Patent Application: US-25025399-A

Abstract:
tissue cells are preserved for infrared spectroscopic analysis by soaking a fresh cellular specimen in a 0 . 5 to 3 . 0 % concentration inorganic salt solution , removing excess salt solution by centrifuging , placing the remaining damp specimen on an infrared optical window and then drying the specimen in a flow of room temperature air such that a dried , spectrally preserved specimen is obtained within 2 minutes . alternatively , a cellular specimen in wet and fresh form is placed on the surface of a crystal water - soluble inorganic salt in the form of an infrared window to dissolve some of the salt and thereby cover the specimen with the dissolved salt solution and then drying the specimen as described above in less than 2 minutes . in both cases , the drying results in the formation of a salt crystal film covering the surface of the cells for spectral preservation . another feature is an infrared spectroscopic method in which polymorph effects are avoided by obtaining an infrared spectrum of pure polymorph cells and subtracting this from superimposed infrared spectra of tissue cells and polymorphs .

Description:
in this specification , fresh biological tissue or cells in natural or cultured form means biological tissue or cells newly obtained , cultured tissue or cells , or cells exfoliated from fresh biological tissues which are substantially free of degradation by exposure to room temperature . a typical room temperature is in the range of about 15 - 28 ° c . the specimens may be prepared from fresh microtome sections of tissue biopsy , punched or needle tissue biopsy , cultured cells or exfoliated cells . typical of the exfoliated cells are papanicolaou smears , cervical specimens , endocervical specimens , ectocervical specimens , vaginal specimens , uterus specimens or bronchial specimens . a typical tissue may be liver tissue , and the anomaly an indication of malignancy in the liver tissue . typical cells may be ovarian epithelial cells . when the specimen is tissue , the tissue may be cervical tumor tissue and the anomaly an indication of malignancy in the tissue . when the specimen is exfoliated cells , the specimen may be obtained from scraping , brushing , washing , secretions , exudates or transudates from various organs and tissues . fig1 shows an infrared spectra in the frequency region 950 to 1500 cm − 1 obtained from a fresh , normal healthy , exfoliated cervical cells and after air drying the specimen at 28 ° c . for 10 minutes ; fig2 shows an infrared spectra in the frequency region 2800 to 3020 cm − 1 obtained from a fresh , normal health , liver tissue cell specimen that was not treated with a preservative and after keeping the specimen at room temperature and measured after the passage of various time periods ; fig3 shows a similar infrared spectra to that shown in fig2 but for normal healthy and malignant specimens ; fig4 shows an infrared spectra in the frequency region 900 to 1500 cm − 1 obtained from a specimen of fresh , exfoliated cervical cells which was diagnosed as high grade dysplasia and after keeping the specimen at room temperature for three days in saline ; fig5 shows an infrared spectra in the frequency region 950 to 1500 cm − 1 obtained from a specimen of fresh , normal , exfoliated cervical cells and a specimen of pure polymorphs isolated from human blood ; fig6 shows an additional infrared spectra in the frequency region 950 to 1500 cm − 1 obtained from the addition of the spectrum of a specimen of fresh , normal , exfoliated cervical cells and the spectrum of pure polymorphs with various ratios between the cervical cells and polymorphs ; fig7 shows an infrared spectra in the frequency region 950 to 1500 cm − 1 obtained from a specimen of fresh , normal , exfoliated cervical cells and after keeping the specimen at room temperature for four hours in 1 % nacl solution ; fig8 shows an infrared spectra in this frequency region 950 to 1500 cm − 1 obtained from a fresh , normal cervical cell specimen and immediately after suspending the specimen in 5 % nacl solution , or saline ; fig9 shows an infrared spectra in the frequency region 950 to 1500 cm − 1 obtained from a fresh , normal cervical cell specimen and after air drying for 30 minutes at room temperature ; fig1 shows an infrared spectra in the frequency region 900 to 1500 cm − 1 obtained from a 1 % nacl solution treated wet specimen of fresh , normal healthy , exfoliated cervical cells and after the wet specimen was air dried at room temperature on a sample holder for two minutes ; fig1 shows an infrared spectra in the frequency region 900 to 1800 cm − 1 obtained from a preservative treated and air dried specimen of fresh , normal , healthy , exfoliated cervical cells and after keeping the specimen at room temperature for thirty six months ; fig1 shows an infrared spectra in the frequency region 900 to 1800 cm − 1 obtained from a preservative treated and air dried specimen of fresh , exfoliated cervical cells which has been diagnosed as low grade dysplasia , and after keeping the specimen at room temperature for thirty six months ; fig1 shows an infrared spectra in the frequency region 950 to 1500 cm − 1 obtained from a preservative treated specimen ( by the second method ) of fresh , exfoliated cervical cells which has been diagnosed as moderate dysplasia , and after keeping the specimen at room temperature for thirty six months ; fig1 shows an infrared spectra in the frequency region 950 to 1500 cm − 1 obtained from a fresh , exfoliated cervical cell specimen which has been diagnosed as moderate dysplasia and immediately after suspending the specimen in 0 . 