Determination of iohexol and iothalamate in serum and urine by capillary electrophoresis.

Estimation of glomerular filtration rate (eGFR) is essential to assess kidney function. Iodine-containing contrast agents detection by HPLC has been proposed as a safe alternative for inulin or radioactive compounds. However, HPLC is a time-consuming and labor-intensive method. The aim of this study was to develop an assay for iohexol and iothalamate using capillary electrophoresis. Iohexol and iothalamate were directly analyzed by CE in serum and urine, using photometric detection (246 nm). Serum peak height was proportional to iohexol and iothalamate concentrations. Detection limits for iohexol and iothalamate were 10 and 5 mg/L. Limits of quantification were 13.0 and 15.0 mg/L. Within-run CVs were 4.9 and 6.5%; between-run CVs 3.1-9.9% and 3.8-13.7%. A good correlation was observed between CE and HPLC: y = 1.1703x + 5.017 (iohexol) and y = 0.7807x + 11.01 (iothalamate; (y = concentration obtained by CE [mg/L], x = concentration obtained by HPLC [mg/L]). In addition, CE allowed to determine urinary iohexol concentration. Although the detection limit for CE was higher than for HPLC, CE can still be used for eGFR determination. Advantages of this high-throughput method are the absence of sample pretreatment and a minimal sample volume requirement.

Long-term stability of laboratory tests and practical implications for quality management.

BACKGROUND: Long-term stability of analytical performance is required for adequate patient management. We investigated the use of patient data to document test stability, and the relevance of observed instabilities on a surrogate medical outcome. We used multiyear patient and internal quality control (IQC) data from two laboratories for tests to monitor chronic kidney and thyroid disease. METHODS: We plotted moving means of the 50th percentiles of stratified patient data and of the daily IQC means. We evaluated observed instabilities based on goals inferred from the analytes' biological variation and investigated their effect on classification of results against reference intervals. RESULTS: Patient and IQC data generally matched well, except for analytes, for which other than analytical variation sources prevailed. Analytical instabilities were predominantly due to reagent/calibrator lot changes, however, for immunoassays also to within-lot instabilities, urging frequent recalibrations. The relevance of biased results on medical decisions ranged from negligible to very pronounced, indicating the need for assessment of analytical performance in relation to quality goals inferred from biological variation. CONCLUSIONS: Patient percentiles offer great potential to assess/monitor the medium- to long-term analytical stability of a test within certain constraints. Differences in analytical quality between assays can significantly affect medical outcome.

"Good samples make good assays" ­ the problem of sourcing clinical samples for a standardization project.

Clinical samples are the cornerstone in all aspects related to in vitro diagnostic testing. They are particularly valuable in the process of establishing/validating metrological traceability, because they eliminate commutability issues potentially associated with artificial calibrators. Therefore, they are essential for IFCC standardization projects. However, sourcing clinical specimens is particularly challenging. It mostly turns out that only dedicated supply sources can accommodate the varying specifications within reasonable timelines. Here we describe the torturous experience in this regard of the IFCC Working Group for Standardization of Thyroid Function tests (since transformed into a Committee). We always focused on obtaining high quality samples in sufficient volume to serve all project participants. We applied a step-up approach: in phase I, we used high volume (200 mL of plasma/serum) single donations from apparently healthy individuals, and switched in phase II and III to medium-sized volume clinical samples (15 ­ 30 mL) from well-defined patient categories. In the first two phases we observed for some assays a sample-related discrepant analytical performance for total/free triiodothyronine and thyroid stimulating hormone (TSH), whereas in phase III we faced a severe delay in obtaining the relevant panels for free thyroxine (FT4) and TSH (n = 90 and n = 100, respectively). Additional experiments only allowed us to exclude hypothesized causes of the observations. We believe that there would be merit in a collaborative effort by chairholders of similar projects to establish a sample procurement infrastructure based on a solid relationship with commercial supply sources with the support of a significant number of committed clinicians.

IFCC international conventional reference procedure for the measurement of free thyroxine in serum: International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Working Group for Standardization of Thyroid Function Tests (WG-STFT)(1).

The IFCC Working Group for Standardization of Thyroid Function Tests proposes a candidate international conventional reference procedure (RMP) for measurement of the amount-of-substance concentration of free thyroxine in plasma/serum at physiological pH 7.40 and temperature (37.0°C). The unit for reporting measurement results is, by convention, pmol/L. The RMP is based on equilibrium dialysis isotope dilution-liquid chromatography/tandem mass spectrometry (ED-ID-LC/tandem MS). The rationale for proposing a conventional RMP is that, because of the physical separation step, it is unknown whether the measurement truly reflects the concentration of free thyroxine (FT4) in serum. Therefore, the ED part of the RMP has to strictly adhere to the following conditions: use of a dialysis buffer with a biochemical composition resembling the ionic environment of serum/plasma as closely as possible; buffering of the sample to a pH of 7.40 (at 37.0°C) before dialysis, however, without additional dilution; dialysis in a device with a dialysand/dialysate compartment of identical volume and separated by a membrane of regenerated cellulose and adequate cut-off; thermostatic control of the temperature during dialysis at 37.0°C±0.50°C. The convention does not apply to the ID-LC/tandem MS part, provided it is eligible to be nominated for review by the Joint Committee for Traceability in Laboratory Medicine. Here, we describe the ED procedure, inclusive its validation and transferability, in greater detail. We recommend a protocol for successful calibration, measurement and monitoring of the accuracy/trueness and precision of the candidate conventional RMP. For details on our ID-LC/tandem MS procedures, we refer to the Supplement.

Traceability to a common standard for protein measurements by immunoassay for in-vitro diagnostic purposes.

BACKGROUND: There is a lot of confusion about the meaning of "measuring a protein in biological fluids" for in-vitro diagnostic purposes. This is due to a lack of understanding of metrological concepts and of acceptance of a pragmatic metrological concept for mixture analysis. METHODS: We describe the metrological concepts that apply to measurement of proteins. We propose a pragmatic reference measurement system for protein analysis. We investigate the feasibility of the approach with TSH as example. RESULTS: The reference measurement system for a protein should be viewed as a dynamic continuum from discovery to translation into an SI-component. A quasi surrogate component-mixture may be defined by recommendations for epitopes that immunoassays should recognize. The all-procedure trimmed mean is proposed as surrogate reference measurement procedure. Traceability is established by transfer of the IU of an WHO standard to a panel of commutable sera. Investigation of the TSH-example showed that the approach may be feasible. CONCLUSIONS: The proposed pragmatic concept would be a major step towards traceability of protein measurements by immunoassay. It would allow a staged introduction of standardization during the continuum from discovery of a protein to full scientific understanding and transformation to the SI-unit.