Abdullah A. Ahmadzai

Histology Verification Demonstrates That Biospectroscopy Analysis of Cervical Cytology Identifies Underlying Disease More Accurately than Conventional Screening: Removing the Confounder of Discordance

Background Subjective visual assessment of cervical cytology is flawed, and this can manifest itself by inter- and intra-observer variability resulting ultimately in the degree of discordance in the grading categorisation of samples in screening vs. representative histology. Biospectroscopy methods have been suggested as sensor-based tools that can deliver objective assessments of cytology. However, studies to date have been apparently flawed by a corresponding lack of diagnostic efficiency when samples have previously been classed using cytology screening. This raises the question as to whether categorisation of cervical cytology based on imperfect conventional screening reduces the diagnostic accuracy of biospectroscopy approaches; are these latter methods more accurate and diagnose underlying disease? The purpose of this study was to compare the objective accuracy of infrared (IR) spectroscopy of cervical cytology samples using conventional cytology vs. histology-based categorisation. Methods Within a typical clinical setting, a total of n = 322 liquid-based cytology samples were collected immediately before biopsy. Of these, it was possible to acquire subsequent histology for n = 154. Cytology samples were categorised according to conventional screening methods and subsequently interrogated employing attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy. IR spectra were pre-processed and analysed using linear discriminant analysis. Dunn’s test was applied to identify the differences in spectra. Within the diagnostic categories, histology allowed us to determine the comparative efficiency of conventional screening vs. biospectroscopy to correctly identify either true atypia or underlying disease. Results Conventional cytology-based screening results in poor sensitivity and specificity. IR spectra derived from cervical cytology do not appear to discriminate in a diagnostic fashion when categories were based on conventional screening. Scores plots of IR spectra exhibit marked crossover of spectral points between different cytological categories. Although, significant differences between spectral bands in different categories are noted, crossover samples point to the potential for poor specificity and hampers the development of biospectroscopy as a diagnostic tool. However, when histology-based categories are used to conduct analyses, the scores plot of IR spectra exhibit markedly better segregation. Conclusions Histology demonstrates that ATR-FTIR spectroscopy of liquid-based cytology identifies the presence of underlying atypia or disease missed in conventional cytology screening. This study points to an urgent need for a future biospectroscopy study where categories are based on such histology. It will allow for the validation of this approach as a screening tool.

Identification of benzo[a]pyrene-induced cell cycle-associated alterations in MCF-7 cells using infrared spectroscopy with computational analysis

Chemical contaminants, such as benzo[a]pyrene (B[a]P), may modulate transcriptional responses in cells via the activation of aryl hydrocarbon receptor (AhR) or through responses to DNA damage following adduct formation. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy can be employed in a non-destructive fashion to interrogate the biochemical signature of cells via generation of infrared (IR) spectra. By applying to generated spectral datasets subsequent computational approaches such as principal component analysis plus linear discriminant analysis (PCA-LDA), derived data reduction is achieved to facilitate the visualization of wavenumber-related alterations in target cells. Discriminating spectral variables might be associated with lipid or glycogen content, conformational protein changes and phosphorylation, and structural alterations in DNA/RNA. Using this approach, we investigated the dose-related effects of B[a]P in MCF-7 cells concentrated in S- or G₀/G₁-phase. Our findings identified that in PCA-LDA scores plots a clear segregation of IR spectra was evident, with the major spectral alterations associated with DNA/RNA, secondary protein structure and lipid. Dose-related effects were observed and even with exposures as low as 10⁻⁹ M B[a]P, significant (P ≤ 0.001) separation of B[a]P-treated vs. vehicle control cells was noted. ATR-FTIR spectroscopy with computational analysis is a novel approach to identify the effects of environmental contaminants in target cells.

