SU611598A3 - Method of counting leucocytes - Google Patents

Description

one

The invention relates to the field of medicine and can be used in diagnostic practice for determining the content of leukocytes in the blood.

There is a method for counting leukocytes by fixation, enzymatic staining and hemolysis 1.

However, the known method does not provide differential counting of leucomas from other blood corpuscles.

The aim of the invention is the differential count of leukocytes from other blood cells, for example, by means of photometry.

This goal is achieved by fixing whole blood with monoaldehyde followed by hemolysis and staining with a cytochemical substrate mixed with a chromogenic conjugating reagent that does not form extracellular particulates, adding a buffer mixture to adjust the pH and heating the resulting mixture from 20 to 55 ° C. for 1-10 min.

In addition, when staining neutrophils and EOSINOFI.IOV, peroxide is used as a cytochemical staining substrate, 4-chloro-1-naphthal is used as a chromogenic conjugate reagent, and alpha naphthyl butyrate is used as a cytochemical staining substrate, and chromogenic conjugate is used. Hexazonium - pararosaniline.

The method is carried out as follows.

An otologic fixative reagent is added to a solution containing white blood cells (for example, whole blood) to kill the contained blood cells and immobilize the contained catalytic cells. Preferred are body fluid samples, such as whole blood and cerebrospinal fluid. After the addition of fixing reagent, the solution is treated with a special cytochemical substance, chromogenic precipitating copulating reagent and buffer.

The cytological fixative solution is a predominantly 0.2-40% aqueous solution of monoaldehyde, such as formaldehyde, butyraldehyde, propionaldehyde, and acetaldehyde. Dialdehydes are unsuitable because they cause the mesh structure and cause extracellular precipitation.

The concentration of monoaldehyde should be high enough to kill the cells, however, without disrupting the activity of the enzyme.

Low concentrations at low temperatures require additional time. For example, at a concentration of formaldehyde in a solution of 2%, at 4 ° C it takes 12 hours, and at 4% concentration at 50 ° C it takes 2 minutes.

Since equal volumes of body fluid and monoaldehyde solution are usually mixed, the strength of the aldehyde solution is double compared to the concentration during fixation of the aldehyde in solution.

A 37% formaldehyde solution is preferred.

 When the body fluid containing leukocytes is whole blood, hemolysis of leukocytes is necessary, since there is a danger that the coinciding passage of two or more red cells through a photometric counting can be mistakenly taken as painted leukocytes or abnormal cells. For this purpose, hemolysis of red blood cells is carried out by adding the reagent to the cell suspension in order to cause the red cells to be discharged and their contents (for example, hemoglobin) to be discharged into the solution.

Hemolyzing reagents should not curtail suspended cells and should not interfere with the flow of any subsequent histochemical reactions by, for example, interacting with any compounds; used for staining cells in the post stages.

Hemolysis is carried out either before or after staining. If hemolysis is carried out after staining, this should not change color or change stained or unstained white blood cells.

Hemolysis is carried out by introducing into a solution containing red and white blood cells an aqueous solution of aliphatic acid with a pH value of | 3.0-5.5. Satisfactory results are obtained by using acetic, propionic, butyric, and lactic acids in a concentration of 1–10%.

Acetic acid solution of about 7% is preferred. The solution can be heated to temperatures ranging from 40 (within 8 minutes) to 55 ° C (within 1 minute), which results in inactivation of the catalysis enzyme, reduction of the hemoglobin pseudoperoxidase activity and acceleration of hemolysis. At lower temperatures, reaction takes longer. The use of even higher temperatures inactivates peroxidase enzymes and accelerates clotting after the introduction of the hemolyzing agent.

Cell staining requires careful selection of reagents. They use a lot of quality

known cytochemical substrates. Thus, a mixture of inorganic or organic peroxide and 4-hporo-1-naphthal is used to stain cells containing peroxidase, such as eosinophils or neutrophils. Peroxide and 4-chloro.1-naphthal serve as enzyme substrates, and 4-chloro-1 naphthal also serves as a copulating reagent. 4-Chlrro-1-naphthal produces a dark blue stain inside the peroxidase-containing cell without the introduction of a separate chromogenic precipitating copulating reagent. It is also possible to use a mixture of (7-tolidine and 4-chloro-baphthal to produce a purple-red dye if the formalreside used in the fixation step is inactivated.

The pH of the solution is monitored for selective staining. If the pH of the solution is below 3.0, then heavy precipitates of the dye are formed only in the rosinophils. If the pH of the solution is in the range of 3.0-5.0, then heavy precipitates of the dye are formed only in neutrophils. If the pH is in the range of 5.0-7.0, then heavy precipitates of the dye are formed only in eosinophils and neutrophils.

IF stained with lipase-containing cells,

those. monocytes, then combinations of an ester, naphthol, such as 1-naphthylacetate or 1-naphthylbutyrate and a diamonium salt, such as hexazonium — pararosanilium, which is a chromogenic precipitating copulating reagent; The pH for this dyeing reaction is 5.56, 5, preferably about 6.0, at this pH, heavy precipitates of dyes are formed only in monocytes.

For the purpose of adequate color painting

in monocytes in a short period of time, for example, in 5 minutes, the concentration of the substrate should exceed about 0.5 mg / ml in the final incubation solution. Naphthal-AS esters and their derivatives and 1-naphthylbutyrate are less soluble than 0.5 mg / ml, therefore 2,2-oxydiethanol (diethylene glycol) or other aqueous alcohols or ethers, for example ethylene glycol, propylene glycol, dimethyl ether, are introduced into them. ethylene glycol

to diethylcarbitol.

Thereafter, 1-naphthylbutyrate and 1-naphthyl acetate can be dissolved at concentrations up to 1 mg / ml. Others may also be dissolved.

substrates, such as naphthyl chloroacetate, indoxyl esters, such as esters of indoxyl acetate, indoxyl propionate and indoxyl butyrate, esters of 8-hydroxychino; tina, such as 8-hydroxyquinoline, 7-hydroxyiiioliniropioate

and 8-hydroxyquinoline butarate. Under the same conditions, only about 0.1 mg / ml of naphthal-AS-acetate will be dissolved, which leads to a significantly weaker color staining. Other ester derivatives of the afatal-AS are even less soluble.