HU197450B - Reagent and method for detecting cells of phagocyte activity - Google Patents

Abstract

The invention relates to a neutral red-containing reagent solution for detecting cells with phagocytic activity. The reagent solution according to the invention comprises 0.2-0.8 % by weight of neutral red, 0.1-0.25 % by weight of lysozyme, 0.8-2.0 % by weight of sodium chloride, 0.1-0.3 % by weight of a saccharide and optionally 0.02-0.6 % by weight of a preservative in an aqueous solution with a pH of 3.0-8.0, preferably 3.5-5.0. The invention also relates to a method for detecting cells with phagocytic activity by utilizing a reagent solution of the above composition.

Description

The present invention relates to a reagent and a method for detecting cells having phagocytic activity.

One of the main goals of modern medicine is to diagnose fetal malformations as early as possible in pregnancy.

It is known that tubal disorder can be diagnosed by cell staining, since cells with phagocytic activity in amniotic fluid can be detected by color reaction in the amniotic fluid. Neutral red is used as the cell staining reagent (Medical Weekly 125: 9, 1984); N. Eng. J. Med. 310, 1463 (1984)].

However, difficulties have arisen in the routine clinical diagnostic application of the method. It was found that the reliability of the reaction is greatly influenced by the quality of the neutral red, that is to say, significant differences were found in the use of neutral red ink from various production sites. It was also difficult to make a neutral red solution for diagnostic purposes. The neutral red distilled aqueous solution has an acidic pH (1 mg / ml pH 3.1); the pure distilled aqueous solution severely damages the cells due to the acidic or hypo-osmotic effect. None of the various aqueous buffer and nutrient solutions routinely used in clinical practice proved to be a suitable solvent because precipitation was observed after dissolution. The use of organic solvents has also failed; For example, a neutral color solution using ethanol neutral red solution was not obtained and problems with the sterile filtration of the dimethylsulfoxidoe solution.

It is an object of the present invention to provide a reagent solution and diagnostic method which can be used in practice to detect cells with phagocytic activity with high safety and ease.

In our experiments, it was recognized that the neutral red-containing cytological reagent solution should be approximately iso-osmotic relative to potentially abnormal cells. It was also essential that the mixture of reagent solution and test sample (amniotic fluid) be approximately neutral. Surprisingly, it has also been found that the presence of the lysozyme enzyme significantly enhances the dye uptake of the cells while also enhancing the stability of the dye solution. Finally, it has been recognized that the reagent solution must also contain saccharide as an energy source for the cells. If a reagent solution is needed for a longer period of time, it is advisable to add a preservative to the reagent solution.

The present invention therefore relates to a solution of a neutral red reagent for detecting cells with phagocytic activity. The reagent solution of the invention provided 0.2-0.8 wt.% Neutral red, 0.8, 25 wt.% Lysozyme enzyme, 0.8-2.0 wt.% Sodium chloride, 0.1-0.3 wt.% Saccharide in aqueous solution. in the case of 0.02-0.6% by weight of a preservative; the pH of the aqueous solution is between 3.0 and 8.0, preferably between 3.5 and 5.0.

The invention further relates to a method for detecting cells with phagocytic activity by staining with neutral red vein. According to the invention, 0.2 to 0.8% by weight of neutral red, 0.1 to 0.25% by weight of lysozyme, 0.8 to 2.0% by weight of sodium chloride, 0.1 to 0.3% by weight of glucose are used. and optionally 0.02-0.6% by weight of a preservative in water, adjusting the pH of the solution to 3.0 to 8.0, preferably 3.5 to 5.0, filtering the solution to sterile 1 volume of reagent solution is mixed with 3-8 volumes of test sample, preferably amniotic fluid, and the resulting cell suspension is assayed in a manner known per se in a hemocytometer.

The reagent solution of the present invention may contain mono- or disaccharides, particularly preferably glucose, as a source of energy for the cells as an azaccharide. Preferably, benzoic acid and / or polyvinyl alcohol is used as a preservative.

The hemocytometric assay of the cell suspension was performed as described in Medical Weekly 125, 9 (1984) and N. Eng. J. Med. 310, 1463 (1984). During the hemocytometric assay, the cell suspension treated with the reagent solution is centrifuged after incubation, the supernatant is discarded, the pellet is mixed with the washing dilution solution and the color reaction is examined in a Bürker chamber.

