is almost completely reabsorbed by the proximal renal tubular cells so that little is normally excreted in the urine. The reciprocal of plasma cystatin C concentration has been shown to be correlated with GFR. Cystatin C has significant advantages over creatinine as a marker of renal function, since increases in serum concentration become apparent with mild renal impairment, such as GFR of 60–90 mL/min, and may be more useful than creatinine in detecting acute kidney injury. Like creatinine, the measurement of cystatin C suffers to some extent from problems with standardization, in particular with reported differences [2] in measured concentrations using turbidimetric versus nephelometric assays by different manufacturers, presumably due to the use of different antibody and detection systems. The availability of an international reference preparation (ERM–DA471/IFCC) is likely to lead to greater agreement between cystatin C methods.
Calculated estimates of GFR
In order to overcome the limitations of measuring creatinine and cystatin C concentrations alone, calculations have been devised. These involve corrections for body surface area, sex, age and ethnicity. Among the most well known are the 4- or 6-parameter MDRD (Modification of Diet in Renal Disease study), Cockroft–Gault (which estimates creatinine clearance) and CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) equations for adults and the Schwartz and the Counahan–Barratt prediction equations for children.
The Cockroft–Gault equation [3] provides an estimate of creatinine clearance. Devised in the 1970s, the equation was calculated by examining the mean of two 24-hour creatinine clearances from 249 adult men.
The 4-variable MDRD equation [4] (incorporating creatinine, age, sex and race) is the most widely used in the UK, being reported by laboratories in the form of eGFR, and forms the basis of the staging system for CKD. The formula was developed from the Modification of Diet in Renal Disease study, using data from 1628 patients. The patients had already been diagnosed with CKD, and the study aims were to slow the progression of CKD with a low protein diet and blood pressure control. However, significant limitations to the use of the equation exist, mainly connected with the use of creatinine as a marker of GFR. These include inaccuracies of the equation at extremes of body type such as malnourished or amputee patients and the unsuitability of the equation for use in pregnancy. The formula includes a correction factor for use in African American populations but its validity in other ethnic groups has not been established. The MDRD formula tends to underestimate function at normal GFR, therefore slight reductions in eGFR should not be over-interpreted and reporting eGFR >90mLs/min/1.73m2 is not recommended.
The CKD-EPI equation was developed in 2008 [5] and updated in 2012 [6]. The 2008 equation was developed using data from over 8000 patients from 10 studies. The 2012 versions used over 5000 patients from 13 studies. Importantly, both studies included patients with normal GFR, as well as those with CRF. It was found that the CKD-EPI equations performed better than the MDRD equation, particularly at higher levels of estimated GFR. The limitations of the studies include a limited number of elderly patients and those from ethnic groups other than black race.
In terms of estimation of GFR in children, the Schwartz [7] and Counahan–Barratt [8] equations were developed in the 1970s and have been widely used to estimate GFR from serum creatinine and height (length in infants). The Schwartz equation was modified in 2009 [ 9, 10]. Based on new studies of iohexol clearance, the original formula was found to overestimate GFR. It was postulated that this was in part due to the use of new, standardized creatinine assays. In 2009 the equation was updated, based on studies of 349 patients aged 1–16 years with mild to moderate chronic renal disease. The formula uses creatinine determined using an enzymatic method, urea, height and cystatin C.
Various modifications of these equations, using creatinine, cystatin C [11] or both [12] have been published and optimized in various patient cohorts. When using creatinine-based assays, it is important to know which creatinine assay is being used, since equations have been devised which include different coefficients depending on the methodology used.
