Cyanide-Nitroprusside Colorimetric Assay: A Rapid Colorimetric Screen for Urinary Cystine.

BACKGROUND: Cystinuria is an autosomal recessive disorder resulting in poor proximal tubule reabsorption of cystine in the nephron, increasing the risk of cystine stone formation. A fast, inexpensive assay to screen for urinary cystine is needed because cystine stones are difficult to noninvasively differentiate from more common calcium-containing ones. Tandem mass spectrometry (MS/MS) is sensitive and specific but is labor-intensive and costly. Alternatively, a colorimetric assay is fast and cost-effective; however, creatinine interference is an issue. METHODS: A published cyanide-nitroprusside colorimetric assay was modified for a high-throughput microplate format. Creatinine interference was reduced using 0.1 mol/L PBS and a standard reaction time of 60 s and was further corrected using a formula derived from the slope of multiple creatinine standard curves. RESULTS: The limit of blank was determined to be 2.6 mg/L, the limit of detection 11.9 mg/L, and the limit of quantitation 15.3 mg/L. The analytic measurement range was established as 15.3-100 mg/L cystine. Intraassay and interassay CV was calculated to be 9.6% and 8.0%, respectively, for a high-level cystine concentration (83.6 mg/L). Low-level cystine (36.4 mg/L) intraassay and interassay CV was determined to be 18.1% and 17.6%, respectively. Passing-Bablok regression analysis of colorimetric vs LC-MS/MS results revealed a slope of 1.10 and y intercept of -7.14 mg/L, with an overall bias of 2% by Bland-Altman plot analysis. CONCLUSIONS: We analytically validated a rapid colorimetric assay suitable to quantify urinary cystine. The effect of thiol drugs on this assay remains to be determined.

Creatinine-Based and Cystatin C-Based GFR Estimating Equations and Their Non-GFR Determinants in Kidney Transplant Recipients.

BACKGROUND AND OBJECTIVES: eGFR equations have been evaluated in kidney transplant recipients with variable performance. We assessed the performance of the Modification of Diet in Renal Disease equation and the Chronic Kidney Disease Epidemiology Collaboration equations on the basis of creatinine, cystatin C, and both (eGFR creatinine-cystatin C) compared with measured GFR by iothalamate clearance and evaluated their non-GFR determinants and associations across 15 cardiovascular risk factors. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: A cross-sectional cohort of 1139 kidney transplant recipients >1 year after transplant was analyzed. eGFR bias, precision, and accuracy (percentage of estimates within 30% of measured GFR) were assessed. Interaction of each cardiovascular risk factor with eGFR relative to measured GFR was determined. RESULTS: Median measured GFR was 55.0 ml/min per 1.73 m(2). eGFR creatinine overestimated measured GFR by 3.1% (percentage of estimates within 30% of measured GFR of 80.4%), and eGFR Modification of Diet in Renal Disease underestimated measured GFR by 2.2% (percentage of estimates within 30% of measured GFR of 80.4%). eGFR cystatin C underestimated measured GFR by -13.7% (percentage of estimates within 30% of measured GFR of 77.1%), and eGFR creatinine-cystatin C underestimated measured GFR by -8.1% (percentage of estimates within 30% of measured GFR of 86.5%). Lower measured GFR associated with older age, women, obesity, longer time after transplant, lower HDL, lower hemoglobin, lower albumin, higher triglycerides, higher proteinuria, and an elevated cardiac troponin T level but did not associate with diabetes, smoking, cardiovascular events, pretransplant dialysis, or hemoglobin A1c. These risk factor associations differed for five risk factors with eGFR creatinine, six risk factors for eGFR Modification of Diet in Renal Disease, ten risk factors for eGFR cystatin C, and four risk factors for eGFR creatinine-cystatin C. CONCLUSIONS: Thus, eGFR creatinine and eGFR creatinine-cystatin C are preferred over eGFR cystatin C in kidney transplant recipients because they are less biased, more accurate, and more consistently reflect the same risk factor associations seen with measured GFR.

