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theses

Spectrophotometric measurements, both manual and automated, are extensively used in the clinical laboratory. Thousands of such measurements per day on rather diverse equipment are made in different laboratories across the country. Erroneous data reported to a physician may adversely affect patients. Inapparent errors in the spectrophotometric measurement step in an analytical procedure are critical and cause a false result that leads to the wrong assumption, intervention, or treatment.

The research objective is to explore the replacement of spectrophotometers in protein measurement and cell viability assays. In this study, the protein dye-binding methods (Bromocresol Purple and the Bradford assays) were used to test the validity of the proposed method. Four solutions were used with each method (N=4). A digital camera was used to take a picture of the reaction well instead of a spectrophotometer. Data were extracted from the digital image and used to drive the polynomial regression equation. The equation was used to estimate the concentration of the unknown samples. The results of the Bromocresol experiment was compared to a gold standard (Siemens VISTA 500).
The cell viability experiment was conducted using the images collected from an experiment performed by Shashi et al. The experiment used breast tissue and Paclitaxel. Matlab was used to count the number of viable cells in the cell culture. The results of the image analysis were compared to the results obtained from the manual cell count using trypan blue assay and the spectrophotometric count using the MMT dye assay.

The proposed protein measurement method was equivalent to the gold standard and within the allowable total error of 10%. The average error index (y-x)/TEA was -0.24, with a range of -0.88 to 0.00. The largest error-index occurred at a concentration of 4.00 g/dl. Both methods correlated well with a correlation coefficient of R=0.999.
The Digital Image Processing (DIP) results were compared to the MTT assay results to determine whether the methods are equivalent within the allowable total error (TEA) of 10 %. The difference between the two methods was within allowable error for 3 of 6 specimens (50.0%). The average error index (Y-X)/TEA was 0.87, with a range of 0.00 to 1.68. The largest error-index occurred at 53.21%. A similar comparison study was performed between the DIP and the trypan blue assay. For the trypan blue and DIP, the methods were equivalent within the allowable total error of 10 %. The difference between the two methods was within allowable error for 4 of 6 specimens(66.7%). The average error index (Y-X)/tea was 0.59, with a range of 0.00 to 1.40. The largest error-index occurred at a concentration of 37.90 %.

The results obtained from the proposed image analysis technique are equivalent to the results obtained using the spectrophotometric assay in the protein measurement experiment. The proposed Digital Image Processing technique for the cell viability experiment exhibited improvement in the number of counted viable cells when compared to the conventional biochemical methods. In conclusion, digital image analysis can be used to replace spectrophotometers in protein and cell viability measurement experiments.

ETD_11070

doi:10.7282/t3-wjvv-6v55

Ph.D.

Includes bibliographical references

ETD doctoral

The author owns the copyright to this work.