Iohexol clearance: a gold standard method in routine use
Oxford University Hospitals NHS Trust has been routinely offering an iohexol clearance service for the measurement of GFR since June 2011. The service is widely used by the hospital’s pediatric service, and is used in a variety of clinical situations, including determining renal function in surgical patients and for chemotherapy dosing. Before the introduction of this service, GFR was assessed using 51Cr EDTA clearance. Although this provided an accurate measurement of GFR, a long waiting list and difficult patient preparation made this technique suboptimal for use in children. In addition, where GFR results are required before administration of chemotherapy agents, timely result availability is critical to prevent delayed treatment. With iohexol clearance we have been able to improve GFR turnaround time compared to 51Cr-EDTA clearance and audit of our first 2 years of service demonstrated that 89% of results were reported within 2 working days and 99% within 3 working days. Iohexol clearance also avoids the use of radioactive isotopes, reducing exposure for patients, carers and hospital staff. Therefore the service has several distinct advantages over conventional isotopic clearance methods (Table 1).
Iohexol clearance is measured according to a standardized protocol, as described previously [13]. In summary, the patient is cannulated, a baseline blood specimen is collected and a standard dose of Omnipaque containing 300 mg iodine/mL is administered through the cannula, (2 mL in patients weighing <40 kg, 5 mL in patients weighing >40 kg). Blood (1 mL) is collected into lithium heparin tubes at 120, 180 and 240 min after iohexol administration.
The iohexol concentration in each specimen is measured by a UPLC (ultra-high performance liquid chromatography) method involving precipitation of plasma samples with equal volumes of perchloric acid and injection of the supernatant onto a Waters 50 x 2.1 mm 1.8 μm reversed phase column with isocratic acetonitrile-based solvent elution. The assay uses a one-point (604 mg/L) calibration through zero. The assay is straighfoward, reliable and rapid; chromatography time is 6 min per specimen. The assay demonstrates excellent inter-assay CVs: 2.2%, 1.9% and 1.9% at 39, 163 and 322 mg/L respectively. The laboratory participates in external quality assessment through the Scandinavian EQUALIS scheme.
The two-point model is used to calculate iohexol clearance, according to the Brochner–Mortensen method [14]. The model assumes a one-compartment model where iohexol is cleared by first-order kinetics. The estimated clearance (Cl ) of the GFR marker is expressed as:
Cl= Q• b/c1 (ml/min)
Where Q = amount of injected marker, b = disappearance rate of marker (min−1), c1= intercept on the y-axis.
The clearance correction for non-immediate mixing of the tracer substance is expressed as:
Cl = 0.990778 x estimated Cl−0.001218• estimated Cl2
Alternatively, single point estimates of GFR at appropriate time points can be calculated using the Jacobsson model. This method can be used in cases where the two-point method cannot be used, for example when contamination has occurred due to poor flushing of lines between sample collection. Here:
GFR = ln(D/VCt) / (t/V + 0.0016)
Where V = volume of distribution in mL (V = 187 x weight in kg + 732), t = time of sampling in min, D = dose of iohexol administered, Ct = concentration of iohexol at time t.
Iohexol clearance is corrected for actual body surface area, using the Dubois and Dubois method, calculated as 0.007184 x (weight in kg)0.425 x (height in cm)0.72, to yield a measure of GFR in mL/min/1.73 m2.
Since initiating the iohexol GFR service in 2011, we have analysed over 400 sets of patient samples. The use of UPLC over HPLC allows for smaller sample volumes to be collected, making the technique particularly suited to pediatrics. Ongoing close liaison with clinicians and nursing staff has revealed that patients and their families have responded favourably to the new procedure and in particular prefer being able to remain on the Daycare Ward throughout the investigation, which is a child-friendly environment.
Iohexol clearance has the added benefit of being significantly less costly than 51Cr-EDTA clearance. The approximate cost is £80 compared to £250 for 51Cr-EDTA.
Conclusions
The measurement of glomerular filtration rate, GFR, is important in a number of clinical scenarios. Much work has been undertaken to develop methods of estimating GFR in order to avoid the time-consuming and relatively invasive formal measurement of renal function using clearance methods. These include the development of eGFR equations based on factors such as plasma creatinine, cystatin C, age, race and body surface area. Equations such as the MDRD and CKD-EPI formulae for adults, and Schwartz and Counahan–Barratt equations for children have been widely used in clinical practice.
However, despite their widespread use, limitations of these equations have been described, including the problems of creatinine-based equations tending to underestimate GFR at normal GFR levels and the issue of creatinine-based formulae being unsuitable for use in patients with non-normal muscle mass.
