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BME 115L Lab 4 Gauge R&R Analysis (Statistics) Purpose: In this lab we apply statistical analysis techniques, specifically to determine measurement repeatability and reproducibility and process capability. These are crucial metrics for metrology and are used in a wide range of industries, not just biomedical. In addition to applying statistical analysis techniques, the lab aims to highlight and quantify sources of experimental and/or measurement errors in science. In this lab, we will be evaluating the performance of two different measurement techniques using fluorescence microscopy. In this manner we can compare both the gauge and the process. The gauge is the tool used to make a measurement and is composed to two parts: the instrument and the user. The process is the technique used to manufacture the sample. Repeatability and reproducibility refer to characteristics of the gauge. Key Concepts:

Accuracy Capability Gauge Operator

Precision Repeatability Reproducibility Variance

Laboratory Skills: 1. Develop facility with bright-field microscopy 2. Apply digital and analog measurement techniques 3. Evaluate the capability of a measurement technique 4. Identify errors attributable to different measurement instruments and operators via gauge R&R analysis 5. Perform a capability analysis to evaluate performance of a manufacturing process Background Bright-field microscopy is the most widely known and used type of microscopy. The subject is illuminated via a broad-spectrum source and transmitted or (less often) reflected light is observed. In many cases it is desirable to highlight certain portions of a sample that are of interest either due to their chemical or morphological significance. In these cases, people often make use of fluorescence microscopy. In fluorescence microscopy, a sample is labeled with some photosensitive chemical and interrogated with light of a particular wavelength. This initial light is called the excitation source. It excites electrons within the fluorophor compound. When these electrons return to their original energy state, they emit photons with less energy and a longer wavelength. This emission light can be separated from the excitation light using special light filters and mirrors called dichroic mirrors. We will be using two different gauges to make our measurements: Labsmith inverted epifluorescence microscopes and a Nikon inverted fluorescence microscope. Both tools are research grade equipment and should be treated with appropriate care and respect. You can damage these tools if you are not paying attention!

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Gauge analysis is used to evaluate the ability of the measurement process to precisely measure a sample. The gauge is the measurement instrument (e.g., a ruler is a gauge used to measure length), but can also include the overall technique (e.g., spectrophotometry used to measure concentration). Accuracy is how close a measurement result is to the true value; precision is how much variability there is in the results. A measurement with high accuracy will get very close to the true value; a measurement with high precision will have very low scatter around the mean (low variance). The two primary values used to characterize a measurement technique are repeatability and reproducibility. Repeatability is the variation in measurements made by the same operator on the same instrument of the same or replicate sample. Reproducibility is the variation in measurements made by different operators or on different instruments of the same or replicate sample. (Different instruments does not mean by different techniques; the instruments are duplicates, but manufacturing tolerance means that there may be slight variation. We do not mean, for example, that we are using a ruler for making the first measurement and a tape measure for making the second.) Laboratory 1. The Labsmith SVM-340 synchronized video microscope is set up to capture fluorescence images from your samples. Familiarize yourself with the objective positioning controls and the illumination controls prior to beginning your experiments. 2. Position your slide on the stage and locate your samples. You will need to place a box or other shade over the sample to prevent background illumination from saturating your images. 3. Using the Labsmith uScope software, measure and record the width of you image (in microns). This will be used as a reference to calibrate your images in postprocessing. 4. Using the Labsmith uScope software, capture 10 images for post-processing. 5. In post processing, you will open the images in ImageJ and perform 10 measurements. Make sure to set your global calibration first. Record all of your measurements and the order in which they are made. Each group member should repeat their measurements. 6. All group members should repeat these measurements. Each measurement should be made in the same order or on the same part in each image. You must keep track of which measurement corresponds to which part in each image. Post-lab 1. Perform a gauge R&R analysis in Minitab. In order to perform this analysis, you will need to format your data properly: every measurement should be on a separate row with columns for the measurement value, measurement number, operator, and part number.

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Table 1: Example data format for use in Minitab Measurement Measurement # Part 1234.5678 1 1 1256.890 2 1 1245.876 1 2

Operator Jill Jill Jill

The rule of thumb for samples in gauge analysis is to have 10 measurements taken by at least 2 different operators and have at least 2 replicates. 2. Select Gage R&R Study (Nested) and select the appropriate columns for measurement, part, and operator. The Nested option is necessary because we are assuming that the operators are measuring different parts. The results of this analysis will be several plots that will show the measurement variance that is attributed to the gauge (microscope) repeatability and the operator reproducibility. There are additional components, such as standard deviation of the repeatability and reproducibility variance. 3. The Gage R&R Study (Nested) function in Minitab applies an ANOVA-type analysis. For comparison, manually calculate the Repeatability and Reproducibility according to the table included in the Appendix. For additional information on this analysis, please see the attached paper posted on Canvas. 4. Perform a process capability analysis for both stents and microspheres. The capability analysis reflects how well a process meets its specifications. To this end, you will need to specify upper and lower limits. For microsphere measurements, choose limits that are 10% and 50% on either side of the mean. The capability analysis will return three parameters: C , C , and sigma. In Minitab, select Process Capability Sixpack. This analysis reports C , C , sigma, and an analysis of the normality of the data. Normality is a critical analysis parameter because the assumption of normality is implicit in the above analysis.
p pk p pk

C is the process capability and is calculated assuming that the mean lies equidistant between the upper and lower specification limits (USL and LSL, respectively).
p

Cp =

USL ! LSL 6!
pk

where sigma is the standard deviation. C is a similar calculation, except that it does not assume that the mean is centered between the specification limits:

"USL ! ! LSL % C pk = min # , & $ 3! 3! '

where is the mean. Minimum process capabilities depend on the process maturity and application, but in general, if C is greater than 1.0, the process is minimally capable of reaching its specifications.
p

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Appendix Table A.1: Example data for manual gauge R&R study.

Note: WR or Within range is the absolute value of the difference between measurements 1 and 2 for a given operator and part.

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Table A.2: Summary of equations used for gage R&R study assuming the data follow a standard distribution. The equations are based on one standard deviation.