JPH0746101B2 - Cell fixative / preservative solution with mucolytic properties - Google Patents

Description

TECHNICAL FIELD The present invention relates to a cell-fixing / preserving solution, and in particular, a cell-fixing / preserving solution having a mucolytic property, which is suitable for fixing and preserving a mucinous cytological specimen. Regarding

[Conventional technology]

Mucus samples taken with a swab from sputum or vagina contain a myriad of cells, which should be examined for precancerous atypical cells and cancer cells. Is a cytology, and this cytology plays an important role in early detection of modern cancer. Although a microscope has been used for this cytodiagnosis, in recent years, in addition to this, flow cytometry (flow cytometry) is an automatic cell analysis / separation device that measures by flowing cells at a speed of several hundreds per second or more.
Automated cytology using cytometry is also about to be applied. In either case of cytodiagnosis using a microscope or flow cytometry, it is extremely important that the cells in the specimen be fixed in good condition in order to accurately diagnose the cells In this case, cancer cells may be overlooked, or conversely, non-cancer cells may be mistaken for cancer cells.

The general method of fixing the sample for cytodiagnosis is to smear the sample directly on a slide glass and quickly place it in a fixative solution before it dries (wet fixing method of smear),
95% ethanol is used as the fixative. However, when the sputum cytology of lung cancer is performed on a patient who is not hospitalized or a subject who undergoes mass screening, it is not always possible that the sputum can be immediately discharged when the patient visits the hospital. Rather, sputum is more likely to appear when waking up, and this sputum when waking up is most suitable for cytology because it abundantly stores cells released from the lungs during sleep. If the sputum when waking up is brought to the hospital and then fixed, it will take too much time and the cells will be degenerated and the fixation will be poor, resulting in inaccurate cytodiagnosis. In such cases,
If there is a suitable cell fixative for sputum and it is possible to fix the cells immediately, it is convenient because the sputum when waking up can be stored in the fixative at home and brought to the hospital at a later date. But,
When sputum is put into a 95% ethanol fixative solution, the sputum mucus coagulates from the surface layer, and the coagulated surface layer not only prevents the fixative solution from penetrating into the interior, but also impairs the fixation of cells inside, It also prevents the sputum from spreading thinly on a slide glass and smearing it, further worsening the quality of the specimen. In addition, in the case of automated cytodiagnosis by flow cytometry, the measurement error increases due to the non-uniform fixation of cells,
The solidified mucus blockages the nozzle of the device and causes serious problems such as inability to analyze.

Saccomanno (1963) of the United States first introduced a fixative for sputum for lung cancer screening, and this sacomano fluid is often used in Japan. This sacomano liquid has a simple composition in which carbowax (polyethylene glycol 1540) is added to 2% of 50% ethanol water. After sputum is discharged into this liquid and fixed for a certain period of time, it is stirred in a laboratory with a blender to stir vigorously to mechanically disrupt the mucus and release the cells, which are then collected by centrifugation and collection. A smear will be made by smearing on a slide glass, and staining will be performed for microscopic diagnosis. Since this solution has a slightly high ethanol concentration of 50% and contains carbowax, it causes contraction of cells and concentration of nuclei during fixation, which makes it difficult to read the detailed internal structure of cells and makes it difficult to diagnose cells unless used. There is a drawback that causes trouble. In addition, in automated cytodiagnosis by flow cytometry, since this solution does not contain a mucolytic agent, the remaining unmelted mucus clogs the nozzle of the device, making measurement impossible, and collecting cells. Since clumps are likely to occur, it causes serious problems such as the inability to measure individual cells. Therefore, this sacomano fluid is a particularly inappropriate fixative for automated cytodiagnosis by flow cytometry.

In recent years, mucus-soluble fixatives for sputum have been developed and put on the market in Japan, but these are all based on 50% ethanol water containing 2% carbowax, like Sacomano's solution, A mucolytic agent was added. As a mucolytic agent, Tohoku University Mycobacterial Research Institute formula (released by Thermal Co.) is N-acetyl-L cystine (N-acetyl).
-L-cysteine) is used for the Osaka Prefectural Center for Adult Diseases (marketed by Matsunami Glass Co., Ltd.) and dithiotreito.
l), and YM type sputum fixative (Muto Junyaku Co., Ltd., patent pending) is dihydroxy dithiol butane (dihydr
oxy dithiolbuthan) and enzymes have been added.