For the formal measurement of GFR, clearance studies must be performed which have traditionally used radioactive tracers such as 51Chromium-EDTA. However, in recent years the use of iohexol has increased. Thanks to its stability in vitro and relative ease of measurement, the assay of iohexol is rapid, reliable and can be performed on small specimens. This fits in particularly well with the clinical needs of a pediatric service for GFR measurement and is cheaper, safer and more convenient than traditional methods.
References
1. Ng DK, Schwartz GJ, Jacobson LP, Palella FJ, Margolick JB, Warady BA, Furth SL, Muñoz A. Universal GFR determination based on two time points during plasma iohexol disappearance. Kidney Int. 2011; 80: 423–430.
2. Herget-Rosenthal S, Bökenkamp A, Hofmann W. How to estimate GFR-serum creatinine, serum cystatin C or equations? Clin Biochem. 2007; 40: 153–161.
3. Cockcroft DW, Gault MH. Prediction of creatinine clearance from serum creatinine. Nephron 1976; 16: 31–41.
4. Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D. A more accurate method to estimate glomerular filtration rate from serum creatinine: A new prediction equation. Modification of Diet in Renal Disease Study Group. Ann Intern Med. 1999; 130: 461–470.
5. Levey AS, Stevens LA, Schmid CH, Zhang YL, Castro AF III, Feldman HI, Kusek JW, Eggers P, Van Lente F, Greene T, Coresh J, for the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI). A new equation to estimate glomerular filtration rate. Ann Intern Med. 2009; 150: 604–612.
6. Inker LA, Schmid CH, Tighiouart H, Eckfeldt JH, Feldman HI, Greene T, Kusek JW, Manzi J, Van Lente F, Zhang YL, Coresh J, Levey AS, for the CKD-EPI Investigators. Estimating glomerular filtration rate from serum creatinine and cystatin C. N Engl J Med 2012; 367: 20–29.
7. Schwartz GJ, Haycock GB, Edelmann CM Jr, Spitzer A. A simple estimate of glomerular filtration rate in children derived from body length and plasma creatinine. Pediatrics 1976; 58: 259–263.
8. Counahan R, Chantler C, Ghazali S, Kirkwood B, Rose F, Baratt TM. Estimation of glomerular filtration rate from plasma creatinine concentration in children. Arch Dis Child 1976; 51: 875–878.
9. Schwartz GJ, Muñoz A, Schneider MF, Mak RH, Kaskel F, Warady BA, Furth SL. New equations to estimate GFR in children with CKD. J Am Soc Nephrol. 2009; 20: 629–637.
10. Schwartz GJ, Work DF. Measurement and estimation of GFR in children and adolescents. Clin J Am Soc Nephrol 2009; 4: 1832–1843.
11. Grubb A, Nyman Un, Björk J, Lindström V, Rippe BG, Christensson A. Simple cystatin C–based prediction equations for glomerular filtration rate compared with the modification of diet in renal disease prediction equation for adults and the Schwartz and the Counahan–Barratt prediction equations for children. Clinical Chemistry 2005; 51: 1420–1431.
12. The Swedish Council on Health Technology Assessment (SBU). Methods to estimate and measure renal function (glomerular filtration rate). Stockholm: The Swedish Council on Health Technology Assessment (SBU). 2012; Yellow 214.
13. James TJ, Lewis AV, Tan GD, Altmann P, Taylor RP, Levy JC. Validity of simplified protocols to estimate glomerular filtration rate using iohexol clearance Ann Clin Biochem. 2007: 44; 369–376.
14. Bröchner-Mortensen J. A simple method for the determination of glomerular filtration rate. Scand J Clin Lab Invest. 1972; 30(3): 271–274.