El desempeño de las ecuaciones de estimación del índice de filtrado glomerular basado en cistatina-C y creatinina depende de las características del paciente / Performance of Cystatin C- and Creatinine-Based Estimated Glomerular / Filtration Rate Equations Depends on Patient Characteristics

Antecedentes: La guía Kidney Disease Improving Global Outcomes (KDIGO) recomienda el uso de un filtrado glomerular estimado (IFGe) basado en cistatina-C para confirmar un IFGe basado en creatinina entre 45 y 59 mL ∙ min-1 ∙ (1,73 m2)-1. Estudios anteriores han demostrado que comorbilidades tales como trasplantes de órganos sólidos influyen fuertemente en la relación entre el índice de filtrado glomerular (IFG) medido, creatinina y cistatina-C. Nuestro objetivo fue evaluar el desempeño de ecuaciones de IFGe basadas en cistatina-C en comparación con IFG medido e IFGe basada en creatinina en diferentes situaciones clínicas. Métodos: Comparamos el rendimiento de la ecuación CKD-EPI 2009, ecuación de IFGe basada en creatinina (IFGeCr), y las nuevas ecuaciones CKD-EPI 2012 basadas en cistatina-C (IFGeCys y IFGeCr-Cys), con el IFG medido (depuración renal de iotalamato) en poblaciones de pacientes totalmente definidas. Los pacientes (n=1.652) fueron clasificados como receptores de trasplantes (de riñón, n=568 u otro órgano, n=319), pacientes con enfermedad renal crónica (ERC) conocida (n=618), o potenciales donantes de riñón (n=147). Resultados: IFGeCr-Cys mostró el desempeño más consistente a través de diferentes poblaciones clínicas. Entre los potenciales donantes de riñón sin ERC [estadio 2 o superior; IFGe> 60 mL ∙ min-1 ∙ (1,73 m2)-1], IFGeCys y IFGeCr‑Cys demostraron significativamente menor sesgo que IFGeCr; sin embargo, las 3 ecuaciones subestimaron substancialmente IFG cuando IFGe fue <60 mL ∙ min-1 ∙ (1,73 m2)-1. Entre los receptores de trasplante con ERC estadio 3B o mayor [IFGe <45 mL ∙ min-1 ∙ (1,73 m2)-1], IFGeCys mostró un sesgo significativamente mayor que IFGeCr. No se observaron diferencias claras en el sesgo de IFGe entre las ecuaciones dentro del grupo de pacientes con ERC conocida independientemente del rango de IFGe o en cualquier grupo de pacientes con una IFG entre 45 y 59 mL ∙ min-1 ∙ (1,73 m2)-1. Conclusiones: El desempeño de las ecuaciones de IFGe depende de las características del paciente las cuales son evidentes en su presentación. Entre las 3 ecuaciones CKD-EPI, IFGeCr-Cys tuvo un desempeño más coherente en las poblaciones de pacientes estudiados.

Background: The Kidney Disease Improving Global Outcomes (KDIGO) guideline recommends use of a cystatin C-based estimated glomerular filtration rate (eGFR) to confirm creatinine-based eGFR between 45 and 59 mL ∙ min-1 ∙ (1.73 m2)-1. Prior studies have demonstrated that comorbidities such as solid-organ transplant strongly influence the relationship between measured GFR, creatinine, and cystatin C. Our objective was to evaluate the performance of cystatin C-based eGFR equations compared with creatinine-based eGFR and measured GFR across different clinical presentations. Methods: We compared the performance of the CKD-EPI 2009 creatinine-based estimated GFR equation (eGFRCr) and the newer CKD-EPI 2012 cystatin C-based equations (eGFRCys and eGFRCr-Cys) with measured GFR (iothalamate renal clearance) across defined patient populations. Patients (n = 1652) were categorized as transplant recipients (n = 568 kidney; n = 319 other organ), known chronic kidney disease (CKD) patients (n = 618), or potential kidney donors (n = 147). Results: eGFRCr-Cys showed the most consistent performance across different clinical populations. Among potential kidney donors without CKD [stage 2 or higher; eGFR >60 mL ∙ min-1 ∙ (1.73 m2)-1], eGFRCys and eGFRCr-Cys demonstrated significantly less bias than eGFRCr; however, all 3 equations substantially underestimated GFR when eGFR was <60 mL ∙ min-1 ∙ (1.73 m2)-1. Among transplant recipients with CKD stage 3B or greater [eGFR <45 mL ∙ min-1 ∙ (1.73 m2)-1], eGFRCys was significantly more biased than eGFRCr. No clear differences in eGFR bias between equations were observed among known CKD patients regardless of eGFR range or in any patient group with a GFR between 45 and 59 mL ∙ min-1 ∙ (1.73 m2)-1. Conclusions: The performance of eGFR equations depends on patient characteristics that are readily apparent on presentation. Among the 3 CKD-EPI equations, eGFRCr-Cys performed most consistently across the studied patient populations.

