Logistics & Quality Control
Over 20 years of experience in handling thousands of samples on a monthly basis has materialized into optimized procedures for sample handling, software for data integration, and implementation of quality controls. Our procedures and logistics are set up to avoid human errors, minimize preanalytical variation and assay drift, and maintain adequate assay performance in terms of accuracy, precision and low sample consumption. Customers requesting analysis in cell or tissue extracts find information regarding our validation procedures at the end of this page.
1. Sample handling and data integration
The raw data from the separate platforms and analysis sets are integrated, handled and controlled by a specially designed database. The system generates printouts that match the trays installed into the robots carrying out sample handling (Figure 1). This procedure secures correct location of the separate samples in the assay tray. The data files from the separate platforms are merged by name and stored by the system, which calculates spread between parallel runs, and calculates summary statistics. Outliers are flagged by macros, and all import and export functions are automated by scripting.
2. Validation across platforms
Some stable metabolites are measured at two or three platforms (methods), but against the same calibrators. These metabolites are total homocysteine (tHcy), total cysteine (tCys), cystathionine (Cysta), methionine (Met), tryptophan (Trp), kynurenine (Kyn), histidine (His), ornithine (Orn) and trimethylamine N-oxide (TMAO) (Figure 2). For these analytes, the above-mentioned software calculates the ratios between concentrations obtained by different platforms. Analysis on each platform requires separate pipetting of sample and reagents and separate organization of vials into the sample tray. Therefore, analytical errors related to sample identification, handling, pipetting and organization as well as instrumental performance are likely to be discovered as metabolite ratio(s) different from 1. This ensures adequate sample handling and logistics but also minimizes the possibility of assay interference.
3. Quality control
Each set of 96 vials contains 6 vials with calibrators, 3 with control plasma samples with known biomarker concentrations and one vial without biomarker (blank, to control for carry over). The calibrators are diagonally located (from upper left to lover right corner) across the sample tray to verify positioning of the tray in the autosampler. Large stock solutions of plasma calibrator and control plasma are prepared in sufficient amount to last for years, to minimize chance of assay drift over time. These stocks are aliquoted and stored at -80 °C. New stock are calibrated by comparison with the previous validated stock solution by analysing about 1000 parallel samples over one month. BEVITAL participates in external quality control programs for total homocysteine, MMA, cystatin C (DEKS), vitamin D (DEQAS), 24 amino acids, 3-hydroxybutyrate, creatinine (ERNDIM), vitamin K (KEQAS), 5-methyltetrahydrofolate, cobalamin and hsCRP (LABQUALITY).
4. Minimizing sample consumption and analyte degradation
Logistics have been established to minimize consumption of samples to be analyzed on more than one platform and to avoid preanalytical analyte degradation during and after sample thawing.
5. Long-term assay drift (LAD)
LAD refers to systematic rather than random changes in measured analyte concentrations that may take place over months or years. This may result in slight differences in central estimates and/or distribution when comparing values from repeated analyses carried out years apart. LAD most likely occurs when first measurements are carried out shortly after the method has been launched (new method), is detected in large sample series, and is occasionally observed for biobank samples in spite of no detectable changes in levels for longitudinal control samples. Possible explanations include column replacement, altered instrument performance, and method optimization. To avoid bias from LAD, BEVITAL measures sample series for each project within a short time period, when no deliberate changes or optimization in method design are undertaken. BEVITAL strongly discourages composing data sets where samples from comparison groups are analyzed at different time points. Possible bias from LAD may be reduced but not totally avoided by value correction based on overlapping samples or control plasmas.
6. Analysis of metabolites and biomarkers in extract from cells or tissues
The methods used by BEVITAL have been developed and validated for analyses of metabolites in plasma, serum and/or urine. Occasionally, customers request analysis in cell or tissue extracts. In such cases, the method has to be validated and optimized for the actual extract and extraction procedure, to secure adequate performance of the method. Such validation and optimization include the following steps and considerations:
Initially, the customer must provide representative extracts. This will be used to determine the approximate concentration of metabolite. Such information is required for method validation (steps 2 to 3).
Preferably, the extraction should be carried out using the same procedure and precipitating agent as used for the method optimized for serum/plasma. Extraction could be carried out by the customer according to agreed procedure. Alternatively, extraction of frozen cell pellet or frozen tissue specimen could be carried out by BEVITAL. In both cases, the extraction solution (trichloric acid or perchloric acid) should be supplemented with deuterated internals standard for each metabolite, the concentration of which must be similar to the concentration of each metabolite, as obtained in step 1.
The stability of the metabolite prior to extraction is the responsibility of customer, but if rapid changes in concentration are expected, BEVITAL recommends freeze clamping of tissue or treatment cell pellet with liquid nitrogen. Metabolite stability after protein precipitation in extraction solution is expected to be similar for serum/plasma, cells and tissues.
Interference from endogenous compounds may differ between different sample matrices. Interference will be considered by inspection of elution profile, mass fragmentation pattern and comparisons of different ion pairs.
Assessment of linearity is obtained by serial dilution of the extract up to about 30-fold dilution or until Lower Limit of Detection (LOD). In addition, a standard addition procedure should be carried out by adding unlabelled metabolite to obtain a final metabolite concentration of 2 to about 30 times the endogenous levels. An indication of linearity will be obtained by plotting the ratio of ion intensity of metabolite and deuterated internal standard versus dilution for both experiments.
A measure of analytical recovery will be obtained by comparing metabolite concentrations from the standard addition experiment with concentrations detected by adding the same amounts to the extraction solution, assuming 100 % recovery in the absence of cell/tissue components. Comparison of the absolute ion intensities of metabolites with and without cell/tissue extraction, will give a measure of ion suppression.
A 3 points calibration curve covering expected metabolite concentrations will be set up either by adding metabolite to the extraction solution, or, in case of substantial ion suppression (> 50 %) or low analytical recovery (< 70 %) to a cell/tissue extract (standard addition method).
If requested by the customer, assessment of within-day and between-day precision of the method will be done.
The cost for validation and optimization may be difficult to predict, must be covered by customer, and is calculated from man-months required to accomplish tasks 1 to 8. The current rate is 12000 EURO per man-month.