For the purpose of clinical trials, the reagent of the present invention is conveniently marketed as a reagent kit. The reagent kit is comprised of a unit dose reagent solution (preferably filled in ampoules) and a dosage unit wash (preferably filled in ampoules), and the dosage ratio of reagent solution to wash dilution solution is 1: 0.5 (0.5-2).

Wash-dilution solutions which are known per se for cytological studies may be used. Particularly preferred is Hank's solvent (Hank, J.H. and Wallace, R.E. Proc. Soc. Exptl. Biol. Med., 71, 196 (1949)]. It is highly preferred to use a Hank's solution adjusted to pH 7.2 to 7.4 (preferably pH 7.3) with N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid.

Our need for protection extends to reagent kits.

The most important advantage of the present invention is that the solutions used are stable, can be preserved for at least 12 months in the presence of a preservative, and can be formulated immediately. Using the reagent solution or reagent kit of the present invention makes diagnostic work faster and easier than previously known, and the reliability and reproducibility of the detection method is superior to previous solutions.

The invention is further illustrated by the following non-limiting examples.

Example 1

A reagent kit is prepared from the following solutions:

I. Reagent solution:

Dissolve 0.1 g of polyvinyl alcohol in 45 ml of hot distilled water and, after cooling to room temperature, add 0.8 g of sodium chloride, 0.1 g of glucose, 0.02 g of benzoic acid and 0.1 g of lysozyme. . The resulting solution was adjusted to pH 6.9 with 1 M Tris (tris (hydroxymethylaminomethane)) buffer and made up to 50 ml with distilled water. To the resulting solution was added a solution of 0.2 g of neutral red [3-amino-7- (dimethylamino) -2-methylphenosine hydrochloride) in ml of distilled water and, after thorough mixing, the solution was germinated on a G-5 glass sieve. filter and pour the filtrate into 1-ml vials, which are well sealed.

II. Wash-dilution solution:

In 350 ml of distilled water, 4 g of sodium chloride, 0.2 g of potassium chloride, 0.1 g of magnesium sulphate heptahydrate, 0.09 g of calcium chloride dihydrate, 0.03 g of disodium hydrogen phosphate dihydrate, 0.03 g of potassium dihydrogen phosphate and 0.5 g of glucose are dissolved in 0.1 g of benzoic acid. Then, a solution of sodium hydrogen carbonate (0.175 g) in distilled water (12.5 ml) was added. The mixture is thoroughly mixed and the pH of the solution thus obtained (Hank's solution) is adjusted to 7.3 with 5 ml of a 1M aqueous solution of N-hydroxyethylpiperazine-α-2-ethanesulfonic acid.

The solution is then made up to 500 ml with distilled water, filtered through a G-5 glass filter to a germ-free filter and filled in 5 to 5 ml sealed vials.

Example 2

A reagent kit is prepared from the following solutions:

I. Reagent solution:

The composition and preparation of the reagent solution are as described in Example 1.

II. Wash-dilution solution:

In 350 ml of distilled water, 4 g of sodium chloride, 0.2 g of potassium chloride, 0.1 g of magnesium sulphate heptahydrate, 0.03 g of disodium hydrogen phosphate, 0.03 g of potassium dihydrogen phosphate, 5 g of glucose and 0.09 g of calcium chloride dihydrate are dissolved and 0.5 g of benzoic acid is added. Then, a solution of 2.4 g of sodium bicarbonate in 30 ml of distilled water was added. The mixture was thoroughly mixed and the pH of the resulting solution (Hank's solution) was adjusted to 7.3 with 5 ml of 1 N aqueous N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid.

The resulting solution was made up to 500 ml with distilled water, filtered through a G-5 glass filter for germ-free filtration and filled into sealed vials every 5 to 5 ml.

Example 3

A reagent kit is prepared from the following solutions:

I. Reagent solution:

The composition and preparation of the reagent solution is as described in Example 1.