Performance of cystatin C- and creatinine-based estimated glomerular filtration rate equations depends on patient characteristics.

BACKGROUND: The Kidney Disease Improving Global Outcomes (KDIGO) guideline recommends use of a cystatin C-based estimated glomerular filtration rate (eGFR) to confirm creatinine-based eGFR between 45 and 59 mL · min(-1) · (1.73 m(2))(-1). Prior studies have demonstrated that comorbidities such as solid-organ transplant strongly influence the relationship between measured GFR, creatinine, and cystatin C. Our objective was to evaluate the performance of cystatin C-based eGFR equations compared with creatinine-based eGFR and measured GFR across different clinical presentations. METHODS: We compared the performance of the CKD-EPI 2009 creatinine-based estimated GFR equation (eGFRCr) and the newer CKD-EPI 2012 cystatin C-based equations (eGFRCys and eGFRCr-Cys) with measured GFR (iothalamate renal clearance) across defined patient populations. Patients (n = 1652) were categorized as transplant recipients (n = 568 kidney; n = 319 other organ), known chronic kidney disease (CKD) patients (n = 618), or potential kidney donors (n = 147). RESULTS: eGFRCr-Cys showed the most consistent performance across different clinical populations. Among potential kidney donors without CKD [stage 2 or higher; eGFR >60 mL · min(-1) · (1.73 m(2))(-1)], eGFRCys and eGFRCr-Cys demonstrated significantly less bias than eGFRCr; however, all 3 equations substantially underestimated GFR when eGFR was <60 mL · min(-1) · (1.73 m(2))(-1). Among transplant recipients with CKD stage 3B or greater [eGFR <45 mL · min(-1) · (1.73 m(2))(-1)], eGFRCys was significantly more biased than eGFRCr. No clear differences in eGFR bias between equations were observed among known CKD patients regardless of eGFR range or in any patient group with a GFR between 45 and 59 mL · min(-1) · (1.73 m(2))(-1). CONCLUSIONS: The performance of eGFR equations depends on patient characteristics that are readily apparent on presentation. Among the 3 CKD-EPI equations, eGFRCr-Cys performed most consistently across the studied patient populations.

Fecal electrolyte testing for evaluation of unexplained diarrhea: Validation of body fluid test accuracy in the absence of a reference method.

BACKGROUND: Validation of tests performed on body fluids other than blood or urine can be challenging due to the lack of a reference method to confirm accuracy. The aim of this study was to evaluate alternate assessments of accuracy that laboratories can rely on to validate body fluid tests in the absence of a reference method using the example of sodium (Na(+)), potassium (K(+)), and magnesium (Mg(2+)) testing in stool fluid. METHODS: Validations of fecal Na(+), K(+), and Mg(2+) were performed on the Roche cobas 6000 c501 (Roche Diagnostics) using residual stool specimens submitted for clinical testing. Spiked recovery, mixing studies, and serial dilutions were performed and % recovery of each analyte was calculated to assess accuracy. Results were confirmed by comparison to a reference method (ICP-OES, PerkinElmer). RESULTS: Mean recoveries for fecal electrolytes were Na(+) upon spiking=92%, mixing=104%, and dilution=105%; K(+) upon spiking=94%, mixing=96%, and dilution=100%; and Mg(2+) upon spiking=93%, mixing=98%, and dilution=100%. When autoanalyzer results were compared to reference ICP-OES results, Na(+) had a slope=0.94, intercept=4.1, and R(2)=0.99; K(+) had a slope=0.99, intercept=0.7, and R(2)=0.99; and Mg(2+) had a slope=0.91, intercept=-4.6, and R(2)=0.91. Calculated osmotic gap using both methods were highly correlated with slope=0.95, intercept=4.5, and R(2)=0.97. Acid pretreatment increased magnesium recovery from a subset of clinical specimens. CONCLUSIONS: A combination of mixing, spiking, and dilution recovery experiments are an acceptable surrogate for assessing accuracy in body fluid validations in the absence of a reference method.

Serum cystatin C predicts vancomycin trough levels better than serum creatinine in hospitalized patients: a cohort study.

