Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving fixed or stabilized – nonliving microorganism,...
Reexamination Certificate
1999-02-16
2001-08-07
Gitomer, Ralph (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving fixed or stabilized, nonliving microorganism,...
C435S040500, C435S040510
Reexamination Certificate
active
06270986
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to methods for preserving biological tissue specimens to be subject to infrared spectroscopy for detecting a malignant or premalignant anomaly, and for removing the effects of polymorphs for infrared spectroscopic detection of cellular anomalies.
It has been well documented that infrared spectra of various human cancers and precancerous lesions differ substantially from those of the corresponding normal tissues and cells. Several attempts have been made to develop infrared spectroscopic methods for the screening of cytological anomalies, in particular the screening of cervical malignancy or premalignant anomalies. The commonly used cytological screening methods, such as the Pap smear test, are mainly based on the morphological changes of cells, whereas the infrared spectroscopic method is based on the structural changes at the molecular level in cells. Since structural changes at the molecular level occur before morphological changes in abnormal cells, the infrared spectroscopic method is expected to be more accurate than the conventional cytology method in terms of early detection of malignant and premalignant anomalies. A recent clinical study (Ref. 11) has shown that the false negative rate of the infrared spectroscopic method for the screening of the neoplastic cervical cells is about ten times better than the traditional Pap smear test. However, there are still two main obstacles in the infrared spectroscopic method, which cause an extremely high false positive rate and prevent the infrared spectroscopic method from being acceptable for routine cytological screening. These two obstacles relate to problems caused by polymorph effects and in sample preparation.
In many areas of the world, the majority of the cervical specimens exhibit positive results when the infrared spectroscopic method is used. After many years research, we found that this extremely high false positive rate was essentially due to the effects of polymorphs. When a cervical specimen exhibits mild to moderate inflammation, the changes in the molecular arrangement and structure in the cells are insignificant but a considerable amount of polymorph cells are present in the cervical specimen. The presence of a large amount of polymorphs in the cervical cell specimen not only prevents the search for abnormal cervical cells in the specimens under a microscope by the conventional Pap smear test, but also prevents the infrared spectroscopic method from distinguishing a normal cervical specimen from an abnormal one. When polymorphs are present in a cervical specimen, the resulting infrared spectrum is a superimposed spectrum composed of the infrared spectra of cervical cells and polymorph cells. The infrared spectrum of polymorph cells has many features similar to those in the infrared spectra of various precancerous and atypical cells. Consequently, in the presence of polymorphs, a non-neoplastic cervical cell specimen will show an infrared spectrum similar to that of the precancerous or atypia cervical cells, which will lead to a false positive diagnosis.
In many areas of the world, cervical specimens with mild to moderate inflammation are very common place due to the specific environmental effects and sexual behavior in these areas. Therefore, a majority of the cervical specimens from the general public in these areas are accompanied with a significant amount of polymorphs. Consequently, the infrared spectroscopic method may lead to a false positive diagnosis for a majority of the population. This false positive effect of polymorphs must be removed before the infrared spectroscopic method can be adopted to the screening of cervical anomalies for the general population.
Another obstacle in the infrared spectroscopic method is problems in the sample preparation. At the present time, there are several methods to prepare cellular samples for infrared spectroscopic study.
The most common method is the wet process. In this process, exfoliated fresh cells are suspended in saline, centrifuged into a cellular pellet, and some of the wet pellet is then placed on an infrared spectroscopic sample holder for the infrared spectroscopic measurement and analysis.
Benedetti et al., G. Appli. Spectrosc., 44: 1276-1280 (1990) have adopted a method of infrared spectroscopic study for powdered solid samples in which they prepared dry solid powder of fresh biological cells for infrared spectroscopic analysis. In this dry process, fresh lymphocytes were separated from other constituents in the blood by chemicals, and the fresh lymphocytes were dried into a solid. The solid lymphocytes were ground into fine powder with KBr powder. The mixture of solid lymphocyte and KBr was then pressed into a clear solid pellet for infrared spectroscopic analysis. The infrared spectroscopic results obtained by this process are usually inaccurate due to the fact that grinding destroys the cellular form of lymphocytes, creating structural changes at the molecular level which mask those arising from neoplasm and other diseases, and decompose some biomolecules in the cells by the heat generated from the grinding.
In the dry process described by Gal et al., Anticancer Research, 14: 1541-1548 (1994), fresh cultured cells were washed and suspended in normal saline, smeared on a ZnSe window, evaporated for 10 minutes at 37° C. and then the infrared spectrum of the exposed cellular proteins was measured. It is evident from Example 1 that in this process, the intermolecular structure in the cells was destroyed by the hypertonic crenation during the heating and drying process and thus no infrared spectrum of most of the important cellular molecules could be obtained. Only the infrared spectra of the exposed cellular proteins could be measured.
All the present wet and dry sample preparation methods for infrared spectroscopic study of tissue cells are dealing with fresh cells without any preservation. If the fresh cell specimens are not used immediately for infrared spectroscopic analysis, for instance cellular specimens are transported to the pathology laboratory before they are prepared for infrared spectroscopic analysis, the fresh cell specimens must be kept frozen until they are ready for analysis. At room temperature, cellular specimens either in the fresh form or in saline solution will deteriorate very fast. The infrared spectra of deteriorating cells have features similar to those in the spectra of abnormal cells and will lead to a false diagnosis and an increase in the false positive rate.
In many countries, screening of cervical anomalies is done in central laboratories. The transportation of the cervical specimens from the clinics to the central laboratory in the frozen form is extremely impractical. Moreover, in common practices, the cervical cell specimens after preparation are required to be kept for several years for future references in hospitals and clinics. One way to keep the cervical specimens for several years is to store the specimens in a liquid nitrogen tank. For a large number of specimens, such as cervical specimens for screening, to keep the specimens in liquid nitrogen is extremely impractical. The best way to resolve these problems is to fix cellular specimens by preservatives.
The criteria for the selection of preservatives and methods of preservative treatment, which are suitable for the detection of anomalies in tissue cells by infrared spectroscopic technology, are as follows: (1) The preservative must not have any chemical reaction with biomolecules in tissue cells to cause structural changes at the molecular level; (2) The preservative treatment must not damage the intermolecular arrangement and intramolecular structure in cells, which are the basis of detection of anomalies in cells by the infrared spectroscopic method; and (3) The preservative must not have infrared absorption bands at the same frequency regions as the infrared absorption bands of biological tissue cells. Otherwise, the infrared spectra of tissue cells are masked by the infrared spectra of the preserva
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