Biomedical Tracers: Methods for Visualizing Biological Systems

1. Biomedical Tracers Biology 685 University of Massachusetts at Boston created by Kenneth L. Campbell, PhD

2. Introduction Chemical and bioanalytical methods: Make things visible that normally are not This is done by observing energy output from the observed system without viewer intervention, e.g., natural phosphorescence, or after introduction of a source of energy or a means of energy modulation into the system, e.g., radioactivity, laser illumination, X-rays, or x-ray contrast agents, a magnetic field, or heavy isotopes. Tracers change energy output or absorption by the system analyzed.

3. Methods are Dimensional 1D assays may Qualify (Demonstrate), +/-, Y/N Quantify (Measure) Semiquantitative (subjective, dis- continuous, quantal): 0, +1, +2… Quantitative (objective, continuous, calibrated) 2D, 3D, 4D methods use maps that yield spacial and/or temporal information.

4. Dimensionality of Methods (cont.) 2D: qualitative or quantitative result over an area, e.g., gray-scale intensity by pixel in a CRT screen or in a photograph or X-ray film. 3D: qualitative or quantitative result over a volume, e.g., color & color intensity by pixel in a CRT screen or photo; gray-scale intensity by voxel in a hologram. 4D: qualitative or quantitative result over a volume through time, e.g., change in color & color intensity by pixel in a CRT screen over time; or color & color intensity change by voxel in a hologram.

5. Controls All methods require positive & negative controls to define the potential range of response of the method & to verify its proper functioning during use. Quantitative methods also require the use of a set of independently established calibrators to serve as references against which to gauge responses of unknown samples.

6. Method Validation & Operational Parameters LOD Specificity Precision Accuracy Bias Parallelism Biological verification

7. LOD Methods must distinguish a specific signal, s, from background, nonspecific signals or noise, n. The ratio s/n defines method LOD, “limit of detection,” “minimal measurable dose,” “detectability.” Ability to clearly distinguish one level of signal, s1, from the next, distinct level, s2, describes “sensitivity.” It depends on the slope of the analytical response curve.

8. Specificity Uniqueness of the signal for a specific molecule, molecular feature, or molecular transition defines method “specificity.” “Cross-reactivity” & “non-specificity” describe departures from uniqueness & lack of absolute specificity in a method.

9. Precision Reproducibility of measurements across identical samples, subjects, or measurement repetitions defines “precision” of the method. This is normally reported as the %CV, “coefficient of variability,” determined as the ratio, in %, of standard deviation of s over mean of s for a given level of input. It may be presented as a profile of %CV vs level of input, or as a mean for a range of inputs or for the entire method.

10. Accuracy For quantitative methods the principle analytical goal of “accuracy” is achieved by having measurements produce results for calibrator samples or subjects that faithfully describe or repeat known values for these scale-defining materials. Calibrators are established by use of reference methods independent of the method being calibrated.

11. Calibrators These should have a direct link to the defining objects or comparators held by the Bureau of Standards (NIST) or its European counterpart; e.g., a method to quantify a chemical fluorometrically may use calibrators made by dissolving weighed amounts of the chemical into known volumes of a solvent. The scale & pipettes used should be calibrated by weights & volumetric ware prepared to NIST specifications that are compared to reference standard materials.

12. Bias Method “bias” is a systematic departure of method results from those obtained by a different, often prior, “reference,” “gold standard” method; e.g., spectral methods may demonstrate a negative bias compared to gravimetric methods due to slight impurities in the weighed materials & immunoassay results may demonstrate a positive bias compared to mass-spectral results because of a lesser specificity in antibody-based methods.

13. Parallelism Uniformity of analytical slope for response curves or surfaces (s vs input, or dose) for different dilutions of a given sample or calibrator determines “parallelism,” a method characteristic necessary for precise, accurate analytical responses in quantitative methods.

14. Biological Verification If methods are applied to biological materials to ascertain the state of a cellular or physiological function, the analytical test needs to be evaluated for biological accuracy or relevance. This is done using a 2x2 comparison matrix in which independently known biological status, e.g., by subsequent birth, death, or demonstration of disease, is used as a reference for evaluating accuracy of analytical test results.

15. Biological Verification (cont.) State of nature, True biological status  error, chosen P, e.g., 0.05 1-  + Power true +, TP type I false +, FP + Method Result 1- , 1- P type II false -, FN Confidence true -, TN  error, ‘lack of power’ Sensitivity = 100 x TP/(TP+FP) Specificity = 100 x TN/(TN+FN) Efficiency = 100 x (TP+TN)/Total Predictive value of + = 100 x TP/(TP+FP) Predictive value of - = 100 x TN/(TN+FN)