HU208745B - Reagent for staining cells - Google Patents

Abstract

A reagent for staining cells mostly found in urine and esp. blood cells in urine, contains (all wt.%): 0.1-1.0 7-(dimethyl-amino)-3-(methyl-imino)- -3H-pheno-thiazine-hydrochloride and 3,7 bis(dimethyl-amino)-phenothiazinium-chloride in 1:1 wt. proportion, 0.2-3.0 7-(dimethyl-amino)-3- (methyl-imino)- -3H-pheno-thiazine-hydrochloride, 3,7 bis/dimethyl -amino)- phenothiazinium-chloride and 4',5'-dibromo-2',7'-dinitro- -fluorescein-sodium salt in 1:1 wt. proportion and 0.1-2.0 3-amino-7- (dimethyl-amino)-2- -methyl-phenothiazin-5-ium-chloride. These cpds. are dissolved in 1-3 hydric alcohol(s).

Description

Scope of the description: 4 pages (including 1 page figure)

EN 208,745

Field of the Invention The present invention relates to a reagent for staining cells in urine, particularly blood cells, 7- (dimethylamino) -3- (methylimino) -3H-phentiazine hydrochloride, 3,7-bis (dimethylamino) pentiazinium chloride, 4 ' , 5'-dibromo-2 ', 7'-dinitro-fluorescein sodium salt and alcohol content.

In medical laboratory diagnostics, urine sediment testing is one of the most common and most numerous tests. The test, usually performed by doctors, is based on the counting of freshly centrifuged unpainted cells under a microscope. Thus, it is often very difficult to distinguish between white and red blood cells, and the exact identification of certain white blood cell types (neutrophil granulocytes, lymphocytes, monocytes) is almost impossible, although there is a need for this in the diagnosis of kidney disease.

For laboratory diagnosis, Giemsa solution is used to stain protozoa, spirochetes, bacteria and viruses in parasitology, hematology and connective tissue staining (Römpp Chemical Lexicon, Volume II, p. 284, Technical Publisher, Budapest, 1982; Petrányi Autoimmun Diseases, 339). page, Akadémiai Kiadó, Budapest, 1974). The Giemsa solution contains azure II microspheres and azure-eosin microspheres dissolved in a mixture of methanol and glycerol. The Giemsa solution is diluted with a suitable aqueous buffer solution before use. Even after staining with this known reagent, it is very difficult to identify and count cells separated from the urine by centrifugation under a microscope.

SUMMARY OF THE INVENTION The object of the present invention is to provide a reagent which can be clearly distinguished by microscopy by staining cells.

As used herein, the following dyes include the following compounds or mixtures thereof: azure II microspheres: 7- (dimethylamino) -3- (methylimino) -3H-phentiazine hydrochloride and 3,7-bis (dimethylamino) pentiazinium a 1: 1 mixture of chloride; azure II-eosin microsynthesis: 7- (dimethylamino) -3 (methylimino) -3H-phentiazine hydrochloride, 3,7-bis (dimethylamino) pentiazinium chloride and 4 ', 5' dibromo2 A mixture of 7'-dinitro-fluorescein sodium in a 1: 1: 1 weight ratio;

toluidine blue dye: 3-amino-7- (dimethylamino) -2-methyl-phenothiazine-5-ium chloride.

It has been found that the above object is achieved when the solution used for staining cells is

0.1-1.0 wt.% Of a 1: 1 mixture of 7- (dimethylamino) -3 (methylimino) -3H-phenothiazine hydrochloride and 3,7-bis (dimethylamino) pentiazinium chloride. .

0.2-3.0% by weight of 7- (dimethylamino) -3- (methylimino) -3H-phenothiazine hydrochloride, 3,7-bis (dimethylamino) pentiazinium chloride and 4 ', A mixture of 5'-dibromo2 ', 7'-dinitro-fluorescein sodium in a 1: 1: 1 weight ratio;

0.1-2.0% by weight of 3-amino-7- (dimethylamino) -2-methylpentiazin-5-yl chloride

Dissolved in 1-3 alkanol (s).

The invention is based on the recognition that the azure II microspheres, the azure II eosin microspheres and the toluidine blue dyestuff combine well with the core of white blood cells, so that they can be easily identified and counted under a microscope and can be easily distinguished from red blood cells.

In the reagent according to the invention, the dyes are present in one or more alkanol. alkanols

C 1-3 monovalent alkanol such as methanol, ethanol, n-propanol or isopropanol, a divalent alkanol such as ethylene glycol or propylene glycol or trivalent alkanol such as glycerol or mixtures thereof are used.

It has been found advantageous to use a mixture of methanol or ethanol and glycerol.

The reagent of the invention can be stored for a long time. Immediately before use, dilute with a suitable aqueous buffer solution, for example pH 6.8-7.2, buffered with white blood cells separated from the urine by centrifugation, and examine the cells under a microscope after a specific incubation time.

Figure 1 shows a microscope image of protein cells isolated from urine isolated from the known Giemsa solution at 75x magnification.

Figure 2 is a 75-fold magnification of microscope-stained protein cells stained with the reagent of Example 1;

Comparison of Figures 1 and 2 shows that the use of the reagent of the invention makes it easier to detect white blood cells than staining with a known Giemsa solution.

In addition to better distinguishing between white and red blood cells, the reagent of the invention also provides an opportunity to accurately characterize each white blood cell cell. In addition, bacteria and various cylinders present in the urine sediment can be well evaluated using the reagent of the invention.

The invention is illustrated in detail by the following example:

Example

0.8 g of azure II microscope, 3.0 g of azure II eosin microscope and 1 g of toluidine blue are dissolved in 250 g of methanol and diluted with 250 g of 85% glycerol. An aliquot of the resulting reagent is diluted 1: 9 before use with a buffer solution of the following composition:

A solution of 170 g of sodium chloride, 2.7 g of potassium dihydrogen phosphate and 11.0 g of disodium hydrogen phosphate in distilled water, diluted 1: 20 with distilled water before being added to the above reagent. The protein content of urine is tested as follows with the reagent of the invention:

One ml of urine was centrifuged for 10 minutes at 2000 rpm. The supernatant liquid was discarded, and 0.5 ml of the reagent diluted with buffer solution was added to the sediment. The centrifuged pellet is suspended by stirring in the reagent solution and the suspension is kept at room temperature for 10 minutes, followed by a drop of object2.

EN 208 745 Β is placed on a plate, a cover plate is placed and evaluated under a microscope. Under the microscope a

Figure 2 shows an image of 75x magnification.

Claims (1)

Patent Claim Point Reactive staining of cells in urine, especially blood cells, containing 7- (dimethylamino) -3- (methylimino) -3H-phentiazine hydrochloride and 3,7-bis (dimethylamino) pentiazinium chloride, 4 ', 5 Contains' -dibromo-2 ', 7' -dinitrofluorescein sodium salt and alkanol, characterized in that 0.1-1.0% by weight of 7- (dimethylamino) -3-methylimino-3H-phentiazine hydrochloride and 3,7-bis (dimethylamino) pentiazinium chloride in a 1: 1 weight ratio 5 mix, 0.2-3.0% by weight of 7- (dimethylamino) -3 (methylimino) -3H-phentiazine hydrochloride, 3,7-bis (dimethylamino) -fentlazinium chloride and 4 ', 5 '-dibromo2', 7'-dinitrofluorescein sodium salt in a 1: 1: 1 ratio by weight; Contains 0.1-2.0% by weight of 3-amino-7- (dimethylamino) -2-methylpentiazin-5-ium chloride 1-3 in alkanol (s).