UG Laboratorium voor Toxicologie/Ref4U
Abnormal thyroid function has important public health consequences. Disagreement between results from different free thyroxine (fT4) methods has been a problem that limits their clinical utility. Not necessarily for diagnosis and/or monitoring of patients, but e.g. for establishing common reference intervals and clinical decision limits, to develop evidence-based clinical practice guidelines and application of consistent standards of medical care.
To reach a solution, a reference measurement procedure (RMP) for fT4 was developed based on equilibrium dialysis isotope dilution-liquid chromatography-tandem mass spectrometry. The equilibrium dialysis (ED) part of the RMP has to adhere to strictly determined parameters. In this project a robustness study evaluates the performance of different ED materials to prove their suitability for analytical inter-laboratory studies.
A set of parallel tests was performed with the endorsed RMP against the introduction of new ED-materials (i.e. other cell designs or differences in membrane cut-off). The results show that the ratio between the volume of sample per area of exposed membrane has a big influence on the time needed to reach equilibrium. The best suited cells for the procedure – Dianorm Macro 1 S - were the ones with a bigger ratio. An increase in the molecular weight cut-off of the membrane (from 5 kDa to 10 kDa) was also proved possible.
Given the influence of the cell’s design on the dialysis’ time, a study to determine the time needed to reach equilibrium is advised for all different cell-types prior to their use in the RMP.
The aim of this bachelor thesis is to integrate a method for a quantitative determination of GHB in a routine laboratory. The method, which is already developed and validated, has to be transferred to the GC-MS in the routine laboratory. After validation, the determination of GHB can be added to the scope of ISO 17025 accreditation. With this accreditation the Laboratory of Toxicology proves that it works with a good functioning management system and that the obtained results are reliable.
The original method is completely validated. Hence to integrate this method in the routine toxicological laboratory only the instrument specific characteristics have to be validated. Consequently the limit of detection (LOD), limit of quantification (LOQ), linearity, accuracy and precision were tested.
No tests were performed for the LOD and LOQ. The LOD was determinated on 1 µg/ml in the original methode. Because the Laboratory of Toxicology works with a cut-off level of 5 µg/mL no additional experiments were performed. Only the signal to noise ratios for the lowest calibrator and the calibrator closest to the cut-off level were verified in order to confirm that these meet the specifications for LoD and LoQ respectively. The results of the S/N ratio of the LOD and LOQ were both higher than the criteria of respectively three for the LOD and five for the LOQ.
A six-point calibration curve was tested for five days, and its linearity was evaluated. Finaly, we used weighted lineair regression (1/x2) to calculate the results. Note, at each concentration level, the response was corrected for the response of the blanc.
To test the accuracy and precision three quality control (QC) samples were made, independent from the calibration curve. They were tested for five days in a row together with the calibration curve. Quality specifications were: a bias within ± 15% (within ± 20% for the lowest calibrator) and a preciosion, expressed as CV, less than 15% (or 20% for the lowest calibrator).
The results for two of the three QC samples are between the criteria for accuracy, only for the lowest sample, the accuracy is borderline. More measurements are needed to increase the confidence in the result. Also for the precision, although the results are below the specifications, from a statistical point of view more measurements are needed to confirm these observations. Finally, results calculated with the calibration curve were compared with results calculated via 1-point calibration. The rationale was that, the 1-point calibration curve would be the routine labortories first screening test. If a results is higher than the cut-off level, it would be quantitated with using a calibration curve. We could prove that results calcualted in either way are comparable, and the conclusions made for accuracy and precision are similar.
Katleen van Uytfanghe