JPH05164758A - Stabilizing method for saccharide in blood sample - Google Patents

Abstract

PURPOSE:To obtain accurate analysis values by preventing the sugar consumption by the contamination of ferments, bacteria, and microorganisms which takes place in a collected blood sample while the sample is preserved at the time of quantitatively analyzing the blood sample for saccharides, especially for glucose and 1.5 anhydroglucitol. CONSTITUTION:After adjusting the pH of a collected blood sample t 5.0-7.0 by using various kinds of acids, the blood sample is quantitatively analyzed for saccharides by adding hydrogen fluoride salt at a rate of 3mg equivalents of NaF per one ml of blood. Especially, the pH adjustment of the collected blood sample is performed in such a way that an appropriate amount of granules of the mixture of sodium citrate and EDTA-2Na respectively containing citric acid used as an acid and NaF used as the hydrogen fluoride salt are put in a test tube and the blood sample is poured in the test tube.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION This invention relates to glucose in blood,
Changes in the amounts of these components after blood sampling when measuring sugars such as 1,5-anhydroglucitol (polyol sugar),
In particular, the present invention relates to a method for preventing changes caused by microorganisms such as bacteria and obtaining accurate measurement values.

[0002]

2. Description of the Related Art When measuring the value of sugars such as budo sugar in blood for the diagnosis of diabetes and the like, sample blood is collected and analyzed. In that case, if the sample is left as it is at room temperature, the glycolytic reaction by the glycolytic enzyme group in cells originally contained in blood such as red blood cells in blood proceeds. Generally, it takes at least 2-3 hours (in the case of in-house analysis at a hospital, etc.) and, in some cases, several tens of hours (in the case of requesting another place) before the blood sample is collected and analyzed. It was necessary to prevent the sugar reaction.

More recently, for example, Kosei Kawai et al.
"1.5-anhydroglucitol", clinical test vol.
33, No. 8, August 1989, simultaneous measurement of 1,5 anhydroglucitol as a marker for diabetes has come to be performed. The component also decreases over time.

On the other hand, addition of a glycolytic inhibitor mainly containing a fluorine compound or monoiodoacetic acid has been conventionally performed, but the effect was not perfect.

The inventors of the present invention accelerate the glycolysis reaction by the enzyme system by adding acid to the blood collected as a sample to adjust the acidity of the sample so that the pH becomes 5.0 to 7.0. It was discovered that it could be blocked, and Japanese Patent Application No. 60-1001
Application was filed as No. 61 (Japanese Patent Publication No. 61-258174). The invention also includes adjusting the acidity and adding 0.1 to 0.5 mg / ml of NaF (sodium fluoride) to the blood sample to have a longer lasting inhibitory effect.

[0006]

The method of adjusting the pH of the collected blood sample was very effective as a glycolysis inhibition method. However, in practice, it was found that there are rare cases where glycolysis occurs even by this method. For example, Yoshio Kobayashi, “Infectious Endocarditis / Sepsis” Medical Technology
It is pointed out in Vol. 19, No. 10 that hypoglycemia of unknown cause is observed in patients with sepsis and infective endocarditis.
Further, there is a problem that even in the usual measurement of blood glucose level, a glycolytic action rarely occurs and an abnormally inaccurate value may be detected.

[0007]

In view of the above situation, the present inventors have earnestly studied, and as a result, the instability of saccharides in the above-mentioned blood sample showed that instability of saccharides such as red blood cells, white blood cells, and platelets rarely contained in blood. It is due to the consumption of sugar such as budo sugar and 1,5 anhydroglucitol accompanying the growth of bacteria and microorganisms other than the cells originally contained in blood. Therefore, it is originally included to eliminate this instability. It is not enough to prevent the glycolytic action of glycolytic enzymes in the blood cells and the polyol-metabolizing enzyme system by adjusting the pH and other methods, and it is possible to prevent the consumption of sugars due to the growth of other cells and microorganisms. I found it necessary.

That is, the present invention relates to budo sugar in blood,
1.5 The pH of the blood sample collected with various acids is adjusted to 5.0 to 6.8 for the blood sample collected for the analysis and measurement of sugars such as anhydroglucitol, , For example, NaF (sodium fluoride) is a saccharide stabilization method in which 3 mg or more is added per 1 ml of blood, which prevents glycolysis by the enzyme system and the consumption of saccharides due to the growth of bacteria and microorganisms, and always provides accurate analysis. You can get the value. In this case, the same amount of other hydrofluoric acid salt may be used in equivalent conversion.

The collected blood sample is subjected to processing such as container transfer and storage before analysis work. Various bacteria and bacteria are present in the air, and containers and the like may be contaminated by contact with the human body during handling. Blood contaminated by this often contains a large amount of Gram-negative bacilli, Gram-positive cocci, and the like. Further, in the case of patients with bacteremia such as infectious endocarditis and sepsis, it has been confirmed that blood contains gram-positive cocci and other bacteria. If these bacteria and microorganisms are present in the blood, they may easily grow in the blood under normal conditions, depending on the conditions such as temperature, environment, and transportation during the process of blood sampling such as sample preservation. As a result, butote sugar and 1.5 anhydroglucitol are consumed during the growth, which is the cause of the above problems.

