Reference Intervals for Plasma Cystatin C in Healthy Volunteers and Renal Patients , as Measured by the Dade Behring BN II System , and Correlation with Creatinine , Erik

Measurement of biochemical bone markers is commonly used in the management of various metabolic bone diseases (1, 2). The pyridinium crosslinks pyridinoline (PYD) and deoxypyridinoline (DPD) are well-characterized markers for bone resorption that have been available for several years (3 ). Assays to measure the sum of free and peptide-bound urinary PYD or DPD (total PYD or DPD) or free, non-peptide-bound molecules have been developed and described (4 ). Analytical variability of PYD and DPD measurement is a major problem hampering comparability and interpretation of results. As part of the CDC program to develop a reference system to standardize the measurements of PYD and DPD, we conducted a round-robin interlaboratory comparison study to assess the state of analytical variability. We invited laboratorians within the US involved in routine measurement of urinary DPD and/or PYD to participate in this study. Participants were asked to analyze five identical double-blinded sets of six unknown samples for PYD, DPD, and creatinine on 5 days in duplicate. Information was collected from each participant on sample handling and preparation, as well as calibrators, including information on sample hydrolysis. Urine was collected in agreement with CDC Institutional Review Board regulations. We screened individual urine samples for total PYD and DPD concentrations using our in-house HPLC assay (5, 6) and then combined them into three concentrations, from normal to moderately increased pyridinium crosslink concentrations (low-, medium-, and high-pool). We created a fourth urine pool by mixing the low and medium pools (1:1 by volume; mixed pool). One part of the mixed pool was used for the addition of PYD and DPD calibrators (supplemented pool; 638 nmol/L PYD and 219 nmol/L DPD). An aqueous solution of DPD and PYD calibrators (aqueous sample; 306 nmol/L PYD and 555 nmol/L DPD) was included as the sixth sample. We tested immunoreactive, free PYD, and DPD calibrators (obtained from Metra Biosystems, Inc.) for identity and purity by mass spectrometry and spectrophotometry using data described previously (7 ). Concentrations were determined spectrophotometrically with previously described coefficients of absorption (7 ) and were confirmed with our in-house HPLC method. Pools and calibrators were handled under special protective yellow light. All pools were dispensed into brown glass vials and shipped frozen on dry ice (overnight delivery). Bottle-to-bottle variability was tested. We analyzed data separately for immunoassays and HPLC assays. We tested for outliers by calculating the all-laboratory consensus mean 63 SD for each sample and compared each individual result. No result was outside of this range. All evaluations of imprecision and recoveries were based on the mean results over 5 days. The following measures of imprecision were evaluated: amonglaboratory (within-method), within-laboratory (amongpools), and among-run (within-laboratory). We expressed all variations as CV (SD). We calculated the contribution of withinand among-laboratory variability to the total variability using a nested random-effects analysis of variance. We calculated the differences in among-laboratory and within-pool concentrations of PYD and DPD using ANOVA. Results with P .0.01 were considered nonsignificant. Because there were no available analytical reference methods that could be used as accuracy checks, we performed recovery experiments to assess assay accuracy. Recoveries, reported as mean recoveries (SD), were calculated individually for each sample with added DPD and PYD: recovery (%) 5 [(urine with added PYD and DPD) 2 (urine without added PYD and DPD)]/added concentration of PYD and DPD 3 100. Recoveries were also calculated for the mixed sample: recovery (%) 5 measured value/expected value 3 100, with the expected value being the mean of the low pool and medium pool as determined by each laboratory. On the basis of assigned PYD and DPD values for the aqueous sample and the values measured by the laboratories for this sample, a factor was calculated (factor 5 assigned value/measured value). We multiplied this factor by the DPD and PYD values of the pools to normalize the data to the assigned values of the aqueous sample. We used this procedure to estimate the impact of a common calibrator on the among-laboratory variability. Of the 15 laboratories that agreed to participate, 1 laboratory did not report results, and 1 laboratory was excluded because of problems related to assay processing, which did not reflect normal laboratory performance. Of the 13 remaining laboratories, 5 used HPLC assays (4 in-house methods; 1 assay from BIORAD, Inc.), and 8 used immunoassays (Metra Biosystems). The five laboratories performing HPLC assays used four different calibrators. Two different immunoassays were used to analyze either DPD (measured by eight laboratories) or PYD with a cross-reaction for DPD (referred to in the text as “PYD&DPD”; measured by four laboratories). All immunoassays were performed manually with the same calibrator. Creatinine was analyzed in all laboratories with a alkaline picric acid reaction. The mean within-laboratory and among-pool CVs for the immunoassays were 8.1% and 10% for PYD&DPD and DPD, respectively, and for the HPLC assays, 9.0% and 11% for PYD and DPD, respectively. The difference in this variability between both types of assays was not significant (P .0.6; double-sided t-test). The results for the supplemented pool and the aqueous sample varied more than those for the other pools. The mean recoveries for the mixed pool with the immunoassays were 100.1% and 98.6% for DPD and PYD&DPD, respectively, and 98.9% and 100.4% with the HPLC assays for DPD and PYD, respectively. The mean recoveries of the supplemented Technical Briefs