Search

Background: Standardized calibration does not change a creatinine measurement procedure's susceptibility to potentially interfering substances., Methods: We obtained individual residual serum or plasma samples (n = 365) from patients with 19 different disease categories associated with potentially interfering substances and from healthy controls. Additional sera at 0.9 mg/dL (80 μmol/L) and 3.8 mg/dL (336 μmol/L) creatinine were supplemented with acetoacetate, acetone, ascorbate, and pyruvate. We measured samples by 4 enzymatic and 3 Jaffe commercially available procedures and by a liquid chromatography/isotope dilution/mass spectrometry measurement procedure against which biases were determined., Results: The number of instances when 3 or more results in a disease category had biases greater than the limits of acceptability was 28 of 57 (49%) for Jaffe and 14 of 76 (18%) for enzymatic procedures. For the aggregate group of 59 diabetes samples with increased β-hydroxybutyrate, glucose, or glycosylated hemoglobin (Hb A(1c)), the enzymatic procedures had 10 biased results of 236 (4.2%) compared with 89 of 177 (50.3%) for the Jaffe procedures, and these interferences were highly procedure dependent. For supplemented sera, interferences were observed in 11 of 24 (46%) of groups for Jaffe and 8 of 32 (25%) of groups for enzymatic procedures and were different at low or high creatinine concentrations., Conclusions: There were differences in both magnitude and direction of bias among measurement procedures, whether enzymatic or Jaffe. The influence of interfering substances was less frequent with the enzymatic procedures, but no procedure was unaffected. The details of implementation of a method principle influenced its susceptibility to potential interfering substances.

Background: Peptide-based analysis of whole blood using electrospray tandem mass spectrometry (MSMS) in multiple reaction monitoring (MRM) mode enables rapid detection and sequence confirmation of clinically significant hemoglobin (Hb) variants. We applied a similar, quantitative approach to the measurement of delta:beta-globin peptide ratios as potential surrogate markers of HbA(2), a biomarker used in population screening for beta-thalassemia trait., Methods: We studied 163 blood samples with normal HbA(2) (%), 105 with increased HbA(2), 43 with delta-chain variants, and 8 with Hb Lepore. All were tested by HPLC. The samples were also incubated with trypsin for 30 min at 37 degrees C for MSMS with flow injection analysis. MRMs for the delta- (T2, T3, and T14) and beta- (T2, T3, and T13) globin tryptic peptides were acquired for 1 min, and delta:beta peptide ratios were calculated. We used HPLC and MSMS to analyze 26 paired whole blood and dried blood spot samples after storage for 1, 8, and 29 days., Results: Within- and between-assay imprecision values (CVs) were <6.1% and <8.4%, respectively, for the delta:beta peptide ratios. Digests were stable at 10 degrees C for 6 days. Significant correlations (P <0.0001) between MSMS delta:beta-globin peptide ratios and HPLC HbA(2) allowed differentiation between increased HbA(2) concentrations and concentrations within the reference interval and identification of Hb Lepore. This differentiation was repeatable by MSMS, but not by HPLC, after blood spot samples had been stored for 1 month., Conclusion: This study validates the quantitative delta:beta-globin peptide ratio as a surrogate marker of HbA(2) and demonstrates the potential of rapid peptide-based MSMS for multiplexed, high-throughput protein biomarker characterization and quantification.