Hydrofluoric acid salts, especially NaF, have been conventionally used alone or in combination with EDTA and heparin, and the concentration thereof is often in the range of 1 to 10 mg per 1 ml of blood, and as the hydrofluoric acid salt. 3 with NaF
It is usually 1.2 because hemolysis may occur at more than mg / ml.
About 5 mg / ml is used. However, simply adding hydrofluoric acid under these conditions cannot prevent the growth of bacteria and microorganisms mixed in blood.

[0011]

As in the present invention, when the pH of a blood sample after blood collection is lowered (acidified) to 5.0 to 6.8 by acid, Japanese Patent Application No. 60-100161 (Japanese Patent Publication No. 61-258174).
No.), glycolysis by various glycolytic enzymes in blood cells can be immediately blocked. Furthermore, if a fluorine compound (for example, NaF) is added under this condition so as to have a concentration of 3 mg or more per 1 ml of blood, the growth of bacteria and microorganisms will be suppressed only under those conditions. When the acidity is lowered, hemolysis does not occur even if the concentration of NaF is 3 mg / ml or more.

To carry out the present invention, for example, a blood collection test tube is provided with citric acid, sodium citrate, EDTA-2.
A prescribed amount of a granular drug composed of Na and NaF is put in advance, and a prescribed amount of blood collected in the test tube is infused or directly collected, and stored as it is by a conventional means. 5 Anhydroglucitol may be quantified.

[0013]

[Examples] Blood was collected (a) Blood sample with pH adjusted to 5.5 (conventional method), (b) Blood sample with NaF added at 4 mg / ml (conventional method), (c) Sampling Tubes with citric acid, sodium citrate, EDTA-2Na, NaF 16:
1: 1 of granulated drug mixed in the ratio of 34:23:27
Three samples were prepared by mixing 2 mg / ml (per blood) and mixing (method of the present invention). When 10 ⁵ cells / ml (per blood) of Serratia bacteria (a kind of Gram-negative bacillus) were added to these samples, the samples were allowed to stand at 37 ° C and the concentrations of budo sugars were analyzed and measured for comparison. It was on the street. From these results, it can be seen that the method of the present invention enables stable measurement of sugars in blood. Similar results were obtained in the measurement of 1.5 anhydroglucitol.

[0014]

As described above in detail, according to the method of the present invention, an acid, particularly citric acid, is added to a collected blood sample when measuring budo sugar and 1.5 anhydroglucitol in blood. The pH of blood is adjusted to 5.0 to 6.8 and the addition of hydrofluoric acid is used in combination, so that the glycolytic reaction by the glycolytic enzyme group in blood cells in blood and the microorganisms such as bacteria It can prevent consumption (reduction) of saccharides, glucose and 1.5 anhydroglucitol due to their growth in the case of container contamination or contamination from the air, which allows sample collection to analysis work. Accurate analysis values can be obtained even after storage for several tens of hours. Blood glucose level, 1.
This is a very effective method for testing 5 anhydroglucitol, especially when testing a large amount of samples.

[Brief description of drawings]

FIG. 1 is a graph showing time-dependent changes in the glucose level of budo sugar when 10 ⁵ cells / ml (per blood) of Serratia bacteria were added to blood samples subjected to various glycolysis inhibition methods and kept at room temperature. .. In the figure, (a) pH adjustment method (conventional method), (b) method of adding NaF (conventional method), (c) case of the method of the present invention are shown.

Claims (5)

[Claims] 1. A method for analyzing and measuring sugars such as budo sugar and 1.5 anhydroglucitol in blood, wherein p of a blood sample collected by using various acids with respect to the collected blood sample.
Stabilization of saccharides in a blood sample, characterized by adjusting H to 5.0 to 6.8, further adding 3 mg or more per 1 ml of blood by converting the equivalent of hydrofluoric acid into NaF Method 2. Citric acid as an acid, NaF as a fluorine salt
(Sodium fluoride) is used, The method for stabilizing saccharides in a blood sample according to claim 1, wherein 3. A method for stabilizing saccharides in a blood sample according to claim 1, wherein budo sugar, 1.5 anhydroglucitol, or both are analytically measured as saccharides. 4. A blood collecting test tube is preliminarily filled with a specified amount of a granular drug consisting of citric acid, sodium citrate, EDTA-2Na and NaF, and the specified amount of blood collected is injected into the test tube or After collecting directly into a test tube,
The method for stabilizing saccharides in a blood sample according to any one of claims 1 to 3, characterized in that budo sugar and 1.5 anhydroglucitol are quantified. 5. Citric acid, citric acid-sodium, EDT
The method for stabilizing saccharides in a blood sample according to claim 4, wherein a drug which is made into a granule by mixing A-2NA and NaF in a ratio of 16: 34: 23: 27 is used.