D) All of the above
All three factors—sample preparation techniques, laboratory temperature and humidity, and instrument calibration procedures—can significantly affect the reproducibility of analytical results. 1
Sample Preparation Techniques
Sample preparation is a critical source of variability that directly impacts reproducibility:
Pre-analytical handling represents one of the major sources of variability in laboratory measurements, including factors such as time to fixation, duration of fixation, centrifugation parameters (rotor type, speed, temperature, brake use), and freeze-thaw cycles. 1
Centrifugation procedures are particularly problematic—the recovery of samples depends heavily on rotor type, g-force, temperature, brake use, and centrifugation time, which limits the repeatability of measurements. 1
Storage conditions matter substantially: samples should be stored in low protein binding tubes at -80°C rather than -20°C to prevent degradation, and freeze-thaw cycles should be limited to no more than three. 1
Sample homogenization and mixing before measurement is crucial—samples must be completely thawed, thoroughly mixed, and centrifuged before dispensing to minimize particulates that could interfere with instrumentation. 1
Laboratory Temperature and Humidity
Environmental conditions represent often-overlooked but significant sources of variability:
Temperature control is essential for reproducibility, as testing should ideally be performed at the same time of day due to significant diurnal variation, and environmental temperature can affect both sample stability and instrument performance. 1
Housing conditions for biological samples and reagents can dramatically affect results—even factors like subthermoneutral temperatures have been shown to significantly impact experimental outcomes. 1
Instrument Calibration Procedures
Calibration is fundamental to ensuring accurate and reproducible measurements:
Regular calibration checks are advisable and should include physiologic/biologic validation where healthy laboratory staff perform tests at regular intervals, with results compared to a quality control database. 1
Equipment calibration errors are explicitly recognized as a major factor contributing to variability in clinical measurements. 1
Lack of suitable reference materials for calibration and normalization introduces considerable variation and can render quantification inaccurate, affecting reproducibility of research findings. 1
Systematic calibration protocols should include timed expired gas collections or other gold standard methods to validate measurements, typically accurate to within 2-3% when carefully performed. 1
Additional Critical Factors
Beyond the three listed options, other important considerations include:
Operator variability and adherence to procedures, including proper randomization, blinding, and data handling. 1
Batch-to-batch variation in assays and reagents, which requires good process controls and quality assurance programs. 1, 2
Post-analytical factors such as interpretation methods, image analysis parameters, and reporting procedures. 1
The key pitfall is assuming that high precision (reproducibility between replicates) indicates accuracy—a test can be highly precise but systematically inaccurate if there is bias. 1, 3, 4 Therefore, comprehensive quality control must address all three factors simultaneously to ensure truly reproducible analytical results. 1, 2