JPH06507237A - A method for detecting the presence of abnormalities in exfoliated cells using infrared spectroscopy - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] A method for detecting the presence of abnormalities in exfoliated cells using infrared spectroscopy Technical field The present invention provides a method for detecting the presence of abnormalities in exfoliated cells using infrared spectroscopy. Regarding the law.

Background technology For example, secretions, exudates, exudates, scrapings or is a characteristic new tissue thinning in samples of exfoliated tissue or cells from scavenges. It is known that tests for cysts allow diagnosis of diseases. flaking particles Cystology has proven to be of great value in the detection of early cancers of the uterus and cervix. As a routine part of the annual examination for all adult women, , which is usually carried out in the literature: PathologicE xamination, Enfolidative Cytologic 5t udy, Chapter 24, pages 362-365.

Samples of exfoliated tissue or cells may also be used, for example, in the trachea and bronchi, stomach, rectum and Obtained from the colon, ureter, serous sac fluid, cyst fluid, synovial fluid, glandular secretions and exudates, etc. It is possible to Obtain, collect, and, if necessary, preserve these samples. The method used can be varied according to choice.

Each piece of sample collected should not be damaged as a flat layer on a clean microscope slide. For example, it is spread slowly and immersed in an alcohol-ether fixing solution for about an hour. It will be done. After fixing has taken place, the layer is coated with glycerin without drying. , the whole is sandwiched between two glass slides. Then the slide color and microscopic examination by a trained and experienced pathologist or cytologist. He is sent to a laboratory for treatment.

Microscopic diagnosis of sloughed tissue or cells is helpful, but the diagnostic interpretation is: (i) must be carried out by skilled personnel; (ii) e.g. rich in mucus; Not completely reliable due to trichomoniasis infection or atrophy, (iii) Tissues or tissues, e.g., in pre-malignant or early malignant stages; The reason is that it is not possible to accurately detect malignancy in cells.

There is a need for a method to detect the presence of abnormalities in exfoliated cells, and the method In other words, (i) Simple testing of samples and their interpretation does not develop medical proficiency in personnel; can be carried out after a relatively simple course of training by (ii) By taking appropriate precautions, the interpretation of test results can be reliable. Ru, (5) Malignancy in tissues or cells occurs before malignancy or at the stage of early malignancy. This means that it can be detected by

Applicant's Co-pending Canadian Patent Application No. 2008831-1, January 1990 Application filed on the 29th, “Natural or cultured biological tissues and cells by infrared spectroscopic analysis” A method for detecting the presence of abnormalities in J, P, T, T, Wong and B, R igas, living organisms in natural or cultured form by infrared spectroscopy Detecting the presence of abnormalities in tissues or cells (e.g., tissues or cells in a cancerous condition) It has already been proposed to issue

tissue in natural or cultured form containing the cells to be tested or A beam of infrared light is directed at the cells to detect any abnormalities present in the sample, which are characteristic of the anomaly. Has the infrared absorption changed due to the vibration of at least one functional group in the molecule? By determining whether or not an abnormality is detected in at least one frequency range. be done.

The method taught by P. T., T. Wong and B. Rigas is applied to tissue or cultured cells for biopsy procedures. In particular, P, T, T , page 7, line 20 to page 8 of the Wong and B. Rigas patent application. Line 36 is a colonic rectum from the mucosa of the colon immediately after surgical removal of a segment or piece of intestine. Teach the detection of intestinal cancer.

Disclosure of invention According to the present invention, by spectral analysis of infrared absorption characteristics of cells, exfoliated cells can be detected. It has been found that it is possible to detect the presence of abnormalities in. Mucin (viscous) liquid), water, inorganic salts, epithelial cells, red blood cells and fluid white blood cells It does not give rise to any spurious interpretation of the external spectral absorption characteristics.

In the present invention, the presence of abnormalities in exfoliated cells is detected using infrared spectroscopy. A detection method comprising: (a) directing a beam of infrared light onto a specimen of exfoliated cells; and (b) the infrared absorption by the specimen in at least one frequency band. Spectral analysis determines whether anomalies exist in the specimen and determines the infrared absorption characteristics. determining whether at least one change in sex has occurred, the step of determining whether at least one change in sex has occurred; At least one change in external radiation absorption properties is characteristic of the abnormality. is due to vibrations of at least one functional group of molecules present in the specimen Provided is a detection method characterized by comprising the following.