The Syrian hamster embryo (SHE) assay (pH 6.7): mechanisms of cell transformation and application of vibrational spectroscopy to objectively score endpoint alterations

Using morphological transformation as an endpoint, the Syrian hamster embryo (SHE) cell transformation assay (pH 6.7) is an in vitro system with a high sensitivity and specificity for testing the carcinogenic potential of test agents. Advantages of the assay are that SHE cells are metabolically competent, genetically stable and acquire spontaneous transformation with a low frequency; additionally, it detects both genotoxic and non-genotoxic carcinogens. However, in comparison with other short-term mammalian cell assays, it is time consuming, laborious and, most importantly, the visual scoring of morphological transformation might be subjective. In this review, we examine the background to the test and why it has the potential for use in safety risk assessment. Additionally, we propose a novel approach to objectively interrogate and classify SHE colonies using vibrational spectroscopy coupled to a mathematical framework for high-throughput screening. It is our view that this alternative approach has the potential to improve the sensitivity and specificity of the in vitro SHE assay.

Classification of test agent-specific effects in the Syrian hamster embryo assay (pH 6.7) using infrared spectroscopy with computational analysis

The Syrian hamster embryo (SHE) cell transformation assay (pH 6.7) has utility in the assessment of potential chemical carcinogenicity (both genotoxic and non-genotoxic mechanisms of action). The assay uses morphological transformation as an end point and has a reported sensitivity of 87%, specificity of 83% and overall concordance of 85% with in vivo rodent bioassay data. However, the scoring of morphologically transformed SHE cells is subjective. We treated SHE cells grown on low-E reflective slides with benzo[a]pyrene, 3-methylcholanthrene, anthracene, N-nitroso-N-methylnitroguanidine, ortho-toluidine HCl, 2,4-diaminotoluene or D-mannitol for 7 days before fixation with methanol. Identified colonies were interrogated by acquiring a minimum of five infrared (IR) spectra per colony using attenuated total reflection Fourier-transform IR spectroscopy. Individual IR spectra were acquired over a spatial area of approximately 250 × 250 μm. Resultant data were analysed using Fishers linear discriminant analysis and feature histogram algorithms to extract classifying biomarkers of test agent-specific effects or transformation in SHE cells. Clustering of spectral points suggested co-segregation or discrimination of test agent categories based on mechanism of action. Towards transformation, unifying alterations were associated with alterations in the Amide I and Amide II peaks; these were consistently major classifying biomarkers for transformed versus non-transformed SHE cells. Our approach highlights a novel method towards objectively screening and classifying SHE cells, be it to ascertain test agent treatment based on mechanism of action or transformation.

Alterations in the Biomolecular Signatures of Developing Chick Corneas as Determined by Biospectroscopy and Multivariate Analysis

PURPOSE Biospectroscopy tools are increasingly being recognized as novel approaches toward interrogating complex biological structures in a nondestructive fashion. This study was conducted to apply these tools to interrogate alterations in the molecular signatures of developing chick corneas during the onset and development of transparency. METHODS Embryonic chick corneas (n = 46) were obtained at 2-day intervals from embryonic day (E)10 to E18 of incubation and investigated with attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy and Raman microspectroscopy. Resultant spectra were analyzed for variance by using principal component analysis and linear discriminant analysis (PCA-LDA). RESULTS Mean spectra after ATR-FTIR spectroscopy or Raman microspectroscopy derived from corneas at each developmental stage showed some overlap; however, in PCA-LDA scores plots, a clear segregation of spectra was evident, and two-category discrimination indicated that significant molecular alterations occur during tissue morphogenesis. Notable by both techniques was the increasing intensity of DNA signal (1080 cm⁻¹) from E10 onward. Major segregating biomarkers identified by ATR-FTIR spectroscopy between E10 and E18 were in the DNA/RNA (1126 cm⁻¹), glycogen (1045 cm⁻¹), protein (1470 cm⁻¹), and amide II (1512 cm⁻¹ and 1524 cm⁻¹) spectral regions. Raman spectroscopy also identified major distinguishing vibrational modes that included proteins, amino acids (tyrosine, proline phenylalanine, and valine), and secondary structures of proteins (amide I and amide II). CONCLUSIONS The developing chick cornea undergoes significant changes in its biomolecular composition in the E10 to E18 developmental period, with the major changes occurring in the spectral regions associated with DNA/RNA, proteins, glycogen, and secondary protein structures.

