Graeme Clemens

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

There are many approaches to measuring the infrared spectrum of a blood serum sample. Naturally, each approach will have both advantages and disadvantages. We report on the progress of the application of infrared spectroscopy in the field of blood serum analysis towards clinical application, with a focus on prostate cancer. In order to perform a high-powered study with clinical relevance, choosing the most suitable approach must undergo careful consideration. We review the possibilities of using different sample preparation methods and speculate upon the potential pitfalls of both transmission and attenuated total reflectance (ATR) techniques.

The action of all-trans-retinoic acid (ATRA) and synthetic retinoid analogues (EC19 and EC23) on human pluripotent stem cells differentiation investigated using single cell infrared microspectroscopy

All trans-retinoic acid (ATRA) is widely used to direct the differentiation of cultured stem cells. When exposed to the pluripotent human embryonal carcinoma (EC) stem cell line, TERA2.cl.SP12, ATRA induces ectoderm differentiation and the formation of neuronal cell types. We have previously generated synthetic analogues of retinoic acid (EC23 and EC19) which also induce the differentiation of EC cells. Even though EC23 and EC19 have similar chemical structures, they have differing biochemical effects in terms of EC cell differentiation. EC23 induces neuronal differentiation in a manner similar to ATRA, whereas EC19 directs the cells to form epithelial-like derivatives. Previous MALDI-TOF MS analysis examined the response of TERA2.cl.SP12 cells after exposure to ATRA, EC23 and EC19 and further demonstrated the similarly in the effect of ATRA and EC23 activity whilst responses to EC19 were very different. In this study, we show that Fourier Transform Infrared Micro-Spectroscopy (FT-IRMS) coupled with appropriate scatter correction and multivariate analysis can be used as an effective tool to further investigate the differentiation of human pluripotent stem cells and monitor the alternative affects different retinoid compounds have on the induction of differentiation. FT-IRMS detected differences between cell populations as early as 3 days of compound treatment. Populations of cells treated with different retinoid compounds could easily be distinguished from one another during the early stages of cell differentiation. These data demonstrate that FT-IRMS technology can be used as a sensitive screening technique to monitor the status of the stem cell phenotype and progression of differentiation along alternative pathways in response to different compounds.

Introducing discrete frequency infrared technology for high-throughput biofluid screening

Accurate early diagnosis is critical to patient survival, management and quality of life. Biofluids are key to early diagnosis due to their ease of collection and intimate involvement in human function. Large-scale mid-IR imaging of dried fluid deposits offers a high-throughput molecular analysis paradigm for the biomedical laboratory. The exciting advent of tuneable quantum cascade lasers allows for the collection of discrete frequency infrared data enabling clinically relevant timescales. By scanning targeted frequencies spectral quality, reproducibility and diagnostic potential can be maintained while significantly reducing acquisition time and processing requirements, sampling 16 serum spots with 0.6, 5.1 and 15% relative standard deviation (RSD) for 199, 14 and 9 discrete frequencies respectively. We use this reproducible methodology to show proof of concept rapid diagnostics; 40 unique dried liquid biopsies from brain, breast, lung and skin cancer patients were classified in 2.4 cumulative seconds against 10 non-cancer controls with accuracies of up to 90%.

Investigating the use of Raman and immersion Raman spectroscopy for spectral histopathology of metastatic brain cancer and primary sites of origin

It is estimated that approximately 13 000 people in the UK are diagnosed with brain cancer every year; of which 60% are metastatic. Current methods of diagnosis can be subjective, invasive and have long diagnostic windows. Raman spectroscopy provides a non-destructive, non-invasive, rapid and economical method for diagnosing diseases. The aim of this study was to investigate the use of Raman and immersion Raman spectroscopy for diagnosing metastatic brain cancer and identifying primary sites of origin using brain tissue. Through spectral examination, the Raman peaks at 721 cm−1 and 782 cm−1 were identified as being the most distinct for discriminating between the glioblastoma multiforme (GBM), metastatic and normal brain tissue spectra. A ratio score plot of these peaks calculated the classification sensitivities and specificities as 100% and 94.44% for GBM, 96.55% and 100% for metastatic brain, and 85.71% and 100% for normal brain tissue. Principal Component-Linear Discriminant Analysis (PC-LDA) also showed discrimination between normal, GBM and metastatic brain tissue spectra. We also present, for the first time, the use of Raman spectroscopy to investigate primary site of origin for metastatic brain cancer and any biochemical differences between different primary and metastatic cancer using linked samples. This study revealed interesting spectral differences in the amide regions showing changes in the biochemistry of the metastatic brain cancer from the primary cancer.