The at least one change in the infrared absorption property increases the absorption strength at a particular frequency. change in frequency, change in frequency at which a particular absorption occurs, or change in the frequency at which a particular absorption occurs. are different pressures applied to the functional groups, which cause a change in frequency to Is possible.

Functional groups of molecules include the following molecules: carbohydrates, nucleic acids, tissue proteins , or a membrane lipid.

The functional group includes phosphodiester groups in nucleic acids, tissue proteins and and one C-OH group in carbohydrates, or one CH in lipids , could be a group.

The functional groups include carbohydrates, phosphodiester groups in nucleic acids, tissues and One C-0f in proteins and carbohydrates (group, or in lipids) There is a possibility that it is one CH or group.

The specimen may be a Babonicolausmia.

The specimen may be a cervical sample.

The specimen may be an endocervical specimen.

The specimen may be a uterine abdominal specimen.

The specimen may be a vaginal sample.

The specimen may be a uterine sample.

Brief description of the drawing In the accompanying drawings, which illustrate by way of example embodiments of the invention, FIG. Block diagram of an apparatus for detecting the presence of abnormalities in exfoliated cells using analysis, In Figure 2, the dashed lines indicate those obtained from a specimen of normal, healthy, exfoliated cervical cells. It was obtained from a specimen of exfoliated cervix cells, and it is not an actual product. For those diagnosed as malignant by conventional cytological methods, Diagram showing the infrared spectrum in the frequency band from 950 to 1350 cm-' , Figures 3 to 5 show specimens of healthy exfoliated cervical cells and conventional cytology. ~11082C-' and ~115 among specimens diagnosed as malignant by conventional methods. The frequency shift of the infrared band at 5 an-' and ~1025 cm-' and A diagram showing changes in intensity ratio between infrared bands at ~1082an-', Figure 6 shows the enlarged and superimposed band at ~1240an-'. Figure 2 shows the infrared spectrum, and Figure 7 shows the frequency. Regarding the band in the region 1190 to 1275an-', see Figure 2. a diagram illustrating the spectrum of the corresponding third power derivative of the infrared spectrum; Figure 8 shows the frequency of oscillations of PO and groups of nucleic acids in exfoliated cells of cervical tissue. Figure 9 shows a comparison of the pressure dependence of the numbers 1140an-' to 1185an −’ normal, healthy, exfoliated cervical cell specimens and infrared band among specimens diagnosed as malignant by conventional cytological methods. Diagram showing an enlargement of Figure 10 shows the corresponding third power derivation of the infrared spectrum shown in Figure 9. A diagram showing the spectrum of Figure 11 shows the positive Normal, healthy, sloughed cervical tissue specimens and conventional cytological methods were used to detect malignant Frequency of component band of C-0 stretching mode among specimens diagnosed as Figure 12 shows a comparison of the pressure dependence of normal avulsed cervical tissue and conventional methylation in membrane lipids between specimens diagnosed as malignant by cytological methods. Figure 13 is a diagram showing a comparison of the pressure dependence of the CH of the chain and the bending mode. Avulsed cervix diagnosed as normal, malignant, and dysplastic by clinical methods in the frequency band from 950 an-' to 1350 cm-' for the sample Diagram showing a comparison of infrared spectra, Figure 14 shows specimens of healthy, exfoliated tumor cells and Frequencies between 950 and 1350 cm-' obtained from specimens diagnosed as FIG. 3 is a diagram showing an infrared spectrum in several bands.

Mode of carrying out the invention Referring to FIG. 1, an infrared source 1, a lens 2, a sample cell and a holder 4, an infrared A spectroscopic analysis device 6, a computing device 8 and a readout device 10 are shown.

In operation, a sample of exfoliated tissue or cells is placed in the sample cell and holder 4. The infrared beam from the light source 1 is focused by the lens 2, and The sample in the radar 4 is allowed to pass through. Infrared absorption by the sample is measured using an infrared spectrometer 6, and the detection result is calculated by a calculation device and read out by the reading device 10. gives a readout. The reading by the computing device indicates that the sample is normal and healthy. or abnormalities (i.e., benign, dysplastic, or malignant). can be set to display directly.

