WO1988007583A1

WO1988007583A1 - Reagent and method for detecting cells with phagocytic activity

Info

Publication number: WO1988007583A1
Application number: PCT/HU1988/000015
Authority: WO
Inventors: Katalin POLGÁR; György ÁBEL; Ilona HARSÁNYI; László NAGY; Sándor SIPKA; Edit Vaszari; Iván DARÓCZI; Zoltán PAPP
Original assignee: Reanal Finomvegyszergyár
Priority date: 1987-03-27
Filing date: 1988-03-22
Publication date: 1988-10-06
Also published as: EP0345295A1; HUT46145A; HU197450B; JPH03500961A; AU1488588A

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

REAGENT AUD METHOD FOR DETECTIHG CELLS WITH PHAGOCYTIC

ACTIVITY

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

One of the major endeavours of medical science is to recognize developmental anomalies of foetl at the earliest possible stage of pregnancy.

It is well known that disorders of neural tube closure can be diagnostized by cell staining, since when such a disorder exists, cells with phagocytic activity appear in the amniotic fluid which can be detected by colour reaction. As staining agent neutral red is applied [N. Eng. J. Med. 310, 1463 (1984)]. Several problems have been emerged, however, when applying this method in the routine clinical diagnostics. It has been observed that the reliability of detection depends to a great extent on the quality of neutral red, i.e. the results obtained differ significantly when neutral red samples originating from different manufacturers are used. It is also difficult to prepare a solution of neutral red applicable for diagnostic purposes. The solution of neutral red in distilled water is of acidic character (the pH of a solution containing 1 mg/ml of neutral red is 3.1), and, owing to its acidity and hypoosmotic effect, this solution strongly damages the cells. None of the aqueous buffer and nutrient solutions utilized in routine clinical practice proved to be appropriate to dissolve neutral red, since after dissolution precipitation occurs. Organic solvents appeared to be less suitable; thus e.g. neutral red in ethanol solution does not give appropriate staining, whereas the dimethyl sulphoxide solution is difficult to sterilize.

The invention aims at providing a reagent solution and diagnostic method for detecting cells with phagocytic activity which is easy to apply and gives highly reliable results.

We have found that a neutral red-containing cytologic reagent solution should be approximately isoosmotic in relation to the potentially pathologic cells. It is also essential that the mixture of the reagent solu tion formed with the sample to be examined (amniotic fluid) should be almost neutral. It has also been observed that lysozyme enzyme significantly increases the stain uptake of the cells, moreover it Increases the stability of the reagent solution, too. Finally it has been found that the reagent solution should contain a saccharide as an energy source for the cells. When a reagent solution storable for a prolonged period of time is to be prepared, preferably a preservative should also be added to the mixture. Based on the above recognitions, 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 to 8.0, preferably 3.5 to 5.0.

The invention also relates to a method for detecting cells with phagocytic activity by staining them with neutral red. According to the invention one proceeds as follows: 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 (preferably glucose) and optionally 0.02-0.6 % by weight of a preservative are dissolved in water, the pH of the solution is adjusted to 3.0-8.0, preferably 3.5-5.0, the solution is sterilized by filtration, thereafter one part by volume of the resulting reagent solution is admixed with 3-8 parts by volume of a sample to be examined, preferably amniotic fluid, and the resulting cell suspension is examined in a haemocytometer in a manner known per se.

The reagent solution according to the invention may comprise as saccharide any mono- or disaccharide serving as energy source for the cells. Of the saccharides glucose is particularly preferred. As preservative preferably benzole acid and/or polyvinyl alcohol can be used.

The haemocytometrie examination of the cell suspension is performed as described in N. Eng. J. Med. 310, 1463 (1984). According to this method the cell suspension treated with the reagent solution is incubated, thereafter centrifuged, the supernatant is discarded, the sediment is admixed with a washing-diluting solution, and then staining is examined in a Btirker chamber. For clinical examinations the reagent solution according to the invention is presented preferably as a reagent kit. The reagent kit comprises a reagent solution presented in dosage units (preferably filled into vials) and a washing-diluting solution presented in dosage units (preferably filled into vials), the volume ratio of said dosage units being 1 : (0.5-5).

As washing-diluting solution any solution of known composition applied in cytologic examinations for this purpose can be used. Hank's solution [J.H. Hank and R.E. Wallace: Proc. Soc. Exptl. Biol. Med. 71, 196 (1949)] proved to be particularly suitable. According to a particularly preferred method the pH of the Hank's solution is adjusted to 7.2-7.4 (preferably to 7.3) with N-hydroxyethyl-piperazine-N'-2-ethanesulphonic acid. The scope of the Invention also extends to the above reagent kit.

The major advantage of the invention is that the solutions applied in the examination are stable, those containing a preservative can be stored for at least 12 months without change, and can be presented in an immediately applicable form. Utilizing the reagent solution or reagent kit according to the invention diagnostic work can be performed more quickly and easily than before and the reliability and reproducibllity of the method is superior to those of the known ones.