The authors
Zoe Maunsell* MBiochem MSc DipRCPath and Tim James PhD
Department of Clinical Biochemistry, Oxford University Hospitals NHS Trust, Oxford, UK
*Corresponding author
E-mail: zoe.maunsell@ouh.nhs.uk
Monitoring kidney function in aminoglycoside therapy
, /in Featured Articles /by 3wmediaAminoglycosides are antibiotics largely used at the hospital. Their nephrotoxicity imposes therapeutic drug monitoring as well as kidney function monitoring. Creatinine is the most widely used biochemical marker; however, new biomarkers such as neutrophil gelatinase associated lipocalin (NGAL), cystatin C (Cys C) or kidney injury molecule-1 (KIM-1) can allow the detection of acute kidney injury more quickly.
by F. Fraissinet, E. Sacchetto and Dr E. Bigot-Corbel
Background
Aminoglycosides are bactericidal antibiotics used for the treatment of Gram-negative or endocarditis infections. The most important adverse effects of aminoglycosides are nephrotoxicity and ototoxicity. The prevalence of aminoglycoside-associated nephrotoxicity is estimated at 10 to 20 %, although it also depends on the patient’s clinical condition and exposure to nephrotoxic drugs such as cyclosporine, anti-inflammatory or iodinated drugs. In intensive care units, nephrotoxicity is most frequent (60% of patients) and associated with a high rate of mortality [1, 2]. Nephrotoxicity mainly results in nonoliguric acute kidney injury which occurs following 7 to 10 days. This toxicity may manifest as a decrease in the glomerular filtration rate with glycosuria, hypokalemia and hypocalcemia. Aminoglycosides are often administered by intravenous drip and are freely eliminated by glomerular filtration and reabsorbed by the proximal tubule. Of the injected dose, 5% is retained by epithelial cells of the proximal tubule. After endocytosis in tubular cells, these molecules accumulate in lysosomes and induce phospholipidosis, alteration of key cellular components and apoptosis. Aminoglycosides also have glomerular effects: gentamicin stimulates mesangial proliferation, produces mesangial contraction and induces neutralization of negative charges of the glomerulus [3]. Gentamicin also induces a reduction in renal blood flow with an increased renal vascular resistance. These factors contribute to decrease the glomerular filtration rate (GFR). Additional risk factors for nephrotoxicity induced by aminoglycoside have been identified as sepsis, prolonged therapy, renal or liver dysfunction, hypokalemia or hypomagnesemia. Nephrotoxicity is less frequent when aminoglycosides are administered once daily compared with 12 h [4].
Methods of detection of acute kidney injury induced by aminoglycoside therapy
Classic markers: creatinine and creatinine clearance
Creatinine is the most widely used marker in the diagnosis of the acute renal insufficiency. For defining AKI, the Risk Injury Failure Loss End stage kidney disease (RIFLE) classification is based on increase of serum creatinine concentration and decrease of glomerular filtration rate. The introduction of the RIFLE classification has increased the conceptual understanding of AKI syndrome, and this classification has been successfully tested in a number of clinical studies [5].
In spite of an easily accessible dosage, creatinine as a marker of AKI has some drawbacks. Creatinine is filtered by the glomerulus and is not bound to plasma proteins. In standard physiological conditions, the daily rate of creatinine production is constant; however, the rate of creatinine production is affected by conditions of muscular pathology or muscular loss (as occurs in intensive care and cirrhosis). In these patients, AKI does not result in an increase of serum creatinine levels. Other factors, such as age, sex, ethnic group and diet, also influence serum concentrations of creatinine. Creatinine is not the ideal marker to estimate GFR, because it is secreted by renal tubule, which artefacutally increases glomerular filtration rate. At low serum creating concentrations, creatinine is lacks sensitivity to estimate GFR. Large changes in GFR may be associated with relatively small changes in serum creatinine (See Figure 2 in Delanaye et al. [6]). The rise of the creatinine is late (occurring after 3–5 days) and is not specific for nephrotoxicity induced by aminoglycosides, and an increase of creatinine in AKI is a function of the initial concentration of creatinine [7].