1 % nacl solution ; and fig1 is a block diagram of an apparatus for detecting the presence of anomalies in biological specimens using infrared spectroscopy . a typical apparatus for conducting the infrared spectral analyses is shown in fig1 . it includes an infrared source 1 , infrared beam focusing mirrors 2 and 3 , a sample cell and holder 4 , a michelson interferometer 5 , a infrared light detector 6 , a computer 7 , and a readout 8 . in the following tests , the infrared source l , infrared beam focusing mirrors 2 and 3 , the holder of the sample cell and holder 4 , michelson interferometer 5 , and infrared light detector 6 were components of a nicolet magna ir 550 fourier - transform infrared spectrometer obtainable from thermo instruments ( canada ) inc ., mississauga , ontario , canada . the sample cell of the sample cell and holder 4 for measuring the transmission infrared spectra had a single infrared optical window on which the specimen was deposited or two infrared optical windows between which the specimen was placed . the sample cell of the sample cell and holder 4 for measuring atr infrared spectra was a contact samplertm atr accessory ( spectra - tech , inc .) with a znse crystal , obtained from spectra - tech , inc ., stamford , connecticut . in operation , a biological tissue or cell sample was placed in a transmission or atr sample cell of the sample cell and holder 4 , a beam of infrared light from the source 1 , which had been condensed by the focusing mirror 2 , was passed through the sample in the sample cell and holder 4 and was focused to the detector 6 through michelson interferometer 5 by the focusing mirror 3 . any infrared absorption by an anomaly in the specimen was detected by the michelson interferometer 5 and the detector 6 , which , in turn , was computed by the computer 7 to give a readout at the readout 8 . the computer readout may be programmed for the readout 8 to directly indicate whether the sample is a normal healthy one or one which contains an anomaly , which may be , for example , benign , dysplasia , or malignant . in a typical procedure for obtaining fresh , exfoliated cervical cells , a cervical specimen diagnosed as normal and healthy was obtained from a patient by exfoliation with a spatula . within one - two minutes , the spatula containing the specimen was immersed in a centrifuge tube containing an aqueous solution of about 0 . 9 %- 1 . 0 % sodium chloride ( by weight ). prior to immersion , the specimen was in a generally wet , fresh - specimen from characteristic of exfoliated cells that had been freshly removed from a living body . the volume of the aqueous saline solution was about 10 ml . the weight of specimen material was about 40 mg . immediately after immersion , the sample was vortexed to transfer the exfoliated cervical cells from the spatula into the saline solution . after the spatula was removed from the centrifuge tube , the sample was centrifuged for about 4 min . at about 4000 rmp in a standard biological centrifuge unit ( model centra cl2 , available from international equipment company , mass .). the supernatant , excess nacl solution , was decanted leaving the specimen , soaked with nacl solution , at the bottom of the centrifuge tube . a fresh , normal healthy cervical cell specimen was obtained from a patient by exfoliation with a spatula . this was collected by immersion in an aqueous saline solution and centrifuging to obtain a damp saline cell pellet . a portion of the pellet was placed on a silicon infrared window and immediately subjected to infrared spectral analysis . a further portion of the pellet was allowed to sit at 28 ° c . to air dry for 10 minutes , after which it was also subjected to infrared spectral analysis . fig1 shows the infrared spectra in the 950 to 1490 cm − 1 region of the above two specimens . a substantial difference can be seen in the absorption bands due to the hypertonic crenation and degradation of the cells during the long drying and heating process . a fresh , normal healthy liver cell specimen was obtained and tested for deterioration when left for a period of time at room temperature . fig2 shows the infrared spectra in the 2800 to 3000 cm − 1 range of the fresh , normal healthy liver cell specimen , and after keeping the specimen at room temperature for various periods of time ( spectrum 1 is from fresh , normal healthy tissue cell specimen ; and spectra 2 , 3 , 4 , 5 , designate the infrared spectra for the same specimen kept at room temperature for 1 , 2 , 3 , and 4 hours , respectively ). the infrared spectrum of the fresh , normal healthy liver cell specimen kept at room temperature for 4 hours is similar to that of a malignant liver cell specimen as shown in fig3 . these spectra have demonstrated that the infrared spectra of deteriorated cells will give a false positive diagnosis . for this test , fresh , exfoliated cervical cells were obtained which had been diagnosed as high grade dysplasia . fig4 shows infrared spectra in the frequency region 900 to 1500 cm − 1 obtained from these fresh , exfoliated cervical cells , and after keeping the specimen in saline ( 0 . 