JPH0778502B2 - Method for preserving cells in urine - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION "Industrial field of application" The present invention provides accurate measurement by preventing changes in the amount and quality of urinary cells after urine collection when measuring and diagnosing detached or leaked cells in urine. It relates to methods for obtaining measured values and diagnoses.

“Prior art” Urine excreted in urine is a very important clinical test target for diagnosing renal and urinary tract diseases, and is widely used as a screening test for malignant urinary tract tumors. . This test measures the amount of cells in a urine sample collected sequentially (sequential urine) or a sample that accumulates urine of one day (urine collection), or observes the cells with a microscope to determine whether they are malignant. Judgment and diagnosis (cytology) are performed.

By the way, if the urine after collecting urine is left at room temperature as it is, the cells in the urine become undernourished and undergo autolysis (autolysis) to replenish the nutrients and are altered (denatured), or by the bacteria contained in the urine. It is well known that destruction causes a decrease in cell number and degeneration of cells. When this degeneration occurs, it becomes difficult to make a judgment at the time of cytodiagnosis.

The degree of ease of this determination is shown as the determination frequency based on the following criteria.

1. Deterioration is too strong to be judged by cytodiagnosis 2. Deterioration is rather strong and difficult to judge 3. Degeneration is possible, but it can be judged 4. Degeneration is small, judgment is possible 5. Degradation can be judged at 25 ° C even by the inventors' experiments The number of cells in the stored urine decreased from 30 cells / field to 10 cells / field after 12 hours,
The frequency of cytodiagnosis was reduced to 3 or less. In that case, most of the diagnosed cells were denatured, and phagocytic air bubbles were found to spread considerably in the cells. Generally, it takes tens of hours, depending on the case, and twentieth hours from the start of urine collection, depending on the case, until the measurement and diagnosis are performed after successive urine collections, thus preventing the decrease and degeneration of cell number. It is clear that there is a need.

In order to prevent this decrease in cell number and degeneration, in conventional urine, (1) Centrifuge immediately after urine collection, and add formaldehyde or glutardaldehyde as a fixative to the residue (2) Immediately after urine collection. Put it in a low greenhouse and keep it at low temperature For long-term storage in urine, as a preservative (1) Add 2-3 ml / of toluene or xylol,
Mix occasionally with shaking (2) Add 5-10 ml / neutral formalin (3) Add 5 ml / 5% chlorhexidine solution (4) Add commercially available boric acid or paraformaldehyde tablets (5) Put in low temperature room and cool Methods such as holding are considered. However, neither sequential urine collection nor actual urine collection is actually used because the work immediately after urine collection and the transportation and storage of samples are troublesome.

In particular, (1) method for sequential urine, (1) for urine collection
In the method (4), a highly toxic substance that is extremely dangerous to the human body must be used. In addition, although the method (1) of sequential urine can withstand measurement, it has a drawback that it is difficult to determine by diagnosis. In cases (1) to (4) of storing urine, it suppresses bacterial growth in urine as an antiseptic action. However, it cannot suppress the autolysis of cells.

"Problems to be solved by the invention" There are the following two causes of destruction and degeneration of living cells in urine. That is to say, one is that cells that have been exfoliated or leaked from the living body continue to carry out metabolic action, and at the time of undernutrition, digest the self with intracellular phagocytic bubbles and supply the nutrients necessary for metabolism.
Eventually, it is autolyzed, and secondly, bacteria that are resident or pathologically present in urine propagate and directly attach to cells or destroy living cells by their toxins.

At present, there is a problem that there is no safe storage method that prevents both of these actions, does not cause troublesome labor.

“Means for Solving the Problems” In view of such circumstances, the inventors of the present invention have conducted earnest research to solve the drawbacks of the conventional method for preserving cells in urine, and as a result, it was confirmed that the cells of It was found that metabolism can be suppressed, autodigestion can be suppressed, and bacterial reproduction and metabolism can be suppressed at the same time. Furthermore, it was discovered that the addition of EDTA.2Na (ethylenediaminetetraacetic acid disodium salt) significantly inhibits the metabolism and growth of Gram-negative bacteria often contained in urine due to its antibacterial effect. It is what you have done.

That is, the present invention is a buffer that can keep the pH acidic such as citric acid when measuring and diagnosing cells in urine, 0.4 ~ 4.0 mg
This is a method for preserving cells in urine by adding / ml (per urine) EDTA · 2Na (ethylenediaminetetraacetic acid disodium salt) to the collected sample urine to adjust the pH to 4.5 to 6.5.