Classification of agents using Syrian hamster embryo (SHE) cell transformation assay (CTA) with ATR-FTIR spectroscopy and multivariate analysis

The Syrian hamster embryo (SHE) cell transformation assay (pH 6.7) has a reported sensitivity of 87% and specificity of 83%, and an overall concordance of 85% with in vivo rodent bioassay data. To date, the SHE assay is the only in vitro assay that exhibits multistage carcinogenicity. The assay uses morphological transformation, the first stage towards neoplasm, as an endpoint to predict the carcinogenic potential of a test agent. However, scoring of morphologically transformed SHE cells is subjective. We treated SHE cells grown on low-E reflective slides with 2,6-diaminotoluene, N-nitroso-N-ethylnitroguanidine, N-nitroso-N-methylurea, N-nitroso-N-ethylurea, EDTA, dimethyl sulphoxide (DMSO; vehicle control), methyl methanesulfonate, benzo[e]pyrene, mitomycin C, ethyl methanesulfonate, ampicillin or five different concentrations of benzo[a]pyrene. Macroscopically visible SHE colonies were located on the slides and interrogated using attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy acquiring five spectra per colony. The acquired IR data were analysed using Fishers linear discriminant analysis (LDA) followed by principal component analysis (PCA)-LDA cluster vectors to extract major and minor discriminating wavenumbers for each treatment class. Each test agent vs. DMSO and treatment-induced transformed cells vs. corresponding non-transformed were classified by a unique combination of major and minor discriminating wavenumbers. Alterations associated with Amide I, Amide II, lipids and nucleic acids appear to be important in segregation of classes. Our findings suggest that a biophysical approach of ATR-FTIR spectroscopy with multivariate analysis could facilitate a more objective interrogation of SHE cells towards scoring for transformation and ultimately employing the assay for risk assessment of test agents.

Determination Using Synchrotron Radiation-Based Fourier Transform Infrared Microspectroscopy of Putative Stem Cells in Human Adenocarcinoma of the Intestine: Corresponding Benign Tissue as a Template

The epithelial-cell layer lining the two morphologically and functionally distinct segments of the mammalian intestinal tract, small intestine, and colon is constantly being renewed. This renewal is necessitated by a harsh lumen environment and is hypothesized to be driven by a small population of stem cells (SCs) that are believed to reside at the base of intestinal crypts. A lack of specific markers has hampered previous attempts to identify their exact location. We obtained tissue sections containing small intestine and colon crypts derived from normal (benign) or adenocarcinoma (AC) human intestine. The samples were floated onto BaF2 windows and analyzed using synchrotron radiation-based Fourier transform infrared microspectroscopy via an aperture size of 10 × 10 μm. Derived infrared (IR) spectral data was then analyzed using principal component analysis and/or linear discriminant analysis. Hypothesized cell types (as a function of aperture location along the length of individual crypts) within benign crypts were classed based on exploratory unsupervised IR spectral point clustering. Scores plots derived from individual small intestine crypts consistently generated one or two distinct spectra that clustered away from the remaining cell categories; these were retrospectively classed as “distinct base region” spectra. In these plots, a clear progression of locations along crypt lengths designated as from putative stem cells (SCs) to transit-amplifying (TA) cells to terminally differentiated (TD) cells was observed in benign small intestine and colon crypts. This progression of spectral points was crypt specific, pointing away from a unifying cell lineage model in human intestinal crypts. On comparison of AC-derived spectra versus corresponding benign, a subpopulation of AC-derived spectra suggested a putative SC-like spectral fingerprint; remaining IR spectra were classed as exhibiting TA cell-like or TD cell-like spectral characteristics. These observations could point to a cancer SC phenotype; an approach capable of identifying their in situ location has enormous therapeutic applications.