9 % nacl ) at room temperature for three days . it is evident from fig4 that after the cervical cell specimen was kept in saline at room temperature for three days , the shape , frequency and intensity of various absorption bands in the entire spectrum were changed dramatically , which indicated that the cervical cells had deteriorated dramatically and cervical cells in a 0 . 9 % nacl solution without a drying process are not preserved . the purpose of this test was to compare the infrared spectrum of pure polymorphs and a normal cervical specimen . the pure polymorphs were extracted from the blood of the patient . the infrared spectrum of pure polymorph cells is characteristic of strong absorption of the nucleic acid band near 1240 cm − 1 and weak absorption of the c - oh band and the glycogen band at 1155 cm − 1 and 1025 cm − 1 , respectively . the infrared spectrum of normal cervical cells is opposite to that of the polymorphs with strong intensities of the 1155 cm − 1 and 1025 cm − 1 bands and weak intensity of the 1240 cm − 1 band . this example shows the removal of polymorph effects on the infrared spectroscopic screening method . a combination of an infrared spectrum of normal cervical cells with various ratios of the infrared spectrum of polymorphs is illustrated in fig6 . the combination of an infrared spectrum of normal cervical cells with an infrared spectrum of pure polymorphs give rise to an intensity increase in the 1240 cm − 1 band and an intensity decrease in the 1155 and 1025 cm − 1 bands . these resulting infrared spectra in fig6 are comparable with those of various precancerous cervical cells and lead to a false positive diagnosis . to remove the polymorph effects on the infrared spectroscopic screening method , the infrared absorption of polymorphs in the infrared spectra of cervical cells are removed by a digital subtraction of the infrared spectrum of pure polymorph cells from the superimposed infrared spectra of tissue cells and polymorphs . the subtraction is performed stepwise with increasing scales of the infrared spectrum of polymorphs until the intensity of the 1240 cm − 1 band reaches the intensity level of normal cervical cells . then inspection is made for the spectral parameters of other regions of the spectrum . if the spectral parameters of the other regions of the spectrum after subtraction are comparable with those of normal cervical cells , then the diagnosis of this cervical specimen is negative from neoplasm . on the other hand , if the infrared spectral parameters and absorption intensities of other regions of the spectrum after subtraction are higher or lower than those of normal cervical cells , then the diagnosis of the specimen is abnormal ( neoplasm ). all these subtraction , calculation and comparison can be performed automatically by building all these features into a software . a test of this process for a groups of 650 cervical specimens in a general population has shown that the false positive rate was reduced from 516 to 4 . 1 % after the polymorph effects were removed . tests were conducted to determine the effect of a 1 % sodium chloride solution on normal cervical cells . the infrared spectrum of normal cervical cells was recorded immediately after freshly removing from a living body and placing in wet pellet form and this was compared with the infrared spectrum of the same normal cervical cells after they were suspended in a 1 % sodium chloride solution for 4 hours at room temperature . the results are shown in fig7 . the spectrum of the normal cervical cells after they were suspended in 1 % sodium chloride without the specific drying process for four hours changed dramatically and is similar to the infrared spectra of abnormal cervical cells . this change in the infrared spectrum indicates that 1 % sodium chloride contains insufficient sodium chloride to preserve the normal cells and the cells in 1 % sodium chloride solution are deteriorated in 4 hours at room temperature . if a salt solution of a very high concentration is used , it may preserve the cells from deterioration . however , it is well known that in a salt solution of a high concentration , biological cells undergo hypertonic crenation , which results in dramatically changes in the molecular arrangement and structure in cells . these changes will certainly alter the infrared spectrum from that of normal cells . a further test was conducted to determine the effect of salt solutions having other concentrations . for this test a specimen of fresh , normal cervical cells was obtained in wet pellet form . part of the specimen was immediately subjected to infrared spectral analysis . also a portion was suspended in 5 % sodium chloride solution and a further portion was suspended in saline solution ( 0 . 