II. Wash-dilution solution:

In 350 ml of distilled water, 4 g of sodium chloride, 0.2 g of potassium chloride, 0.1 g of magnesium sulphate heptahydrate, 0.03 g of disodium hydrogen phosphate dihydrate, 0.03 g of potassium dihydrogen phosphate, Dissolve 0.5 g of glucose and 0.09 g of calcium chloride dihydrate, and add 0.6 g of sodium nzoate to the solution. The solution is then thoroughly mixed with 0.130 g of sodium bicarbonate solution in 10 ml of distilled water and the resulting solution (Hank's solution) is brought to pH 5 with 1 ml of aqueous N-hydroxyethylpiperazine-N'-2- adjust to 7.3 with ethanesulfonic acid solution.

The resulting solution was made up to 500 ml with distilled water, filtered through a G-5 glass filter for germination, and filled in 5 to 5 ml sealed vials.

Example 4

A reagent kit is prepared from the following solutions:

I. Reagent solution:

The reagent solution was adjusted as described in Example 1, except that 0.8 g of neutral wick was used. After filtration, the reagent solution thus obtained is filled into 1-ml sealed vials.

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II. Wash-dilution solution:

The composition and preparation of the wash-dilution solution were as described in Example 1.

1-4. One set of ampoules reagent solution and one ampoule of molar diluent are required to perform one assay of the reagent kits of Example 1A.

Example 5 To 1 ml of amniotic fluid 1 ampoule (1 ml) of the reagent solution of Example 1 was added. After 15 minutes of incubation, the mixture was centrifuged, the supernatant was discarded, and 0.5 to 2.0 ml of washer-dilution solution was added to the pellet. The stained sample was examined and evaluated in a Bürker chamber.

for comparison, 5 ml of amniotic fluid per ml, see Medical Weekly 125, 9 (1984) and N. Eng. J. Med., 310, 1463 (1984) was treated with a freshly prepared neutral red solution as described in the articles using HAM F-10 (manufactured by Gibco, UK).

In comparative experiments, the dye uptake and the color persistence of cells were examined and evaluated. According to the observed data, the dye uptake by the reagent solution of the present invention was increased by 25-30% compared to the solution of the known composition, and the color durability was approximately ten times that of the solution of the known composition.

The reagent solution of known composition was stored at room temperature for 1 week and then used for cell staining. The solution became unstable for cell staining. In contrast, the reagent solution of the present invention, after one year of storage, was able to be used for staining with unchanged results.

Claims (7)

PATENT CLAIMS CLAIMS 1. A reagent solution containing neutrophil for detecting cells with phagocytic activity, characterized in that 0.2 to 0.8% by weight of neutral butter, 0.1 to 0.25% by weight of lysozyme, 0.8 to 2.0% by weight of sodium chloride 0.1-0.3% by weight of saccharide and optionally 0.02-0.6 5% by weight of a preservative in the form of an aqueous solution of 3.0 to 8.0, preferably 3.5 to 5.0. The reagent solution of claim 1, wherein the saccharide is a It contains a mono- or disaccharide useful as an energy source for cells, preferably glucose. 3. Reagent solution according to claim 1 or 2, characterized in that the preservative is benzoic acid and / or polyvinyl alcohol. 4. Procedure for detecting cells with phagocytic activity by staining with neutral red, characterized in that 0.2-0.8 wt. 0 neutral red, 0.1-0.25% * lysozyme, 0.8-2.0% * sodium chloride, 0.1-0.3% * saccharide and optionally 0.02-0.6% * the preservative is dissolved in water, the pH of the solution being between 3.0 and 8.0, preferably between 3.5 and Adjust the pH to 5.0, filter sterile, mix 1 volume of reagent solution thus obtained with 3-8 volumes of test sample, preferably amniotic fluid, and examine the resulting suspension of θ cells in a manner known per se in a hemocytometer. 5. A reagent kit for detecting cells with phagocytic activity, characterized in that it is packaged in dosage units according to claims 1-3. A dosage ratio of reagent solution to detergent dilution solution in any one of claims 1 to 5, and the dosage ratio of reagent solution to wash dilution solution is 1: 0.5 (0.5-2). ⁰ 6. A reagent kit according to claim 5, wherein the reagent solution is filled in ampoules in a unit dose of 1 ml and a solution in detergent diluted in ampoules in a volume of 5 ml. A reagent kit according to any one of claims 5 or 6 wherein the wash dilution solution is Hank's pH adjusted to 7.2 to 7.4 with N-hydroxyethylpiperazine-N'-2-ethanesulfonea. solution containing glaze.