Investigating optimum sample preparation for infrared spectroscopic serum diagnostics

Biofluids, such as serum and plasma, represent an ideal medium for the diagnosis of disease due to their ease of collection, that can be performed worldwide, and their fundamental involvement in human function. The ability to diagnose disease rapidly with high sensitivity and specificity is essential to exploit advances in new treatments, in addition the ability to rapidly profile disease without the need for large scale medical equipment (e.g. MRI, CT) would enable closer patient monitoring with reductions in mortality and morbidity. Due to these reasons vibrational spectroscopy has been investigated as a diagnostic tool and has shown great promise for serum spectroscopic diagnostics. However, the optimum sample preparation, optimum sampling mode and the effect of sample preparation on the serum spectrum are unknown. This paper examines repeatability and reproducibility of attenuated total reflection (ATR) compared to transmission sampling modes and their associated serum sample preparation with spectral standard deviation of 0.0015 (post pre-processing) achievable for both sampling modes proving the collection of robust spectra. In addition this paper investigates the optimum serum sample dilution factor for use in high throughput transmission mode analysis with a 3-fold dilution proving optimum and shows the use of ATR and transmission mode spectroscopy to illuminate similar discriminatory differences in a patient study. These fundamental studies provide proof of robust spectral collection that will be required to enable clinical translation of serum spectroscopic diagnostics.

Design and biological evaluation of synthetic retinoids: probing length vs. stability vs. activity

All trans-retinoic acid (ATRA) is widely used to direct the differentiation of cultured stem cells. When exposed to the pluripotent human embryonal carcinoma (EC) stem cell line, TERA2.cl.SP12, ATRA induces ectoderm differentiation and the formation of neuronal cell types. We report in this study that novel polyene chain length analogues of ATRA require a specific chain length to elicit a biological responses of the EC cells TERA2.cl.SP12, with synthetic retinoid AH61 being particularly active, and indeed more so than ATRA. The impacts of both the synthetic retinoid AH61 and natural ATRA on the TERA2.cl.SP12 cells were directly compared using both RT-PCR and Fourier Transform Infrared Micro-Spectroscopy (FT-IRMS) coupled with multivariate analysis. Analytical results produced from this study also confirmed that the synthetic retinoid AH61 had biological activity comparable or greater than that of ATRA. In addition to this, AH61 has the added advantage of greater compound stability than ATRA, therefore, avoiding issues of oxidation or decomposition during use with embryonic stem cells.

Preclinical screening of anticancer drugs using infrared (IR) microspectroscopy

High-throughput label-free technologies such as IR microscopy can objectively assess the effect of drugs upon cellular systems, offering the potential of a valuable preclinical tool that can aid in the drug development process.

Elemental and molecular profiling of licit, illicit, and niche tobacco

The recognition of differences between regulated large-scale mass manufactured products and the uncontrolled cultivation of tobaccos for illicit purposes plays a significant role within identification of provenance. This research highlights X-ray fluorescence and Fourier transform infrared spectroscopy as useful analytical techniques for the rapid identification of tobacco samples of unknown provenance. Identification of key discriminative features within each technique allowed for the development of typical characteristic profiles for each type of tobacco. Analysis using X-ray fluorescence highlights chlorine, potassium, calcium and iron as key elemental indicators of tobacco provenance. Significant levels of chlorine seen within Snüs samples prompted attempts to visualise chlorine containing regions and structures within the sample. Scanning electron microscopy images showed crystalline structures visible within the Snüs tobacco, structures which Energy dispersive X-ray spectroscopy qualitatively confirmed to contain chlorine. Chloride levels within Snüs samples were quantified using ion chromatography with levels found to range between 0.87mgmL(-1) and 1.28mg. Additionally, FTIR indicated that absorbances attributed to carbonyl stretching at 1050-1150cm(-1), alkane bending at 1350-1480cm(-1) and amide I stretching at 1600-1700cm(-1) highlighting a spectral fingerprint region that allowed for the clear differentiation between different types of tobaccos using PCA analysis, but was limited by differentiation between provenance of cigarettes and hand rolled tobacco. X-ray fluorescence and Fourier transform infrared spectroscopy yielded different information with regards tobacco discrimination and provenance, however both methods overall analysis time and cost reduced indicating usefulness as potential handheld analytical techniques in the field.