To estimate GFR, formulas that use creatinine plasma concentration, such as the Modified Diet in Renal Disease formula (MDRD), Chronic Kidney Disease Epidemiology collaboration formula (CKD-EPI) or estimation of clearance creatinine by Cockroft–Gault (CG) equation, were derived in subjects with chronic, not acute, kidney disease. A limitation of the MDRD equation was an underestimation of GFR in the high range. The CKD-EPI equation performs better at high GFR levels (GFR >60 mL/min/1.73 m²). Use of serum creatinine concentration to estimate GFR supposes a steady-state between creatinine production and excretion [8]. In spite of the use of correction factors, it is more difficult to estimate GFR in Asian or African populations as well as in elderly or obese patients [9].
New biomarkers
Cystatin C
Cystatin C (CysC), a 13-kDa endogenous cysteine proteinase inhibitor, plays an important role in intracellular catabolism of various peptides and proteins. CysC is considered to be a good biomarker of decreased kidney function because it is produced at a relatively constant rate and released into plasma, and is filtered by glomeruli without tubular secretion. The influence of muscular mass is less than for creatinine, and CysC allows diagnosis of AKI 48 h before serum creatinine [10]. Equations with serum CysC concentration can also estimate GFR. If GFR is great than 60 mL/min/m², CysC measurement is more powerful than the MDRD equation. CysC is a useful biomarker for early detection of AKI in the pediatric population and for patients in the intensive care unit, as CysC determination can be performed in serum and/or in urine. In spite of efforts to standardize the procedure, there is no reference method. Production of CysC also depends on hormonal factors, so CysC cannot be used in cases of thyroid dysfunction.
Neutrophil gelatinase associated lipocalin
Neutrophil gelatinase associated lipocalin (NGAL) is a protein of 25 kDa protein of the lipocalin family and is covalently bound to matrix metalloproteinase-9. NGAL is expressed early in ischemic kidney impairment in animal models. During AKI, NGAL expression is induced in distal nephron epithelia resulting in elevated plasma and urinary levels of NGAL (Fig. 1) [2]. NGAL determination can be performed on serum and/or urine by immunoturbimetric or immunofluorimetric assays. There is a general agreement on a cut-off value of >150 ng/mL, but a clear cut-off NGAL concentration for AKI has not been reported. Several studies show the importance of NGAL in cardiac surgery or critically ill patients for predicting AKI. NGAL is also useful for the detection of nephrotoxicity induced by contrast agents and has prognostic value for mortality or initiation of renal replacement therapy. Plasma NGAL measurements may be influenced by a number of coexisting variables as chronic hypertension, systemic infections, inflammatory conditions or hypoxia. Changes in NGAL values are potentially associated with septic state or aminoglycoside therapy [11].
Kidney injury molecule-1
Kidney injury molecule-1 (KIM-1) is a glycoprotein localized in the apical membrane of the proximal tubule of kidney, and KIM-1 expression can be induced by nephrotoxic drugs. Urine KIM-1 is a promising biomarker of proximal tubular injury. As with NGAL, urinary KIM-1 levels predicted adverse clinical outcomes such as dialysis requirement and mortality. In a previous study, urinary KIM-1 is correlated with AKI severity in non-critically ill children treated by aminoglycosides [12].
Conclusion
Patients treated with aminoglycosides must be carefully monitored for nephrotoxicity. Creatinine has been the most used biochemical marker of AKI, but new biomarkers, such as NGAL and KIM-1, have been developed in recent years.
References
1. Oliveira JF, Silva CA, Barbieri CD, Oliveira GM, Zanetta DM, Burdmann EA. Antimicrob Agents Chemother. 2009; 53(7): 2887-2891.
2. Schmidt-Ott KM. Nephrol Dial Transplant. 2011; 26(3): 762-764.
3. Lopez-Novoa JM, Quiros Y, Vicente L, Morales AI, Lopez-Hernandez FJ. Kidney Int. 2011; 79(1): 33-45.
4. Rybak MJ, Abate BJ, Kang SL, Ruffing MJ, Lerner SA, Drusano GL. Antimicrob Agents Chemother. 1999; 43(7): 1549-1555.
5. Ricci Z, Cruz DN, Ronco C. Nat Rev Nephrol. 2011; 7(4) :201-208.
6. Delanaye P, Cavalier E, Maillard N, Krzesinski JM, Mariat C, Cristol JP, et al. [Creatinine: past and present]. Annales de Biologie Clinique 2010; 68(5): 531-543 (in French).
