Jagannathan Ramesh

Diagnostic potential of Fourier-transform infrared microspectroscopy and advanced computational methods in colon cancer patients

Colon cancer is the third leading class of cancer causing increased mortality in developed countries. A polyp is one type of lesion observed in a majority of colon cancer patients. Here, we report a microscopic Fourier transform infrared (FTIR) study of normal, adenomatous polyp and malignant cells from biopsies of 24 patients. The goal of our study was to differentiate an adenomatous polyp from a malignant cell using FTIR microspectroscopy and artificial neural network (ANN) analysis. FTIR spectra and biological markers such as phosphate, RNA/DNA derived from spectra, were useful in identifying normal cells from abnormal ones that consisted of adenomatous polyp and malignant cells. However, the biological markers failed to differentiate between adenomatous polyp and malignant cases. By employing a combination of wavelet features and an ANN based classifier, we were able to classify the different cells as normal, adenomatous polyp and cancerous in a given tissue sample. The percentage of success of classification was 89%, 81%, and 83% for normal, adenomatous polyp, and malignant cells, respectively. A comparison of the method proposed with the pathological method is also discussed.

Novel spectral method for the study of viral carcinogenesis in vitro.

Fourier transform infrared (FTIR) spectroscopy is a unique technique for the laboratory diagnosis of cellular variations based on the characteristic molecular vibrational spectra of the cells. Microscopic FTIR was used to investigate spectral differences between normal and malignant fibroblasts transformed by retrovirus infection. A detailed analysis showed significant differences between cancerous and normal cells. The contents of vital cellular metabolites were significantly lower in the transformed cells than in the normal cells. In an attempt to identify the cellular components responsible for the observed spectral differences between normal and cancerous cells, we found significant differences between DNA of normal and cancerous cells.

A comparative study of gallstones from children and adults using FTIR spectroscopy and fluorescence microscopy

BackgroundCholelithiasis is the gallstone disease (GSD) where stones are formed in the gallbladder. The main function of the gallbladder is to concentrate bile by the absorption of water and sodium. GSD has high prevalence among elderly adults. There are three major types of gallstones found in patients, White, Black and Brown. The major chemical component of white stones is cholesterol. Black and brown stones contain different proportions of cholesterol and bilirubin. The pathogenesis of gallstones is not clearly understood. Analysis of the chemical composition of gallstones using various spectroscopic techniques offers clues to the pathogenesis of gallstones. Recent years has seen an increasing trend in the number of cases involving children. The focus of this study is on the analysis of the chemical composition of gallstones from child and adult patients using spectroscopic methods.MethodsIn this report, we present FTIR spectroscopic studies and fluorescence microscopic analysis of gallstones obtained from 67 adult and 21 child patients. The gallstones were removed during surgical operations at Soroka University Medical Center.ResultsOur results show that black stones from adults and children are rich in bilirubin. Brown stones are composed of varying amounts of bilirubin and cholesterol. Green stones removed from an adult, which is rare, was found to be composed mainly of cholesterol. Our results also indicated that cholesterol and bilirubin could be the risk factors for gallstone formation in adults and children respectively. Fluorescence micrographs showed that the Ca-bilirubinate was present in all stones in different quantities and however, Cu-bilirubinate was present only in the mixed and black stones.ConclusionsAnalysis based on FTIR suggest that the composition of black and brown stones from both children and adults are similar. Various layers of the brown stone from adults differ by having varying quantities of cholesterol and calcium carbonate. Ring patterns observed mainly in the green stone using fluorescence microscopy have relevance to the mechanism of the stone formation. Our preliminary study suggests that bilirubin and cholesterol are the main risk factors of gallstone disease.

FTIR microspectroscopy of malignant fibroblasts transformed by mouse sarcoma virus

Fourier transform infrared microspectroscopy (FTIR-MSP), which is based on the characteristic molecular vibrational spectra of cells, was used to investigate spectral differences between normal primary rabbit bone marrow (BM) cells and bone marrow cells transformed (BMT) by murine sarcoma virus (MuSV). Primary cells, rather than cell lines, were used for this research because primary cells are similar to normal tissue cells in most of their characteristics. Our results showed dramatic changes in absorbance between the control cells and MuSV124-transformed cells. Various biological markers, such as the phosphate level and the RNA/DNA obtained, based on the analysis of the FTIR-MSP spectra, also displayed significant differences between the control and transformed cells. Preliminary results suggested that the cluster analysis performed on the FTIR-MSP spectra yielded 100% accuracy in classifying both types of cells.