[Problems to be Solved by the Present Invention]

In recent years, the demand for sputum cytology has increased sharply in association with the rapid increase in lung cancer patients, and when the revision of the Geriatric Medical Law requires adult lung cancer screening from 1987, the demand for sputum cytology will further increase. It is an accelerating situation. However, sputum cytology for lung cancer screening is significantly more laborious than microdiagnosis for uterine cancer screening because of the features of significantly higher number of normal cells and lower content of abnormal cells. In addition to the work, there are few cytologists and instructors who are familiar with this technique. Therefore, in order to save labor, the development of automated cytodiagnosis using flow cytometry has been strongly promoted in recent years. This is because the dissociated cells are fluorescently stained and suspended in a solution, and while these cells are flowing through the device at high speed, a laser beam is applied to cause the fluorescent color to develop, and the fluorescent amount from each cell is measured. Since the cells are quantified by measurement, in the case of a mucous specimen such as sputum, a fixative that can dissolve the mucus and fix the cells in a suspended state (floating fixation) is indispensable. Also,
When collecting sputum of a large number of subjects in fixatives in mass screening, it is usually stored in fixative for several days, so there is also a risk that cell degeneration will progress during this period. is there. In such a case, a fixative / preservative solution that has good cell fixability and cell structure preservability is desired. With such a liquid, it can be conveniently used for mass screening for lung cancer, and also in mass screening for uterine cancer, the medical doctor does not have to smear each swab on the slide glass on the spot. Since it is sufficient to add the liquid to the fixative, it is convenient to save time and to avoid the serious obstacle of drying the smear, which is likely to occur during smearing.

The quality of cell fixation / preservation has a significant effect on the diagnostic accuracy of cells, which may result in a diagnosis failure or misdiagnosis for defective specimens. High interest. The items required for such a cell fixing / preserving solution are as follows.

1. Mucus solubility: Mucus in a sample generally coagulates when it comes into contact with the fixative, and the coagulated mucus inhibits the permeation of the fixative into the mucus mass and causes improper cell fixation, resulting in errors in cell analysis. Birth and mucus block the flow cytometry nozzles, rendering analysis impossible. Therefore, a fixative is desired in which mucus is dissolved during fixation.

2. Immobilization of DNA and protein: The size of the nucleus, its internal structure, and the nucleus / cytoplasm ratio are the targets for diagnosis under a microscope, and the number of the nucleus is determined during automated cytodiagnosis by flow cytometry.
Since the DNA amount and the DNA amount / protein amount ratio of cells are the objects of quantification, both the nucleus (DNA) and the cytoplasm (protein) must be well fixed and preserved.

3. Prevention of contraction and aggregation of cells due to fixation: Although fixation of cells must be reliable, if fixation is strengthened, cells contract or concentrate, making microscopic diagnosis difficult, or agglomeration of cells. To make a cell mass,
This can clog the flow cytometry nozzles and render the measurements impossible, so the strength of the fixation should be moderate.

4. Toxicity of fixative: During mass screening for lung cancer, sputum fixative is placed in a container and distributed to the subjects, but the fixative distributed at home is mistakenly taken by children. In consideration of an accident, the fixative must have low toxicity.

5. Preservation of fixed cells: During mass screening, a large amount of specimens are collected at one time and these are examined sequentially, so the cell structure is preserved in the fixative for several days to two weeks. Must be done.

In recent years, various sputum fixatives that have been marketed in Japan have been examined, but a fixative of the Tohoku University Mycobacteria Research Institute-type fixative in which 0.2% acetylcystine was added to 50% ethanol water showed mucus solubility as a result of additional tests. Although favorable, the present applicant reacted with hematoporphyrin and acetylcystine, which are components of a fluorescent staining solution for simultaneous quantification of DNA proteins developed for automated cytodiagnosis by flow cytometry, and caused a brown wavy precipitate. However, it is a disadvantage that the dyeing is caused and the dyeing by the dyeing solution is disturbed. Next, we examined a YM sputum fixative using dihydroxydithiol butane (Muto Pure Chemicals Co., Ltd., patent pending), which is considered to be equivalent to a fixative using N-acetyl L-cystine in terms of mucolytic solubility. However, there is a peculiar foul odor, and when I put my mouth close to the container to spit out sputum,
The stench of rotten eggs makes me sick. According to a group medical examination in a certain area, some of the subjects who were given this thought that the fixative was rotten due to the malodor of the fixative, and some had discarded the fixative. . Dihydroxy
There is also a problem in that the toxicity of dithiolbutane is unknown, and because it uses an enzyme, it has an effective period of about 3 months and is expensive. We have not conducted an experiment on the sputum fixative of the Osaka Adult Disease Center type using dithiothreitol, but dithiothreitol is a drug used as an expectorant like N-acetyl-L-cystine, so its toxicity is low. There seems to be no problem, but there are problems in that it is expensive and in terms of price.

