Standardization and quality control of quantitative microscopy in pathology

Standardization and quality control of quantitative microscopy techniques are distinct but related concerns. The first deals with the great variety of quantitative methods, measured features, and even response variables used in investigation of biological or clinical processes. The latter deals with reproducibility of results from those investigations across time and test performance sites. Though distinct, efforts for standardization and quality control are inherently interactive. Consensus on standard methods, instrumentation, and data analysis is hard to achieve in fields developing as rapidly as quantitative microscopy. Consensus is possible, however, on the issues that affect test performance and interpretation. For example, issues of speciment type, fixation, processing, and staining affect image cytometry just as they do flow cytometry. Raw data acquisition issues include area sampling rules and fidelity of optical and sensor systems (light wavelength, glare/stray light, lens aberrations, numerical aperture, depth of focus, scan precision, pixel spacing and depth, sensor linearity, and stability). Intermediate data issues are primarily related to image foreground/background segmentation techniques—automated versus manual, object‐specific versus field‐based. Data reduction and interpretation procedures also provide many roads for divergence from uniformity. Each of these issues must be considered in terms of its effect on comparability and utility of quantitative microscopy results. Quality control for quantitative microscopy is as important as standardization for its use in research programs and with clinical specimens. The sine qua non of quality control is comparison of experimental results against a known “correct” value to estimate accuracy, and against other experimental results to estimate precision. Intralaboratory quality control often uses internal standards, but can also use analysis of separate specimens with feature values known to a specified precision. Such separate specimens can also be used for interlaboratory, or “survey,” quality control efforts. In any of these settings, limits must be established by which to declare a test in or out of control. The proper values of those limits depend on the accuracy and precision required for confident use of test results for a specific purpose. Standardization and quality control are challenging requirements for effective multicenter use of cytometry or any other technology to establish surrogate endpoints of disease progression.