Signal-to-noise contribution of principal component loads in reconstructed near-infrared Raman tissue spectra.

The overall quality of Raman spectra in the near-infrared region, where biological samples are often studied, has benefited from various improvements to optical instrumentation over the past decade. However, obtaining ample spectral quality for analysis is still challenging due to device requirements and short integration times required for (in vivo) clinical applications of Raman spectroscopy. Multivariate analytical methods, such as principal component analysis (PCA) and linear discriminant analysis (LDA), are routinely applied to Raman spectral datasets to develop classification models. Data compression is necessary prior to discriminant analysis to prevent or decrease the degree of over-fitting. The logical threshold for the selection of principal components (PCs) to be used in discriminant analysis is likely to be at a point before the PCs begin to introduce equivalent signal and noise and, hence, include no additional value. Assessment of the signal-to-noise ratio (SNR) at a certain peak or over a specific spectral region will depend on the sample measured. Therefore, the mean SNR over the whole spectral region (SNR(msr)) is determined in the original spectrum as well as for spectra reconstructed from an increasing number of principal components. This paper introduces a method of assessing the influence of signal and noise from individual PC loads and indicates a method of selection of PCs for LDA. To evaluate this method, two data sets with different SNRs were used. The sets were obtained with the same Raman system and the same measurement parameters on bladder tissue collected during white light cystoscopy (set A) and fluorescence-guided cystoscopy (set B). This method shows that the mean SNR over the spectral range in the original Raman spectra of these two data sets is related to the signal and noise contribution of principal component loads. The difference in mean SNR over the spectral range can also be appreciated since fewer principal components can reliably be used in the low SNR data set (set B) compared to the high SNR data set (set A). Despite the fact that no definitive threshold could be found, this method may help to determine the cutoff for the number of principal components used in discriminant analysis. Future analysis of a selection of spectral databases using this technique will allow optimum thresholds to be selected for different applications and spectral data quality levels.

Raman spectroscopy of bladder tissue in the presence of 5-aminolevulinic acid.

Raman spectroscopy has the ability to provide differential diagnosis of different cancers with high sensitivity and specificity. A major limitation in its clinical application is the weak nature of Raman signal, which inhibits scanning large surface areas of tissues. In bladder cancer diagnosis, fluorescence-guided endoscopy with 5-aminolevulinic acid (5-ALA) has gained interest as a technique that can provide such spatial differentiation, thus improving early detection and more complete removal of superficial tumors. However, several studies have demonstrated the poor specificity of this modality. Combining fluorescence with Raman spectroscopy could improve its diagnostic capability. However, little is known about the effect of agents such as 5-ALA on Raman spectra of tissue. In this paper, we present measuring Raman spectroscopy from benign and malignant bladder tissues in the presence of 5-ALA and attempt to evaluate the potential to discriminate between different pathologies. Raman spectra were recorded from 92 bladder biopsies without 5-ALA and 38 biopsies with 5-ALA using a Raman microspectrometer system at 830nm excitation. Empirical and multivariate statistical techniques were used for data analysis. Algorithms were developed to determine the effect of 5-ALA on tissue and its influence on the prediction ability of a preliminary benign/malignant prediction model. In samples with 5-ALA, an overall decrease in Raman intensity was observed when compared to the Raman spectra from samples without 5-ALA. Additionally, differences in relative intensities at 1270 and 1330cm(-1) were also noted. However, significant differences were observed in the Raman spectra of benign and malignant samples with 5-ALA indicating the potential of using Raman spectroscopy for discriminating bladder cancer in the presence of 5-ALA. The Principal-Component fed Linear-Discriminant Analysis (PCA/LDA) algorithm derived from biopsies in the absence of 5-ALA used to predict biopsies in the presence of 5-ALA resulted in an overall sensitivity and specificity of 42.6% and 71.1%, respectively. This suggests the presence of 5-ALA in tissue affects the Raman spectra. A PCA/LDA algorithm based on fluorescence information (i.e. PpIX fluorescence positive or negative) and the Raman spectrum of 5-ALA biopsies, had a sensitivity and specificity of 100% and 80.8%, respectively. This study demonstrates that applying 5-ALA affects the Raman spectra of bladder tissues. However, benign/malignant differentiation can be accomplished with a preliminary PCA/LDA algorithm, suggesting the potential of a combined diagnostic modality in vivo.

Reduced specificity of 5-ALA induced fluorescence in photodynamic diagnosis of transitional cell carcinoma after previous intravesical therapy.

OBJECTIVE: Photodynamic diagnosis (PDD) for the detection of bladder cancer has become a diagnostic tool in several hospitals. Several studies have reported different rates of false positive biopsies using 5-aminolevulinic acid induced fluorescence. In this study we evaluated the effect of previous intravesical therapy on the false positive biopsy rate. METHODS: Two hours prior to endoscopy 1.5g ALA dissolved in 50ml 1.4% NaHCO(3) solution was instilled intravesically. For fluorescence excitation a blue light source (D-light, Karl Storz) was used. Under white and fluorescence light guidance, tumor locations were recorded, cold cup biopsies were taken and tumors were resected. Patients were divided into 3 groups, last intravesical therapy (IVT) less than 6 months prior to PDD, last IVT longer than 6 months before PDD and no previous IVT. RESULTS: In total 917 biopsies were taken in 249 procedures of fluorescent and non-fluorescent areas. White light endoscopy revealed 270 and PDD 378 of in total 390 tumors, resulting in a sensitivity of 97% and specificity of 49% for PDD. Pathologic evaluation considered 270 fluorescent biopsies as false positive. The rate of false positive biopsies was 25.7% in the group No IVT, 30.6% in the group PDD-IVT >6 months, whereas in the group "within 6 months after intravesical therapy" the rate was 39.6% (p<0.025). When premalignant lesions such as dysplasia II are considered tumor the difference between the groups is even more significant (p<0.001). CONCLUSIONS: The procedure has a high sensitivity for superficial bladder cancer and decreases the number of overlooked lesions. Recent intravesical therapy results in significantly more false positive fluorescent biopsies. Since patient outcome might predominantly be determined by the early detection and subsequent treatment of (pre)malignant tissue we suggest that PDD is justified even shortly after intravesical therapy.