It is an object of the present invention to provide a cell-fixing / preserving solution having mucus solubility, which does not have the above-mentioned drawbacks.

[Means for solving problems]

The present inventor found methyl cystine as a result of various studies on mucolytic agents that do not react with the staining solution, while developing automation of cytodiagnosis by flow cytometry and fluorescent staining of cells. Methylcystine hydrochloride is chemically chemically converted to methyl β-mercaptoalanine hydrochloride (methylβ-mercury).
−mercaptoalanine hydrochloride), HSCH ₂ CH (NH ₂ ) CO
OCH ₃ · HCl, once used as an expectorant. As a result of examination using sputum of a large number of patients, it was found that 0.1-0.2% of methyl cystine was added to the fixative,
The mucus was well dissolved, the cell fixing effect was good, the fixative did not have a particularly bad odor, and neither deterioration nor efficacy decrease was observed during long-term storage.

In addition, the mucolytic agent methylcystine is a drug that is also used as an expectorant, and since the amount used in this fixative is very small, it does not pose a problem in terms of toxicity.

That is, the present invention is a cell-fixing / preserving solution containing methylcystine as a mucolytic agent,
This is highly useful as a fixative / preservative solution for mucus cytological specimens such as sputum, and since the mucolytic agent used has the characteristic that it does not inhibit fluorescence staining, flow cytometry and cell fluorescence It is also suitable for automated cell hearts using light staining.

The main ingredient and other components of the fixative / preservative solution are not particularly limited, but if the buffer solution containing ethyl alcohol and salt and either sucrose or propylene glycol newly found by the present inventor is used, cell contraction and aggregation will occur. It can be used as a fixative / preservative solution that can fix the nucleus and cytoplasm in a state suitable for cytodiagnosis without causing the above. An example of such a fixing / preserving solution is 0.01 mol phosphate buffer containing 0.8 to 0.9% sodium chloride (0.01 mol phosphate buffered saline, phosphate buffered).
0.2% methyl cystine, 2% sucrose or propylene glycol, 10 ppm water-soluble antifoam, 40% ethanol in a saline solution) (hereinafter abbreviated as PBS).
%, And some were added so that the final concentrations were obtained.

Furthermore, during analysis by flow cytometry, the fixative containing the sample is vigorously shaken with a high-speed blender for the purpose of mucus lysis and cell dispersion, but innumerable fine air bubbles are generated in the layer, which causes mucus clumps. The antifoam, which is a silicone resin formulation, was added as an antifoaming agent, as it covered the syrup and was entangled in the mucus lump to prevent contact between the mucus and the mucolytic agent, and good results were obtained. Silicone resin is used as an antifoaming agent in the concentration, fermentation, and distillation processes of various foods such as beer, budo, soy sauce, dairy products, and fruit juices, and is useful for streamlining work and making products uniform. Therefore, the residual amount of this product in food is specified to be 50 ppm or less. As a result of the experiment, the defoaming action of the silicone resin was sufficiently exhibited at 10 ppm, and at 30 ppm or more, the insoluble portion of the silicone resin remained, which was wasted.

How to use this fixative When fixing sputum, spit out sputum into this fixative and shake it well by hand. After fixing for 2 days to 1 week,
Shake for about 2 minutes in a high-speed blender, centrifuge to precipitate, and in the case of preparation of a microscope specimen, transfer the residue to a slide glass with a pipette, dry overnight, and refix with 95% ethanol or spray method. In the case of automated cytodiagnosis by flow cytometry, the residue is washed once with a phosphate buffer solution and then subjected to fluorescent staining for analysis.

〔Example〕

Experimental Example 1 Experiment to determine the concentration of methylcystine, a mucolytic agent for sputum: To know the proper concentration of the mucolytic agent methylcystine to be added to the fixative solution for mucus dissolution of sputum, 30% ethanol / PBS, methylcystine were used. To 0, 0.05, 0.1, 0.2, 0.5
% Each of them was prepared, and sputum of the patient was put therein, and the one left at room temperature for 3 days was shaken by hand for about 20 illnesses, and the solubility of mucus was visually observed.