In tests proving the invention, the infrared spectrum of sloughed cervical cells was A specimen was obtained as described with reference to FIG. Surveillance Papanicolaou test (cervical The data were collected from 25 women undergoing cytology. These specimens are above skin cells, inflammatory cells, red blood cells, and bacteria that are part of the normal flora It consists of a.

The tests were present in varying numbers in all specimens and were performed in half by two observers. The quantified non-epithelial cells are shown in the infrared spectrum in the spectral region under study. to no significant extent in the control sample. The hemolysis of red cells studied in the spectral region under investigation is It was shown that there was no significant change in torque.

In further tests, cells from exfoliated scrapings obtained using a brush , collected by gently shaking the brush in a solution of normal salts. This thin The two halves of the cell suspension are pelleted separately by centrifugation. surface floaters is removed, one pellet is frozen in liquid nitrogen, which According to the present invention, infrared absorption will be studied later by spectroscopic analysis. , while a divisible number of other pellets are spread out on a microscope slide, It is fixed and stained with Papanicolaou stain and diagnosed in a conventional manner.

Each specimen was evaluated independently by two experienced physicians, and between these evaluations A complete agreement was reached. The infrared absorption spectrum is as described above with reference to FIG. As in, you got it. Cells are smeared onto the sample holder and after sample application , the spectral pattern was stable for many hours. automatic kale blotting The generator was set to automatically normalize the peak intensities.

The tests carried out are classified into two groups, namely: (i) atmospheric pressure tests; It is described in U.S. Patent No. 4,980,551 dated December 25, 1990 and patented those in which the claimed specimen holder has been found useful; It has been found useful that the sample holder described and claimed in No.

Cervical specimen evaluated as a normal healthy specimen based on conventional cytological evaluation displayed essentially identical infrared spectra.

The spectrum of a cytologically diagnosed malignant specimen is different from that of a normal specimen. There was a marked difference. Results for normal healthy cervical tissue in Figure 2 The results for malignant cervical tissue are shown as a dashed line, and the results for malignant cervical tissue are shown as a solid line. are representative spectra for normal, healthy and malignant cervical specimens. Ru.

The most notable differences involve findings in malignant specimens, such as:

(a) The intensity of the bands at ~1025on-' and ~1047an-' Severe changes in (~1025an-'looks like a shoulder on the band),~ 1082an-', ~1155an-', ~124Jan-', and ~13 03cm-', (b) ~1082cm-', ~1155cm-', and ~ a significant shift of the 1244 cm-' peak; (C) ~970an-' New Imperial Area Peaking Figures 3 to 5 , white circles indicate normal, healthy sloughed cervical tissue, black circles indicate malignant sloughs. These are the results for the uterine cervix tissue. For all of the specimens studied, 4 and averaging over 12 wavenumbers, ~1082an-' and ~115 The shifts in the two peaks at 5ao-' are shown in Figures 3 and 4. ing. Figure 5 shows that between the ~1025an-' and ~1082an-' bands. The results show that the ratio of peak intensities between normal and cancerous tissues differs greatly.

The bands at ~1025 cm-' and ~1047 cm-' are due to carbohydrates. It was found that glycogen contributed overwhelmingly to the strength of these bands. It turned out that it would be donated. ~1082an-' and ~1244an-' The bands are due to symmetric and asymmetric phosphate (PO2−) stretching modes. It turned out to be something. These are caused by vibrations of phosphodiester groups in cell nucleic acids. It turned out to be mainly based. The band at ~1155an-' C- residues of the amino acids serine, threonine, and tyrosine in proteins OH group and carbohydrate C-0) 1 group C-0 stretching mode It turned out that it was caused by de.

The band at ~1303an-' consists of many overlapping bands, and its allocation It has not been confirmed. Finally, the band at ~970an-' is partially As observed in the infrared spectra of model phosphorylated proteins, Due to vibrational modes of anionic phosphate groups, in part due to the vibrational modes of DNA and/or or by RNA molecules.

Referring to Figure 6, Figure 6 is an enlargement of a portion of Figure 2, and the corresponding third power The derived spectrum of - is shown in Figure 7, with a symmetric phosphate stretching band. A more detailed study of the malignant exfoliated uterus, shown by the solid line in Figure 7, revealed that In cervical tissue, it consists of two overlapping bands, one of which is the lower frequency band. area, which is significantly increased compared to normal exfoliated cells. asymmetric PO 2-Stretching zone is the PO2-group at the time the test is performed. is ~1220 an-' when all are hydrogen bonds, and there are no hydrogen bonds. It was known to be larger than ~1240 an-'.

