FTIR spectroscopy of biofluids revisited: an automated approach to spectral biomarker identification

Abstract

The extraction of disease specific information from Fourier transform infrared (FTIR) spectra of human body fluids demands the highest standards of accuracy and reproducibility of measurements because the expected spectral differences between healthy and diseased subjects are very small in relation to a large background absorbance of the whole sample. Here, we demonstrate that with the increased sensitivity of modern FTIR spectrometers, automatisation of sample preparation and modern bioinformatics, it is possible to identify and validate spectral biomarker candidates for distinguishing between urinary bladder cancer (UBC) and inflammation in suspected bladder cancer patients. The current dataset contains spectra of blood serum and plasma samples of 135 patients. All patients underwent cytology and pathological biopsy characterization to distinguish between patients without UBC (46) and confirmed UBC cases (89). A minimally invasive blood test could spare control patients a repeated cystoscopy including a transurethral biopsy, and three-day stationary hospitalisation. Blood serum, EDTA and citrate plasma were collected from each patient and processed following predefined strict standard operating procedures. Highly reproducible dry films were obtained by spotting sub-nanoliter biofluid droplets in defined patterns, which were compared and optimized. Particular attention was paid to the automatisation of sample preparation and spectral preprocessing to exclude errors by manual handling. Spectral biomarker candidates were identified from absorbance spectra and their 1(st) and 2(nd) derivative spectra using an advanced Random Forest (RF) approach. It turned out that the 2(nd) derivative spectra were most useful for classification. Repeat validation on 21% of the dataset not included in predictor training with Linear Discriminant Analysis (LDA) classifiers and Random Forests (RFs) yielded a sensitivity of 93 ± 10% and a specificity of 46 ± 18% for bladder cancer. The low specificity can be most likely attributed to the unbalanced and small number of control samples. Using this approach, spectral biomarker candidates in blood-derived biofluids were identified, which allow us to distinguish between cancer and inflammation, but the observed differences were tiny. Obviously, a much larger sample number has to be investigated to reliably validate such candidates.

Improved protocols for vibrational spectroscopic analysis of body fluids

“…[62,[67][68][69] and limitations due to the presence of water are often quoted in recently published materials [70]. Therefore, in the present study, different approaches will be described and proposed as alternatives in order to demonstrate and highlight the feasibility to record high quality data without the requirement for water free samples.…”

“…A commonly employed approach to analyzing the composition of biological fluids using FTIR is to deposit a drop of the solution on a suitable substrate such as CaF 2 and air dry the sample before collection of spectra in transmission mode [62,63,70,71]. The process concentrates the analytes from the solution, potentially allowing better signal to noise, but results in a physically and chemically inhomogeneous sample and, as demonstrated in the previous section, the spectra of the molecular components can be significantly altered in the condensed form.…”

Assessing the challenges of Fourier transform infrared spectroscopic analysis of blood serum

“…Recently, Ollesch et al have successfully addressed the issue of reproducibility of dried blood-related materials by the application of a robotic spotting system, in combination with vacuum drying [6]. A preparation technique for a high volume of dried samples offers the ability of rapid screening.…”

“…Over the years, there have been frequent reports of both transmission and attenuated total reflectance (ATR) measurements of blood-derived samples for diagnostic applications such as bovine spongiform encephalopathy (BSE) [4], cardiology [5], bladder cancer [6], brain cancer [7] and ovarian cancer [8]. On first inspection, the application of infrared spectroscopy for the analysis of blood serum may seem straight forward to the uninitiated.…”

Vibrational spectroscopy of biofluids for disease screening or diagnosis: translation from the laboratory to a clinical setting

“…In the handling of some biofluids such as blood, there remain challenges to be overcome [98]. Whilst there is a large body of compelling evidence to support the application of vibrational spectroscopy to biofluids such as blood to triage patients for particular conditions such as myocardial infarction [99,100], the integration of these techniques into standard clinical practice remains some way off [101] and definitive clinical trials are needed to define their true diagnostic accuracy [102]. The enormous advantage of a real-time analytical monitoring device is that in the face of emerging disease such as neurodegeneration, one could take simple medications to slow down the progress of the pathology [103].…”