FTIR Microscopic Studies on Normal, Polyp, and Malignant Human Colonic Tissues

Fourier-Transform Infrared Spectroscopy (FTIR) employs a unique approach tooptical diagnosis of tissue pathology based on the characteristic molecularvibrational spectra of the tissue. The biomolecular changes in the cellularand sub-cellular levels developing in abnormal tissue, including a majorityof cancer forms, manifest themselves in different optical signatures, whichcan be detected in infrared microspectroscopy. This report has two parts. Inthe first part, we report studies on normal, premalignant (polyp) andmalignant human colonic tissues from three patients with different stages ofmalignancy. Our method is based on microscopic infrared study (FTIR-microscopy)of thin tissue specimens and a direct comparison with traditional histopathologicalanalysis, which serves as a “gold” reference. The limited dataavailable showed normal colonic tissue has a stronger absorption thanpolypoid tumor and cancerous types over a wide region in a total of 100measurements. Detailed analysis showed that there is a significant decreasein total carbohydrate, phosphate and possibly creatine contents for polyp andcancerous tissue types in comparison to the controls. The same trend is maintainedin seven other patients studied. The second part consists of an analysis showingthe influence of various independent factors such as age, sex and grade of malignancy. Ourpreliminary results suggest that among the above three factors, age and gradeof malignancy have significant effect on the metabolites level, but sex has onlyminor effect on the measured spectra. Initial results on Linear DiscriminantAnalysis (LDA) showed good classification between normal and malignant cellsof human colonic tissues.

Application of FTIR microscopy for the characterization of malignancy: H-ras transfected murine fibroblasts as an example

Recently, microscopic FTIR is widely used in the field of biology and medicine. FTIR can detect biomolecular changes in the cells and tissues responsible for various disorders. In this report, we characterize the H-ras transfected fibroblasts and its normal control using microscopic FTIR. The intensity of the normal fibroblasts was higher than that of H-ras transfected fibroblasts. Our studies showed significant differences occur in the concentration of vital metabolites upon transformation. The DNA and carbohydrates level decreased in the transformed cells compared to the controls. A linear correlation could be found between the levels of carbohydrates and phosphate, while the RNA/DNA ratio varied inversely with glucose/phosphate levels.

Novel methodology for the follow-up of acute lymphoblastic leukemia using FTIR microspectroscopy

In this report, we present a novel spectroscopic method of follow-up during chemotherapy treatment for B- and T-cell childhood leukemia patients. We isolated peripheral lymphocytes from blood drawn from patients before and after the chemotherapy and collected Microscopic FTIR (FTIR-MC) spectra of the isolated lymphocytes. Our results showed that nucleic acids content decreased in both types of patients. Changes in phospholipids and proteins level could be observed. The overall effects of drugs administered to the patients can be understood at the molecular level using FTIR-MC and these results are expected to stimulate wider applications of spectroscopy in leukemia research.

Preliminary results of evaluation of progress in chemotherapy for childhood leukemia patients employing Fourier-transform infrared microspectroscopy and cluster analysis

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children, but remarkable progress in methods of chemotherapy has increased the cure rate to 80%. The leukemic cells called blasts are eliminated within 7 days of chemotherapy. Clinically, the blast count is monitored directly with the use of blood smears on the basis of specific genetic markers and immunophenotyping methods such as flow cytometry. In this article, we present preliminary results, obtained with the use of Fourier-transform infrared microspectroscopy and cluster analysis, of an approach to monitoring the progress made with chemotherapy in 1 B-cell and 2 T-cell pediatric ALL patients. Our results indicated that the biological marker derived from the spectra did not provide accurate prediction of the progress made with chemotherapy. However, cluster analysis of FTIR-MSP spectra provided good classification of the samples with and without blasts, which correlate satisfactorily with clinical data. Extensive studies are required to substantiate our findings statistically which may have potential application of FTIM in the diagnosis and follow-up of various types of malignancies.

Application of FTIR microspectroscopy for the follow-up of childhood leukemia chemotherapy

Acute Lymphoblastic Leukemia (ALL) accounts for majority of the childhood leukemia. Outcome of children with ALL treatment has improved dramatically. Sensitive techniques are available today for detection of minimal residual disease in children with ALL, which provide insight into the effective cytotoxic treatment. Here, we present a case study, where lymphocytes isolated from two children before and after the treatment were characterized using microscopic Fourier Transform Infrared spectroscopy. Significant changes in the absorbance and spectral pattern in the wavenumber region between 800-1800 cm-1 were found after the treatment. Preliminary analysis of the spectra revealed that the protein content decreased in the T-lymphoma patient before the treatment in comparison to the age matched controls. The chemotherapy treatment resulted in decreased nucleic acids, total carbohydrates and cholesterol contents to a remarkable extent in both B and T lymphoma patients.

Spectroscopic evidence for site-specific cellular activity in the tubular gland in human intestine.

Abstract. The intestinal crypts contain mucus-secreting goblet cells in large numbers. In the tubular gland (crypt), the cells are generated at the bottom and end their life cycle at the top. Recently, FTIR microspectroscopy (FTIR-MC) has been applied in biology and medicine. The characterization of various cellular types using FTIR-MC and its subsequent application for the diagnosis of cancer is becoming a reality. In this report, we investigate the differential cellular activity in the normal tubular gland using FTIR-MC. Our results indicate that the absorbance for the cells in the bottom of the crypt is always higher than those in the upper portion. There are spectral pattern changes and frequency shifts for cells at the bottom and top sites of the normal crypt. Also, the comparison of a normal crypt with a malignant one has been made. This is the first spectroscopic evidence in the literature showing the difference in the cellular activity at different sites in the tubular gland. The reasons for our observations and their implications are discussed.