This finding shows that in cervical cancer, in contrast to normal sloughed cervical tissue, , many Pot-groups of nucleic acids are known to be hydrogen bonded. . This conclusion is based on the dependence of the frequency of these bands on pressure, as shown in Figure 8. This is further supported by the white circles in Figure 8, which indicate normal, healthy exfoliation. The black circles indicate cancerous sloughed cervical tissue. The results are shown below.

The frequencies in the low frequency band were found to decrease with increasing pressure, but cancer The high frequency band of It was found that the increase in The low-frequency response to this pressure is due to hydrogen bonding. It is known that the pressure is typical for the hydrogen bond group, and the reason is that the pressure This is because it increases the strength of the stretching, thereby decreasing the frequency of PO2-stretching. be.

On the other hand, the influence of pressure on the high frequency PO,-band Although it is common to all normal tissues, it is a characteristic of non-hydrogen bonding functional groups and It is known to be the result of interaction pressure enhancement.

A similar analysis of the C-0 stretching band in 1155an-' 9 and 10, and in FIG. 9 and 1O, the cervical tissue The results for normal healthy exfoliated cells are shown in dashed lines, and the results for normal healthy exfoliated cells in the cervical tissue are The results for exfoliated cells are shown by the solid line, and the same analysis revealed that the three It became clear that it consists of multiple bands, which are ~1153 cm -', ~11 61 an-' and ~1172 an-', but in normal cervical tissue When compared with the corresponding bands, the first two of these display reduced intensity; The third one displays increased intensity.

As shown in FIG. 11, the frequency of the first two increases as the pressure increases. It was found that the frequency of the third one increases with increasing pressure.

Therefore, these findings suggest that ~1153G+1−' and ~1161cm− ’ component band arises from the stretching vibration of C-OH group of hydrogen bond, while , ~1172ao-' is the stretching vibration of non-hydrogen-bonded C-OH groups. I am praying for the rain.

In Figure 12, the white circles indicate the results for normal healthy exfoliated cells in the cervical tissue. The black circles indicate the results for cancerous exfoliated cells in the cervical tissue, but the results for lipid There is no pressure dependence of the frequency of the bending mode of CH in the methylene chain of ~1466 an is shown in the frequency range from ~1476 an-'. in this band of frequencies Pressure dependence is due to interchain backing and order/distribution of lipid bilayers. It has previously been used extensively to study order properties. lipid bilayer odor It was found that this causes an increase in pressure in the second band of frequencies, and the reason for this is explained. The reason is that the induced conformation and orientation of the methylene chain occurs and the chain This is because the interaction between the components increases. In malignant cervical tissue, pressure , induces a smaller shift in this frequency range compared to normal healthy tissue. It turned out that I was going to wake up. This means that in exfoliated cells of cervical cancer tissue, The methylene chains of lipids are more disorganized than in normal, healthy, exfoliated cells of the cervix. I am sorry to hear that you are in this state.

In Figure 13, the results are shown for normal, healthy, and avulsed cervix specimens. The dashed line shows the results for the dysplastic specimen, and the dash-dotted line shows the results for the malignant specimen. The results are shown as solid lines.

In Figure 13, the specimens with dysplasia compared with the normal specimens are shown. The notable difference is (a) ~1025an-', ~1082cm-', ~1244an-', and severe changes in the intensity of the band at ~1303 cm-' and ( b) For frequencies in the band ~1047an-' and ~1082cm-' a significant shift in includes.

Figure 13 also shows the malignant specimen compared to the normal and dysplastic specimens. The notable differences for the specimens are (a) ~970an-', ~1025a n-', ~1047cm-', ~1082an-', ~1155an-', ~1244an-' and ~1303an-' Severe changes and (b) ~1025an-', ~1047an-', ~1082an''1, ~! 155 an-', a significant shift in the frequency of the band.

These studies showed that several important changes were associated with sloughed cervix tissue. It showed that there is. Cancer cells compared to normal, healthy, exfoliated cells in cervical tissue. The amount of glycogen is dramatically reduced in cervical cells with sloughed tissue. It turned out that. This is important for the liver, an organ extremely rich in glycogen. is further confirmed by the fact that they display similar spectral patterns, although the A similar pattern is seen in cancers of the colon, an area of the intestine that is low in glycogen. It is something that cannot be seen.