INTRODUCTION: Serum cystatin C can improve glomerular filtration rate (GFR) estimation over creatinine alone, but whether this translates into clinically relevant improvements in drug dosing is unclear. METHODS: This prospective cohort study enrolled adults receiving scheduled intravenous vancomycin while hospitalized at the Mayo Clinic in 2012. Vancomycin dosing was based on weight, serum creatinine with the Cockcroft-Gault equation, and clinical judgment. Cystatin C was later assayed from the stored serum used for the creatinine-based dosing. Vancomycin trough prediction models were developed by using factors available at therapy initiation. Residuals from each model were used to predict the proportion of patients who would have achieved the target trough with the model compared with that observed with usual care. RESULTS: Of 173 patients enrolled, only 35 (20%) had a trough vancomycin level within their target range (10 to 15 mg/L or 15 to 20 mg/L). Cystatin C-inclusive models better predicted vancomycin troughs than models based upon serum creatinine alone, although both were an improvement over usual care. The optimal model used estimated GFR by the Chronic Kidney Disease Epidemiology Collaborative (CKD-EPI) creatinine-cystatin C equation (R(2) = 0.580). This model is expected to yield 54% (95% confidence interval 45% to 61%) target trough attainment (P <0.001 compared with the 20% with usual care). CONCLUSIONS: Vancomycin dosing based on standard care with Cockcroft-Gault creatinine clearance yielded poor trough achievement. The developed dosing model with estimated GFR from CKD-EPIcreatinine-cystatin C could yield a 2.5-fold increase in target trough achievement compared with current clinical practice. Although this study is promising, prospective validation of this or similar cystatin C-inclusive dosing models is warranted.

Oxalate quantification in hemodialysate to assess dialysis adequacy for primary hyperoxaluria.

BACKGROUND: Patients with primary hyperoxaluria (PH) overproduce oxalate which is eliminated via the kidneys. If end-stage kidney disease develops they are at high risk for systemic oxalosis, unless adequate oxalate is removed during hemodialysis (HD) to equal or exceed ongoing oxalate production. The purpose of this study was to validate a method to measure oxalate removal in this unique group of dialysis patients. METHODS: Fourteen stable patients with a confirmed diagnosis of PH on HD were included in the study. Oxalate was measured serially in hemodialysate and plasma samples in order to calculate rates of oxalate removal. HD regimens were adjusted according to a given patient's historical oxalate production, amount of oxalate removal at dialysis, residual renal clearance of oxalate, and plasma oxalate levels. RESULTS: After a typical session of HD, plasma oxalate was reduced by 78.4 ± 7.7%. Eight patients performed HD 6 times/week, 2 patients 5 times/week, and 3 patients 3 times/week. Combined oxalate removal by HD and the kidneys was sufficient to match or exceed endogenous oxalate production. After a median period of 9 months, pre-dialysis plasma oxalate was significantly lower than initially (75.1 ± 33.4 vs. 54.8 ± 46.6 mmol/l, p = 0.02). CONCLUSION: This methodology can be used to individualize the dialysis prescription of PH patients to prevent oxalosis during the time they are maintained on HD and to reduce risk of oxalate injury to a transplanted kidney.

Urine but not serum soluble urokinase receptor (suPAR) may identify cases of recurrent FSGS in kidney transplant candidates.

BACKGROUND: Recently, serum soluble urokinase receptor (suPAR) has been proposed as a cause of two thirds of cases of focal segmental glomerulosclerosis (FSGS). It was noted to be uniquely elevated in cases of primary FSGS, with higher levels noted in cases that recurred after transplantation. It is also suggested as a possible target and marker of therapy. METHODS: We studied serum and urine suPAR from pretransplantation banked samples from 86 well-characterized kidney transplant recipients and 10 healthy controls to determine its prognostic utility. Causes of native kidney disease were primary FSGS, diabetic nephropathy, membranous nephropathy, immunoglobulin A nephropathy, and autosomal dominant polycystic kidney disease. suPAR was measured using a commercially available enzyme-linked immunosorbent assay kit. Urinary suPAR was indexed to creatinine. RESULTS: Both serum and urine suPAR correlated with proteinuria and albuminuria. Serum suPAR was found to be elevated in all transplant candidates with advanced renal disease compared with healthy controls and could not differentiate disease diagnosis. Urine suPAR was elevated in cases of recurrent FSGS compared with all other causes of end-stage renal disease. Recurrent FSGS cases had substantially higher proteinuria compared with all other cases. However, elevated urinary suPAR showed a trend in providing additional prognostic information beyond proteinuria in the small cohort of recurrent FSGS cases. CONCLUSION: In advanced renal disease, elevated serum suPAR is not unique to FSGS cases. Urinary suPAR appears to be higher in cases of FSGS destined for recurrence and merits further evaluation.