7. Waikar SS, Bonventre JV. J Am Soc Nephrol. 2009; 20(3): 672-679.
8. Nguyen MT, Maynard SE, Kimmel PL. Clin J Am Soc Nephrol. 2009; 4(3): 528-34.
9. Delanaye P, Cavalier E, Mariat C, Krzesinski JM, Rule AD. Kidney Int. 2011; 80(5): 439-440.
10. Herget-Rosenthal S, Marggraf G, Husing J, Goring F, Pietruck F, Janssen O, et al. Kidney Int. 2004; 66(3): 1115-1122.
11. Devarajan P. Nephrology (Carlton) 2010; 15(4): 419-428.
12. McWilliam SJ, Antoine DJ, Sabbisetti V, Turner MA, Farragher T, Bonventre JV, et al. PLoS One 2012; 7(8): e43809.
The authors
François Fraissinet1 BSc, Emilie Sacchetto2 and Edith Bigot-Corbel2* PhD
1Laboratoire de Biochimie, 86021 Poitiers, France
2Laboratoire de Biochimie, CHU de Nantes, Hôpital G et R Laënnec, 44800 Saint-Herblain, France
*Corresponding author
E-mail: edith.bigot@chu-nantes.fr
Anti-PLA2R: the serological biomarker for primary MN
, /in Featured Articles /by 3wmediaAutoantibodies against phospholipase A2 receptors (PLA2R) are a new, highly specific diagnostic marker for primary membranous nephropathy (MN). Detection of anti-PLA2R using easy-to-perform and inexpensive serological assays can indicate primary MN in patients suffering from nephrotic syndrome and secure a differential diagnosis from secondary MN. Anti-PLA2R analysis is also useful for determining the disease activity, assessing the extent of treatment required and monitoring responses to therapy. Anti-PLA2R antibodies can be determined using innovative indirect immunofluorescence and ELISA test systems.
by Jacqueline Gosink, PhD
Primary membranous nephropathy
Primary MN, also known as primary membranous glomerulonephritis or primary MGN, is a chronic inflammatory autoimmune disease of the blood-filtering structures of the kidneys (glomeruli). It is accompanied by a progressive reduction in renal function. The disease manifests the complex nephrotic syndrome, which is characterized by heavy proteinuria, hypoalbuminemia, hyperlipidemia, edema and lipiduria. Primary MN is one of the leading causes of nephrotic syndrome in adults. As proteinuria increases, so does the long-term risk of kidney failure with major morbidity and mortality, especially from thromboembolic and cardiovascular complications. Around a third of patients progress to end-stage renal disease, a third exhibit persistent proteinuria without progression to renal failure, and the remainder experience spontaneous remission. Primary MN is prevalent in all ethnic groups and in both genders, with men over 40 years old being the most frequently affected.
Diagnostic challenge
The diagnosis of primary MN is demanding, as the disease must be differentiated from other nephropathies, especially from secondary MN, which is triggered by an underlying cause such as a malignant tumour, an infection, drug intoxication or another autoimmune disease such as systemic lupus erythematosus or diabetes mellitus type 1. Of all MN cases, 20-30% are of secondary genesis, while the remaining 70–80% are classified as primary. Primary cases with no detectable anti-PLA2R antibodies are subclassified as idiopathic; it has been postulated that these patients may exhibit antibodies against other, as yet unidentified, target antigens. Reliable differentiation of primary and secondary forms of MN is critical because of different treatment regimes: primary MN is treated with immunosuppressants, while therapy for secondary MN is targeted at the underlying disease.