Known spectrum of glycogen (D-glycogen from mammalian liver) is proposed by the applicant in the frequency range ~975 to 1150 cm-'. The results can be compared with those obtained by the method (Figs. 13 and 14).

Furthermore, the hydrogen bonds of the phosphodiester groups of nucleic acids are It was found to increase in exfoliated cells. The discovery about this spectrum is Biopsy specimens of cancer tissues tested by the applicant of this patent, such as colon, liver, etc. Common to those found in other tests on viscera, skin, vagina, and breasts, etc. Met.

In Figure 14, the results for healthy vaginal specimens are shown by the dashed line, and the results for the malignant specimens are shown by the dashed line. The results for are shown by solid lines.

In Figure 14, there are significant differences between the malignant specimen and the normal specimen. teeth, (a) ~1025an-', ~1154an-', ~1240an-', and severe changes in the intensity of the band at ~1300 cm −’ and (b ) ~1025an-', ~1155cm-', and ~1081an-' a significant shift in the frequency of the band at includes.

These and other tests demonstrate that the present invention sloughed tissue or cells from secretions, exudates, and exudates of various organs or tissues. Useful for detecting abnormalities in specimens, especially Papanicolaou cervical smears. i.e. endocervical smear, uterine knee smear, vaginal smear, and uterine smear. It is useful for detecting sloughed cancerous tissue or cells in the body.

Abnormalities in exfoliated cells that can be detected by the present invention include, for example, viruses, bacteria, includes bacterial, fungal, and other infections, as well as non-infectious diseases.

Cellular abnormalities are detected in sloughed cells or cells in sloughed tissue. It is possible.

Frequency, CM-' FIG. 2 Sample number FIG.3 0246 B IQ 121416 Old 20222426 Sample number FIG. 4 FIG, 5 Sample number Frequency, CM-' FIG, 8 Pressure KBAR ++40 1150 1160 1170 1180 Frequency, CM-' Frequency, CM-' pressure KBAR FIG. pressure KBAR FIG. 12 Frequency, CM-' FIG. 13 Frequency, CM-' FIG. 14 Submission of translation of written amendment (Article 184-8 of the Patent Law) June 3, 1993

Claims (11)

[Claims] 1. A detection method that uses infrared spectroscopy to detect the presence of abnormalities in exfoliated cells. There it is, (a) directing a beam of infrared light onto a specimen of exfoliated cells; and (b) at least Spectral analysis of infrared absorption by a specimen in one frequency band Determine whether an anomaly exists in the specimen and detect at least one infrared absorption property. This is a stage to confirm whether a change has occurred, and to determine whether or not a change has occurred. In both cases, one change is a change in the number of molecules present in the specimen that is characteristic of the abnormality. Both of the functions are caused by the vibration of one functional group. detection method. 2. The at least one change in the infrared absorption property is characterized by an increase in absorption at a particular frequency. changes in the strength of absorption, changes in the frequency at which a particular absorption occurs, or With a change in the pressure applied to a functional group causing a change in the frequency at which contraction occurs A method according to claim 1. 3. The functional group of molecules includes the following molecules: carbohydrates, nucleic acids, tissue proteins. 2. The method of claim 1, wherein the protein is present in at least one of a protein or a membrane lipid. 4. The functional group of the molecule is the phosphodiester group in nucleic acids, the tissue group, etc. One C-OH group in proteins and carbohydrates, or in lipids 2. The method of claim 1, wherein the method is one CH2 group. 5. The functional groups of the molecule include: carbohydrates, phosphors in nucleic acids; sulfodiester group, one C-C in tissue proteins and carbohydrates H group, or one CH2 group in a lipid, as claimed in claim 1. How to put it on. 6. 2. The method of claim 1, wherein the specimen is a Papanicolaou smear. 7. 2. The method of claim 1, wherein the specimen is a cervical specimen. 8. 2. The method of claim 1, wherein the specimen is an endocervical specimen. 9. 2. The method of claim 1, wherein the specimen is a uterovaginal specimen. 10. 2. The method of claim 1, wherein the specimen is a vaginal sample. 11. 2. The method of claim 1, wherein the specimen is a uterine sample.