Living cells that have been exfoliated or leaked from the living body in the urine continue to be metabolized, and during malnutrition, phagocytic bubbles in the cells digest themselves to provide the nutrients necessary for metabolism, which results in autolysis. It is known to cause (cell biology, J. Carp, co-translated by Hiroshi Terayama et al., Published by The University of Tokyo Press. 1
See 987.3.25). In addition, most of the metabolic enzymes in these cells, such as glycolysis, citric acid cycle system, fat metabolism, amino acid decomposition system, have an optimum pH of 6.5 or higher, and metabolism is suppressed in an acidic environment. Also known ("Biochemistry of Stryer" translated by Nobuo Tamiya, Tokyo Kagaku Dojin, 1977.3.22 and "Clinical Enzyme Handbook" edited by Shigeaki Baba et al., Kodansha, 1
981.9.10). Also, for example, as "Indispensable for microbiological testing" supervised by the Ministry of Health and Welfare (1961.3.25 published by Japan Public Health Association)
In general, the optimum pH for bacterial growth in most cases is 6.
It is well-known that the growth is suppressed when it is more than 5 and more acidic than this, and it is also known that the extracellular membrane of gram-negative bacteria is destroyed by EDTA and the metabolism and growth functions of the bacteria are stopped. (CASchnaitman. "Effect of Ethylen
ediamine tetraacetic Acid, Trito X−100, and Lysozym
e on the Morphology and Chemical Composition of Is
`` Olated Cell Walls of Esherichiacoli '', Jour. of Bact
eriology, Vol. 108, p553 to 563, 1971). However, these are general knowledge, and when the sample urine is acidified, it has immediate effect on the metabolism and growth inhibition of cells and bacteria, and it preserves living cells in urine by adding acid to urine and keeping it acidic. There is no suggestion on how to do it.

The pH of urine is usually in a wide range of 4.5 to 10.0, but in the present invention, acid is added as a buffer to adjust the pH to 4.5 to 6.5.

The acid used in this invention may be either an inorganic acid or an organic acid.
However, it is preferable to use a powder which is made into a powder with an organic acid and which is put into a sampling tube in advance, and when urine is sampled in the sampling tube, the urine is automatically mixed with the urine so that an appropriate pH can be easily obtained.

Especially, it is convenient to use citric acid at 1.0 to 4.0 mg / ml (per urine) because the pH of urine becomes 4.5 to 6.5. ED to this
The combined use of TA and 2Na improves the effect of preserving cells in urine. EDTA · 2Na is effective in an amount of 0.4 to 4.0 mg / ml (per urine).

In order to carry out the present invention, for example, an amount of the above-mentioned concentration (pH) when urine is mixed with an aqueous solution mainly containing citric acid and EDTA · 2Na is put into a collection tube having a volume of 10 to 30 ml of urine, Moisture is evaporated by some method to make the components powdery and attached to the bottom of the sampling tube. Urine 10 ~
30 ml may be collected and stored as it is for cell measurement and diagnosis.

"Example" Example 1.

Collect urine in a sampling tube to collect citric acid 4 mg / ml (per urine)
Was added and mixed. Urine has a pH of 6.8, and after addition
Was 4.5.

This urine sample was kept at 25 ° C, the number of cells was measured every 12 hours, and the cells were observed under a microscope to evaluate the degeneration of the cells using the above-mentioned judgment frequency. There wasn't. After 48 hours, the number of cells decreased by 8% and the judgment frequency decreased to 3.

Example 2.

Citric acid 4 mg / ml (per urine) and as in Example 1
Citric acid and EDTA · 2Na were added and mixed so that EDTA · 2Na was 3 mg / ml (per urine). The pH of the sample urine used is
The pH was 6.8, and the pH became 5.4 after the addition.

Hold this sample at 25 ℃ and measure the number of cells every 12 hours.
When the cells were observed with a microscope and the judgment frequency was evaluated, there was no change until 24 hours later. After 48 hours, the number of cells decreased by 5% and the judgment frequency decreased to 4.

Example 3.

In the same manner as in Example 1, 1N hydrochloric acid 20 μ / ml EDTA · 2N
3 mg / ml (per urine) of a was added and mixed. The pH of urine
The pH was 6.8, and the pH became 5.4 after the addition.

Hold this sample at 25 ℃ and measure the number of cells every 12 hours.
Moreover, when the cells were observed with a microscope and the judgment frequency was evaluated, it was found that there was no change until 24 hours later. After 48 hours, the number of cells decreased by 5% and the judgment frequency decreased to 4.

"Effects of the Invention" As described in detail above, the present invention adjusts the pH of urine to 4.5 to 6.5 by adding an acid, particularly citric acid, as a buffer to a collected urine sample when measuring and diagnosing cells in urine. The number and denaturation of the cells in the sample urine can be prevented simply by adjusting the value to 1. Therefore, accurate measurement values and diagnosis can be obtained even if the cells are stored for tens of hours before measurement and diagnosis after sample urine collection. It is effective for measuring the number of living cells in urine, such as blood cells, and for cytodiagnostic test, and is a very effective method especially when testing a large amount of sample.

Claims (2)

[Claims] 1. When measuring or diagnosing cells in urine, a buffering agent such as citric acid which can keep pH at an acidic level, and 0.4 to 4.0.
Cell preservation in urine, characterized by adding mg / ml (per urine) of EDTA.2Na (ethylenediaminetetraacetic acid disodium salt) to the collected sample urine to adjust the pH to 4.5 to 6.5 Method. 2. The method for preserving cells in urine according to claim 1, wherein the buffer is 0.4 to 4.0 mg / ml (per urine) of citric acid.