[Results] Methylcystine concentrations of 0 and 0.05%
In the case of the above, the dissolution of mucus was insufficient (Fig. 1), and the mucus dissolution occurred at a concentration of 0.1% or more (Fig. 2).

The photograph in FIG. 1 is a micrograph (100 ×) of a smear-stained specimen of sputum fixed with a fixative (30% ethanol / PBS) containing no methylcystine.

The striae running laterally in the lower third of the screen is mucus, and cells are entangled in it, which is not dispersed.

The photograph in Fig. 2 is a micrograph (100x) of a smear-stained specimen of sputum fixed with a fixative solution (30% ethanol / PBS) containing 0.2% methyl cystine.

The mucus is lysed and the cells are well dispersed. The exceptionally large cells are lung cancer cells.

Experimental Example 2 Experiment to see the effect of a silicone preparation added as an antifoaming agent for the fixative: sputum fixed on the fixative was subjected to a high speed blender together with the container for about 60 minutes per minute for the purpose of mucus lysis and cell dispersion.
Give vibrations 00 times, but at this time the fixative foams strongly,
These air bubbles surround the sputum and prevent it from contacting the fixative. Therefore, we decided to use the antifoaming agent of the silicon preparation, and the fixed solution (2% of which the antifoam which is the product is added at 10 ppm or less, which is less than the concentration approved as a food additive.
30% ethanol / PBS with sucrose added and a fixative without added antiform were prepared, sputum was added to these and shaken for 2 minutes with a high speed blender, and it was revealed that antiform was added. Little bubbles were generated, defoaming was fast, and mucus dissolution was excellent.

Example Fixative (4% containing 0.2% methyl cystine, 2% propylene glycol and 10 ppm antifoam)
Mucus solubility, cell aggregation and cell fixing effect of sputum (0% ethanol / PBS) (Since sputum is exhaled to the fixative solution, saliva is added to dilute the ethanol concentration of the fixative solution, so the ethanol concentration was 4%) Experiment: To see the overall effect of the completed fixative solution, regarding the sputum of 126 patients, when the sputum was shaken by hand immediately after the sputum was put in and when it was shaken with a high speed blender 3 days later, Solubility, cell dispersion and fixation effects were investigated. (Table 1) [Results] With a high-speed blender, vibrating words were found to be mucolytic except for one case of sputum. Mucus dissolution was impossible 1
The example was that the blood was so bloody that it clogged with mucus. Also, microscopically, the cells were well dispersed, and the cytoplasm and nucleus structures were excellent even when stored for 1 month or longer (Figs. 3 to 6).

The photographs in Figures 3-6 are microscopic photographs of lung cancer cells in sputum fixed with the complete fixative solution and stored in the refrigerator for 40 days (40
0 times). The structure of the cytoplasm and the nucleus is very well maintained. 3 and 4 are photographs of lung squamous epithelial cancer cells, and FIGS. 5 and 6 are photographs of lung lung cancer cells.

〔The invention's effect〕

As described above, according to the present invention, by using methylcystine as a mucolytic agent in the cell-fixing / preserving solution, at the time of cell-fixing in cytodiagnosis of a mucous specimen such as sputum, good mucus dissolution is achieved. Therefore, it can be fixed as a suspended state in which cells are dispersed, and a fixing / preserving solution can be provided which has no odor and has no problem in toxicity. Furthermore, in automated cytodiagnosis using flow cytometry and cytofluorescence staining, it also has the effect that the mucolytic agent does not inhibit fluorescence staining.

[Brief description of drawings]

Figure 1 is a photomicrograph (100x) of a smear-stained specimen of sputum fixed with a fixative (30% ethanol / PBS) containing no methylcystine. Fig. 2 shows a fixative containing 30% methyl cystine (30%
Micrograph (100x) of smear-stained specimen of sputum fixed with% ethanol / PBS). 3 to 6 are micrographs (400 times) of lung cancer cells in sputum fixed with the fixative of the present invention and stored in the refrigerator for 40 days.
3 and 4 are photographs of lung squamous cell carcinoma cells, and FIGS. 5 and 6 are photographs of lung adenocarcinoma cells.

Claims (4)

[Claims] 1. A cell-fixing / preserving solution which contains methylcystine as a mucolytic agent. 2. The cell fixing / preserving solution according to claim 1, wherein the content of methylcystine is 0.1 to 0.2%. 3. The cell fixing / preserving solution according to claim 1, further comprising a silicone antifoaming agent. 4. The cell fixing / preserving solution according to claim 3, wherein the content of the silicone antifoaming agent is 10 to 30 ppm.