The invention is elucidated in detail by the aid of the following non-limiting Examples. Example 1

Reagent kit is prepared from the following solutions: I. Reagent solution:

0.1 g of polyvinyl alcohol is dissolved in 45 ml of hot distilled water, the solution is cooled to room temperature, and then 0.8 g of sodium chloride, 0.1 g of glucose, 0.02 g of benzole acid and 0.1 g of lysozyme are added. The pH of the resulting solution is adjusted to 6.9 with 1 molar Trie [tris(hydroaymethylamino-methane)] buffer solution, and then diluted to a final volume of 50 ml with distilled water. A solution of 0.2 g of neutral red (3-amino-7-dimethylamino-2-methyl-phenozine hydrochloride) in 50 ml of distilled water is added to the above solution, the mixture is well stirred, sterilized by passing through a G-5 sintered glass filter, and the filtrate is filled into vials each containing 1 ml of the solution. II. Washing-diluting solution 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 hydrophosphate dihydrate, 0.03 g of potassium dihydrophosphate and 0.5 g of glucose are dissolved in 350 ml of distilled water, and then 0.1 g of benzole acid is added to the solution. The resulting solution is admixed with a solution of 0.175 g of sodium hydrocarbonate in 12.5 ml of distilled water. The mixture is well stirred, then the pH of the resulting solution (Hank's solution) is adjusted to 7.3 with 5 ml of a 1 molar aqueous solution of N-hydroxyethyl-piperazine-N'-2-ethanesulphonic acid.

The volume of the resulting solution is adjusted to 500 ml with distilled water, the solution is sterilized by passing through a G-5 sintered glass filter, and the filtrate is filled into vials each containing 5 ml of the solution.

Example 2

Reagent kit is prepared from the following solutions: I. Reagent solution

The reagent solution has the same composition and is prepared in the same way as described in Example 1.

II. Washing-diluting solution

4 g of sodium chloride, 0.2 g of potassium chloride, 0.1 g of magnesium sulphate heptahydrate, 0.03 g of disodium hydrophosphate, 0.03 g of potassium dihydrophosphate, 0.5 g of glucose and 0.09 g of calcium chloride dihydrate are dissolved in 350 ml of distilled water, and 0.5 g of benzolc acid is added to the solution. The resulting solution is admixed with a solution of 2.4 g of sodium hydrocarbonate in 30 ml of distilled water. The mixture is well stirred, then the pH of the resulting solution (Hank's solution) is adjusted to 7.3 with 5 ml of a 1 molar aqueous solution or N-hydroxyethyl-piperazine-N'-2-ethanesulphonic acid.

The volume of the resulting solution is adjusted to 500 ml with distilled water, thereafter the solution is sterilized by passing through a G-5 sintered glass filter, and the filtrate is filled into vials each containing 5 ml of the solution. Example 3

Reagent kit is prepared from the following solutions: I. Reagent solution

The reagent solution has the same composition and is prepared in the same way as described in Example 1. II. Washing-diluting solution

4 g of sodium chloride, 0.2 g of potassium chloride, 0.1 g of magnesium sulphate heptahydrate, 0.03 g of disodium hydrophosphate dihydrate, 0.03 g of potassium dihydrophosphate, 0.5 g of glucose and 0.09 g of calcium chloride dihydrate are dissolved in 350 ml of distilled water, thereafter 0.6 g of sodium benzoate is added to the solution. The solution is admixed with a solution of 0.130 g of sodium hydrocarbonate in 10 ml of distilled water, and the pH of the resulting solution (Hank's solution) is adjusted to 7.3 with 5 ml of a 1 molar aqueous solution of N-hydroxyethyl-piperazine-N'-2-ethanesulphonic acid. The volume of the resulting solution is adjusted to 500 ml with distilled water, the solution is sterilized by passing through a G-5 sintered glass filter, and the filtrate is filled into vials each containing 5 ml of the solution. Example 4

Reagent kit is prepared from the following solutions:

I. Reagent solution

The reagent solution is prepared as described in Example 1 with the difference that 0.8 g of neutral red is applied. The reagent solution is filtered, and the filtrate is filled into vials each containing 1 ml of the solution.

II. Washing-diluting solution

The washing-diluting solution has the same composition and is prepared in the same way as described in Example 1.

Of the reagent kits described in Examples 1 to 4 one vial of the reagent solution and one vial of the washing-diluting solution are utilized for a single examination. Example 5

5 ml of amniotic fluid are admixed with one vial (1 ml) of the reagent solution described in Example 1. The mixture is incubated for 15 minutes, then centrifuged, the supernatant is discarded, and the sediment is admixed with 0.5 to 2.0 ml of the washing-diluting solution described in Example 1. The stained sample is examined in a Bürker chamber.

Claims

What we claim is:

1. A neutral red-containing reagent solution for detecting cells with phagocytic activity, which 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 to 8.0, preferably 3.5 to 5.0.

2. A reagent solution as claimed in claim 1, in which the saccharide is a mono- or disaccharide serving as energy source for the cells, preferably glucose.

3. A reagent solution as claimed in claim 1 or 2, in which the preservative is benzole acid and/or polyvinyl alcohol.

4. A method for detecting cells with phagocytic activity by staining them with neutral red , characterized in that 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, preferably glucose, and optionally 0.02-0.6 % by weight of a preservative are dissolved in water, the pH of the solution is adjusted to 3.0-8.0, preferably 3.5-5.0, the solution is sterilized by filtration, thereafter one part by volume of the resulting reagent solution is admixed with 3-8 parts by volume of a sample to be examined, preferably amniotic fluid, and the resulting cell suspension is examined in a haemocytometer in a manner known per se.

5. Reagent kit for the detection of cells with phagocytic activity, which comprises a reagent solution as claimed in any of claims 1 to 3 presented in dosage units and a washing-diluting solution presented in dosage units, and the volume ratio of said dosage units is

1 : (0.5-5).

6. A reagent kit as claimed in claim 5, which comprises vials containing 1 ml each of the reagent solution and vials containing 5 ml each of the washing-diluting solution.

7. A reagent kit as claimed in claim 5 or 6, which comprises as washing-diluting solution a Hank's solution the pH of which is adjusted to 7.2-7.4 with N-hydroxyethyl-piperazine-N'-2-ethanesulphonic acid.