MN is diagnosed by kidney puncture followed by histological examination or electron microscopy of the tissue to identify the characteristic glomerular immune deposits. To obtain a definite diagnosis of primary MN, secondary causes must be excluded, which involves additional time-consuming and often invasive procedures, for example tumour screening. Moreover, in some patients, MN appears before the secondary cause is even detectable, adding an extra layer of complexity to diagnosis and therapeutic decision-making. Primary MN must also be differentiated from other autoimmune diseases with kidney involvement, for example lupus nephritis, vasculitides associated with antibodies against neutrophil cytoplasm (ANCA) and Goodpasture’s syndrome. The availability of reliable serological tests to support the diagnosis of primary MN has been elusive until recently due to lack of knowledge about the target antigen.
New pathognomonic marker
Autoantibodies against PLA2R were first discovered and described in patients with primary MN in 2009 (1). PLA2R is a transmembrane glycoprotein (Figure 1) which is expressed in human glomeruli on the surface of podocytes and is involved in regulatory processes in the cell following phospholipase binding (Figure 2). Type M PLA2R has been identified as the major target antigen of autoantibodies. In patients with primary MN, antigen-antibody complexes form deposits in the glomerular basement membrane, where they trigger local complement activation with overproduction of collagen IV and laminin. This causes damage to the podocytes, via destruction of the cytoskeleton and broadening of the basement membrane. As a result protein enters the primary urine, giving rise to proteinuria and other symptoms.
Differential diagnosis
Autoantibodies of class IgG against PLA2R are present in the serum of up to 70-80% of patients with primary MN (1, 2), whereas they are not found in healthy blood donors or patients with secondary MN or other kidney diseases such as lupus nephritis (3) or IgA nephritis. The high predictive value of anti-PLA2R makes this parameter ideally suited as a diagnostic marker (4).
Disease evaluation
Anti-PLA2R antibodies are, moreover, very sensitive markers of clinical disease activity. They reflect the pathogenic immunological activity of the disease, which is responsible for the clinical expression in the form of proteinuria. High antibody titres indicate a severe disease course (2, 5), while low titres are associated with a decreased risk of renal failure and a greater rate of spontaneous remission (6). Thus, the anti-PLA2R titre also serves as a prognostic indicator.
Therapy monitoring
Treatment with immunosuppressants results in a drop in the anti-PLA2R titre, while in relapse the antibody titre increases again. Significantly, changes in the antibody titre typically precede changes in the proteinuria (6, 7). Thus, a titre increase is detectable before proteinuria appears, while a titre decrease is observed before a reduction in the proteinuria. Patients in remission exhibit residual proteinuria months after the anti-PLA2R titre becomes undetectable. Anti-PLA2R measurements are therefore extremely useful for early therapeutic decision-making and for long-term monitoring of responses to immunotherapy.
A recently published prospective study reinforced the value of anti-PLA2R antibodies as a marker of clinical outcome (8). In the study 133 patients with primary MN were tracked over a time period of 24 months. In all cases there was a clear correlation between proteinuria and anti-PLA2R levels. In patients who were given immunotherapy, a significant time lag was observed between the rapid fall in antibody levels and the protracted reduction in proteinuria. Moreover, remission of proteinuria occurred later in individuals with high antibody levels than in those with low levels. In patients who did not receive immunosuppressive therapy, spontaneous remission was also associated with a reduction in anti-PLA2R, while individuals who did not achieve remission showed continued elevated antibody levels. Thus, anti-PLA2R proved a reliable biomarker for immunological and clinical activity in primary MN.
Risk assessment
Up to 40% of patients with primary MN experience a relapse after kidney transplantation. The risk of recurrent primary MN is particularly high if anti-PLA2R antibodies are found prior to transplantation. In a study on a patient with primary MN, who exhibited high anti-PLA2R levels before and three months after transplantation (7), it could be shown that immunotherapy resulted in a drop in the antibody concentration and also the level of proteinuria. Other studies have shown that anti-PLA2R or PLA2R deposits are detected more often in transplant patients with recurrent MN than in those with de novo MN. A retrospective analysis of fifteen transplant patients revealed that a persistently positive anti-PLA2R activity at six months or later after transplantation was a significant risk factor for relapse, especially in patients on a weak immunosuppressive regimen (9). Thus, the anti-PLA2R antibody titre is useful for assessing the risk of relapse after transplantation and the extent of immunotherapy needed to prevent a recurrence.
