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.

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.

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.

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.