Raman Spectroscopic Detection and Quantification of Macro- and Microhematuria in Human Urine

Hematuria refers to the presence of blood in urine. Even in small amounts, it may be indicative of disease, ranging from urinary tract infection to cancer. Here, Raman spectroscopy was used to detect and quantify macro- and microhematuria in human urine samples. Anticoagulated whole blood was mixed with freshly collected urine to achieve concentrations of 0, 0.25, 0.5, 1, 2, 6, 10, and 20% blood/urine (v/v). Raman spectra were obtained at 785 nm and data analyzed using chemometric methods and statistical tests with the Rametrix toolboxes for Matlab. Goldindec and iterative smoothing splines with root error adjustment (ISREA) baselining algorithms were used in processing and normalization of Raman spectra. Rametrix was used to apply principal component analysis (PCA), develop discriminate analysis of principal component (DAPC) models, and to validate these models using external leave-one-out cross-validation (LOOCV). Discriminate analysis of principal component models were capable of detecting various levels of microhematuria in unknown urine samples, with prediction accuracies of 91% (using Goldindec spectral baselining) and 94% (using ISREA baselining). Partial least squares regression (PLSR) was then used to estimate/quantify the amount of blood (v/v) in a urine sample, based on its Raman spectrum. Comparing actual and predicted (from Raman spectral computations) hematuria levels, a coefficient of determination (R²) of 0.91 was obtained over all hematuria levels (0-20% v/v), and an R² of 0.92 was obtained for microhematuria (0-1% v/v) specifically. Overall, the results of this preliminary study suggest that Raman spectroscopy and chemometric analyses can be used to detect and quantify macro- and microhematuria in unprocessed, clinically relevant urine specimens.