Anti-PLA2R test systems
Anti-PLA2R autoantibodies can be determined easily and reliably using standardized indirect immunofluorescence test (IIFT) and ELISA systems. In the IIFT a BIOCHIP of transfected human cells expressing recombinant PLA2R is used as the antigenic substrate to provide monospecific antibody detection (Figure 3). A second BIOCHIP containing cells transfected with an empty vector serves as a control. The IIFT represents an established test for serodiagnostic screening, providing qualitative and semi-quantitative antibody analysis. The corresponding ELISA is based on purified recombinant PLA2R and shows the same high-quality characteristics as the IIFT. The ELISA is particularly useful for disease and therapy monitoring as it offers precise quantification of antibody levels in patient sera. The IIFT and ELISA are fast and simple to perform and are suitable for use in any diagnostic laboratory. Both procedures can be automated.
Clinical data
The performance characteristics of the Anti-PLA2R IIFT and ELISA have been assessed in a multitude of studies. In a retrospective clinical study (10) the Anti-PLA2R IIFT yielded a prevalence of 52% in a cohort of 100 patients with biopsy-proven primary MN, and a specificity of 100% with respect to healthy controls and patients with secondary MN or non-membranous glomerular injury. In a prospective clinical study (11) the sensitivity amounted to 82% in patients with biopsy-proven MN where no secondary cause could be found. The difference in sensitivities obtained in different study panels is most likely due to factors such as disease remission and the therapy status of the individuals, which can influence the antibody results, especially when studies are performed retrospectively.
Results obtained with the ELISA show a very good correlation with results from the IIFT (Figure 4). In a retrospective study with sera from 198 patients with primary MN and 836 healthy and disease controls, the ELISA showed a sensitivity of 96% with respect to the IIFT, and a specificity of 99.9% with borderline sera included (12). The few discrepant sera that were negative in the ELISA gave only low titres of 1:10 to 1:100 in the IIFT. All sera with titres of over 1:100 in IIFT were also positive in ELISA.
Summary
Antibodies against PLA2R represent a landmark development in nephrological diagnostics. Their detection can secure a diagnosis of primary MN in patients with nephrotic syndrome, offering a convenient, non-invasive alternative to biopsy. Anti- PLA2R determination, moreover, yields information about the disease status and evolvement. Serial measurements are especially useful for monitoring therapy responses over the long term and guiding decision-making on the extent of treatment required for individual patients. Serological tests based on state-of-the-art IIFT and ELISA technology provide simple, quick and highly specific anti-PLA2R antibody detection.
References
1. Beck et al. N. Engl. J. Med. 2009: 361: 11-21
2. Hofstra et al. Clin. J. Am. Soc. Nephrol. 2011: 6: 1286-91
3. Gunnarsson et al. Am. J. Kid. Dis. 2012: 59 (4): 585-6
4. Schlumberger et al. Autoimmunity Reviews 2014: 13: 108-13
5. Kanigicherla et al. Kidney Int. 2013: 83: 940-948
6. Hofstra et al. J. Am. Soc. Nephrol. 2012: 23 (10): 1735-43
7. Stahl et al. N. Engl. J. Med. 2010: 363: 496-8
8. Hoxha et al. J. Am. Soc. Nephrol. 2014: 25: 1137-9
9. Seitz-Polski et al. Nephrol. Dial. Transplant. 2014: under revision
10. Hoxha et al. Nephrol. Dial. Transplant. 2011: 26 (8): 2526-32
11. Hoxha et al. Kidney Int. 2012: 82: 797-804
12. Daehnrich et al. Clin. Chem. Acta. 2013: 421C: 213-8
The author
Jacqueline Gosink PhD
EUROIMMUN AG
Seekamp 31
23560 Luebeck
Germany
E-Mail: j.gosink@euroimmun.de
CLINITEK Status Connect System
, /in Featured Articles /by 3wmediaNew FilmArray GI Panel makes a big splash
, /in Featured Articles /by 3wmedia