Optical spectroscopic methods are highly sensitive in the detection of the biochemical changes occurring in the tissue as it proceeds from normal to dysplastic and malignant conditions [4]. Many groups have studied the fluorescence spectra of cervical tissue under normal and malignant http://www.selleckchem.com/products/ganetespib-sta-9090.html condition [5�C10]. It has been shown that there are noticeable differences in the spectrum, arising from changes in tissue components [11]. In our earlier studies we have analyzed the protein profiles of serum and Pap smear in cervical malignancy, using HPLC-LIF (High Performance Liquid Chromatography-Laser Induced Fluorescence) [12�C14] technique. Our system has been found to give highly reproducible protein profiles and is capable of detecting sub-femtomole quantities of proteins in 20 microlitter of a diluted sample [15].
In the present study we have used the HPLC-LIF protein profiling technique for analysis of cervical tissue samples from normal individuals and cervical cancer patients. The errors from heterogeneous nature of samples were eliminated by homogenizing the entire sample for protein profiling. Possible subjective nature of histo-pathological diagnosis is removed by rigorous mathematical/statistical pattern analysis of the protein profile to give objective diagnosis. The HPV status of the samples was not considered in the present experiments, since the main aim of the study was to see the utility of this method as a preliminary diagnostic technique in small hospitals and clinics, where facilities for such examination may not be available.
Our studies show that the tissue protein profile can be used for early detection and staging of cervical malignancy with high specificity and sensitivity. The results are presented and discussed in this paper.2. Methods2.1. Experimental SetupThe instrumentation has been discussed in detail elsewhere [14, 15]. The HPLC system consists of an HP 1100 gradient system, Rheodyne 7725 Injection port and Biphenyl Reversed Phase narrow bore column (Vydac diphenyl, 2.1 �� 250mm, 5��m, 300?). Protein fluorescence was excited by irradiation of the HPLC effluent in a quartz capillary flow cell with 257.5nm from a frequency doubled Ar+(Innova 90C FreD, Coherent, California, USA) laser.
Protein profiles (Chromatograms) were recorded by measuring the fluorescence intensity of eluted proteins with respect to time using double monochromator (Jobin Yvon DH10 SPEX, New Jersey, USA), Chopper (EG&G model 651), Photomultiplier (Hamamatsu R 453, New Jersey, USA), and Lock-in Amplifier (EG&G model 7265) system interfaced to a computer. The experimental conditions were Laser power: Batimastat 15mW, Chopping Frequency: 20Hz, Monochromator slits: 2mm (Spectral band pass 8nm), Monochromator wave length setting: 340nm, PMT voltage: ?850 volts, Lock-in Amplifier time constant: 2 seconds, and Lock in Amplifier gain: 6dB.2.2.