Measurement of urinary TGF-ß1 in patients with diabetes mellitus and normal controls.

OBJECTIVE: Increasing evidence links TGF-ß1 to progression of renal fibrosis including its association with diabetic nephropathy (DN). Current ELISA assays are not sensitive enough to measure TGF-ß1 in the urine of many clinically healthy individuals, even those with established renal disease. The objective of this study was to validate a sensitive urinary assay for TGF-ß1 and compare levels between healthy controls and patients with established DN. DESIGN AND METHODS: An ELISA method (R&D Systems) was utilized together with an amplification step to assay TGF-ß1 in urine samples from 190 patients with DN and 80 healthy controls. RESULTS: Using an ELAST (Perkin Elmer, Inc) amplification step, the ELISA for urinary TGF-ß1 had a limit of quantification of 15.6 pg/mL and limit of detection of 7 pg/mL. Preliminary studies demonstrated that TGF-ß1 was stable if urine was frozen promptly at -70°C without preservatives. Using this assay, 22/80 controls (27%) had detectable levels of urinary TGF-ß1 (range <7 to 40.9 pg/mL; mean±SD 6.4±11.1 pg/mL). This was significantly lower (p<0.0001) than in the DN group in whom 114/190 (60%) had detectable levels of urinary TGF-ß1 (range <7 to 526.4 pg/mL; mean±SD 20.4±45.8 pg/mL). Urinary protein and TGF-ß1 concentrations demonstrated modest correlation in patients with DN (r=0.47, P<0.001). TGF-ß1 measurement in patients with DN did not demonstrate significant association with progression of proteinuria or increase in serum creatinine during the next 12 months of follow-up. CONCLUSION: We have validated a sensitive ELISA assay for urinary TGF-ß1, and demonstrated correlations with the degree of proteinuria and higher levels in patients with DN compared to controls. Additional study will be necessary in order to determine if serial testing can predict renal prognosis independent of known prognostic factors for patients with DN.

Analytic and clinical validation of a standardized cystatin C particle enhanced turbidimetric assay (PETIA) to estimate glomerular filtration rate.

BACKGROUND: Cystatin C is an alternative biomarker for assessing glomerular filtration rate (GFR), yet lack of standardization could hinder its widespread use. In this study we analytically and clinically validated a newer cystatin C particle-enhanced turbidimetric assay (PETIA) traceable to a certified reference material and compared it to the more commonly used particle-enhanced nephelometric assay (PENIA). METHODS: Samples from four patient cohorts at the Mayo Clinic were studied: 1) clinical convenience samples (n=50); 2) samples from patients undergoing iothalamate urinary clearance testing for clinical indications (n=101); 3) volunteers without kidney disease (n=292); 4) samples from 1999-2000 with previous cystatin C measurements. RESULTS: The cystatin C PETIA was analytically robust between 0.15 mg/L and 8.36 mg/L. PETIA cystatin C values were 27.5% higher than PENIA results. Furthermore, PENIA results were 12.9% lower in 2010 than in 2000. PETIA cystatin C values and existing equations performed reasonably well to estimate GFR with an overall -7.4% bias for all patients analyzed. Age and gender specific reference intervals were established for the PETIA cystatin C. CONCLUSIONS: Cystatin C can be precisely measured by PETIA traceable to the international reference material, ERM-DA471/IFCC, using a routine chemistry autoanalyzer. There are important biases between this assay and the widely employed Siemens PENIA. This study highlights the importance of assay standardization if cystatin C is to be widely used to estimate GFR.

Assessment of clinical performance without adequate analytical validation: A prescription for confusion.

OBJECTIVES: Lp-PLA2 is a biomarker with promise for predicting cardiac risk. The lack of reproducible results has limited its use. In evaluating a new reagent kit, we investigated conditions for optimal reproducibility. METHODS: The Auto-PLAC reagents were evaluated on the Cobas instrument. Performance characteristics, stability, and population ranges were determined. RESULTS: Analytical performance characteristics replicated manufacturer's claims. The stability profile of the analyte was unusual, with increasing results observed with storage at 4°C or -20°C. Only storage at -70°C gave acceptable stability. Population median values with properly preserved samples were much lower than the cut off previously validated for increased risk. CONCLUSIONS: It is postulated that variability in specimen handling was a major contributor to the lack of traceability of the current reagents to the earlier clinical studies demonstrating its utility. We are now unsure how to identify reliable criteria for result interpretation.