9 % nacl ) and these were also immediately subjected to infrared spectral analysis . fig8 shows the infrared spectrum obtained from a specimen of fresh and normal cervical cells in the wet pellet form , and immediately after the cell specimen was suspended in 5 % sodium chloride solution , or saline solution . it is evident that the cervical cells were damaged after the treatment of 5 % sodium chloride and the infrared spectrum of this treated cells changed into a spectrum similar to that of abnormal cells . when the water in a salt solution with suspended tissue cells is evaporated , salt will crystallized and form a solid film on the dried cells . in this case , the cells will certainly be preserved but whether the molecular arrangement and structure in cells are modified by this treatment is unknown . fig9 shows the infrared spectra of a fresh , normal cervical cell specimen and the same specimen after the specimen was suspended in a 1 % sodium chloride solution , centrifuged into pellet and left at room temperature to evaporate its water content for 30 minutes . the infrared spectrum of the cervical cell specimen changed considerably after the cell specimen was treated by 1 % sodium chloride and slowly evaporated at room temperature for 30 minutes . the resulting infrared spectrum is similar to the infrared spectrum of a wet pellet of abnormal cervical cells . this result has demonstrated that the drying time for the cervical cell specimen in the 1 % sodium chloride was too long . during the drying process the concentration of the sodium chloride solution is increased gradually from 1 %. while the cells were still in the wet form , the concentration of the sodium chloride has already increased to such a level to cause the hypertonic crenation of the cells , which resulted in a dramatically change in the molecular arrangement and structure in cells and thus in the infrared spectrum . in order to apply the spectral preservative procedure for the screening of cellular anomalies , the molecular arrangement and structure in the preservative treated and dried cells must not be changed from those of the wet fresh cells . fig1 shows the infrared spectrum of a wet specimen of fresh , normal , healthy , exfoliated cervical cells in wet pellet form and the infrared spectrum of the same specimen after it was spectral preservative treated and dried on an infrared optical window with the procedure described above . it is evident from fig1 that there is substantially no change in the infrared spectrum for the specimen after it was spectrally preserved by air drying at room temperature for 1 - 2 minutes from that of the wet fresh cells . a specimen of fresh , normal healthy , exfoliated cervical cells was obtained and suspended in 1 % sodium chloride solution , then centrifuged to remove excess fluid leaving a damp specimen . portion of the damp sample was placed on a silicon infrared window and this was placed in a stream of room temperature air for 1 - 2 minutes resulting in the drying of the specimen and the formation of a crystal film covering the surface of the cells . this spectrally preserved specimen was subjected to immediate infrared spectral analysis and was kept at room temperature for 36 months after which it was subjected to infrared spectral analysis . fig1 shows the infrared spectra that were obtained in the frequency region 900 to 1800 cm − 1 from the spectral preservative ( 1 % aqueous solution of sodium chloride ) treated and 1 - 2 minutes air dried specimen of fresh , normal healthy , exfoliated cervical cells , and after keeping the dried specimen at room temperature for 36 months . it is evidence from fig1 that there is substantially no change in the spectrum for the spectrally preserved specimen over the period of three years at room temperature . the same procedure was followed as in example 10 , except that the specimen was fresh , exfoliated cervical cells diagnosed as having low grade dysplasia ( cin 1 ). fig1 shows the infrared spectra that were obtained in the frequency region 900 to 1800 cm − 1 from a spectral preservative treated and dried specimen of fresh , exfoliated cervical cells diagnosed as having low grade dysplasia ( cin 1 ), and after keeping the specimen for 36 months at room temperature . it is evident from fig1 that there is substantially no change in the spectra for the treated specimen over the period of three years at room temperature . a specimen of fresh , exfoliated cervical cells diagnosed as having moderate dyplasia ( cin ii ) was obtained . for this test . the wet specimen was placed on the surface of a water - soluble kbr crystal in the form of an infrared optical window and was dried in a stream of room temperature air for less than 2 minutes to obtain a dry sample with a crystal film covering the surface of the cells . fig1 shows the infrared spectrum in the frequency range 950 to 1500 cm − 1 obtained from a preservative treated specimen by this second spectral preservative treatment process and the infrared spectrum of the same specimen after keeping the preservative treated specimen for 36 months at room temperature . it is evident form fig1 that there is substantially no change in the spectra for the treated specimen over a period of three years at room temperature . a specimen of fresh , normal healthy , exfoliated cervical cells which had been diagnosed as moderate dysplasia was obtained . portions were 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