HU216303B - Flow cytometry reagents - Google Patents

Abstract

The object of the application is to differentiate flow cytometric reagent clay strokes for plexiglass flocculation by a laser diffraction principle of 0.1 to 40.0 mmol / l organic and / or inorganic pellet, 0 to 20.0 g / lacillus primer, 0.001 to 4.0 g / l non-sparkling detergent, and contains 0.001 to 2.0 g / l of antioxidant and / or 0.001 to 5.0 g / l of preservative, in addition to 100% by weight of non-aqueous water. ŕ

Description

EXTRACT

The present application relates to flow cytometric reagent for differentiation of leukocytes into five subpopulations by laser diffraction principle containing 0.1-40.0 mmol / l organic and / or inorganic buffer, 0.5-20.0 g / l leukoprotective component, 0.001-4.0 g / l of a nonionic detergent and optionally 0.001 to 2.0 g / l of an antioxidant and / or 0.001 to 5.0 g / l of a preservative and 100% by weight of deionized water.

The scope of the description is 6 pages

HU 216 303 B

HU 216 303 Β

BACKGROUND OF THE INVENTION The present invention relates to a hematological reagent for the operation of laser diffraction hematology analyzers, which is suitable for automated hematology assays: quantitative and qualitative determination of whole blood elements, in particular for differentiation of leukocytes into five subpopulations, and hydrodynamic focus for measurement purposes.

Introducing the leukoprotective and surfactant components of the invention into an optimal osmotic pressure and pH buffer system allows their leukocytes to be differentiated into five subpopulations through their synergistic synergies and improves the stability of these white blood cell fractions.

An important area of medical laboratory diagnostics is hematology, which is now routinely performed on automated measuring devices. Basic parameters to be electronically determined during whole blood tests: red blood cell (RBC, erythrocytes) and platelets (PLT), hematocrit (HCT), hemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (mean corpuscular volume) ', MCH), mean corpuscular hemoglobin concentration (MCHC), and white blood cell (WBC) leukocytes.

Human white blood cells can be divided into lymphocytes, monocytes and polymorphonuclear cells. Polymorphonuclear cells can be further subdivided into neutrophilic, eosinophilic, and basophilic cells according to the staining properties of the cytoplasmic granules. In clinical hematology diagnostics, there is an increased need for qualitative evaluation of leukocytes, ie differentiation into at least 3, but optimally 5 subpopulations.

Electronic automated cell counters are used to measure the aforementioned blood cells. These devices are capable of quickly and accurately measuring the quantity and size of leukocytes, red blood cells and platelets, and measuring hemoglobin concentration.

One type of blood cell counter operates on the principle of flow cytometry. Flow cytometry combines the benefits of microscopic examination and biochemical analysis: rapid and accurate analysis of blood cells using high precision techniques.

In hydrodynamically focused flow cytometric measurements, the cells to be examined are concentrated, using the sheath fluid that circulates the flow line, into the axis of a fast-flowing liquid carrier, whereby the cells form a cluster of particles of small diameter, flowing smoothly. In flow cytometry, the cuvette is illuminated by a laser light emitting monochromatic and coherent photons. Typical laser light sources include: argon-ion laser (UV, blue and green light), helium-cadmium laser (UV and blue light) and helium-neon laser (red light).

The particles flowing through the laser-illuminated measuring cuvette pass through the light beam, the light scattered through the corpuscle and the intensity of the diffracted light scattered at different diffraction angles gives information on the cell size and morphology.

Although the light is scattered in all directions, the intensity (axial diffraction angle) of light scattered in the direction of the illuminating laser beam relative to the axis of the beam is related to the cell size. For example, lymphocytes speak photons at low axial diffraction angles, and are typically distinguished from granulocytes scattering at higher axial diffraction angles, while monocytes exhibit axial diffraction angles similar to granulocytes.

The intensity of the light scattered orthogonally to the axis of the laser light is primarily derived from the internal or surface structure of the light scattering cell and can be interpreted as an index of cellular granularity.

The combination of axial diffraction and orthogonal diffraction parameters provides a more reliable result than any parameter alone for discriminating between different groups of human blood elements.

Differentiation of leukocytes can occur after lysis of erythrocytes (based on different laser diffraction properties) or without haemolysis by staining of leukocytes with fluorescent dye based on scattered and fluorescent signals.

Flow cytometry assays have been the subject of numerous publications (Megla GK: Acta Cytol. 17: 3 (1973); Alpert NL: Foot World 24:40 (1973); Mansberg P., Saunders A.M., Growes W. J.). Histochem. Cytochem. 22: 711 (1974); Shapiro H.M. et al .: J. Histochem. Cytochem. 24: 396 (1976); Shapiro Η. M., et al., J. Histochem. Cytochem., 25: 976 (1977); Gradwohl's Clinical Laboratory Methods and Diagnosis, The C. V. Mosby Company, 1980, 787-792. etc.).

The reagent systems used are described in several patents.

Hypotonic aqueous saline as used in U.S. Patent No. 3,883,247, causes erythrocyte haemolysis. The patent discloses differentiation into 6 leukocyte subpopulations after staining with acridine orange dye based on laser-lighted fluorescence.

U.S. Pat. No. 4,284,412 describes leukocyte differentiation to 3 subpopulations (lymphocyte, monocyte, granulocyte) after erythrocytes on the basis of measured light scattering and fluorescence values.

According to U.S. Patent No. 4,376,620, leukocytes are differentiated after staining with oxazine dye.

According to U.S. Patent 4,801,549, erythrocytes are lysed at 62-72 ° C, leukocytes are fixed with formaldehyde and stained with 4-chloro-1-naphthol.

According to U.S. Patent No. 4,882,284, flow cytometric assays are performed using oxazine fluorescent dye without erythrocyte hemolysis.

HU 216 303 Β

In U.S. Patent No. 5,196,346, the reagents used result in erythrocyte haemolysis and the pH of the lysis reaction is between 2.5 and 3.2.

The reagents used in U.S. Patent No. 5,232,857 haemolyze erythrocytes, and leucocytes are stained with chlorazole black dye.

European Patent Publication No. 177137 lyses erythrocytes at a reaction pH of 1.8 to 2.3.

The present invention is based on the discovery that the components of the invention allow selective stromatolysis of erythrocytes and hydrodynamic focus of the test sample at optimally selected concentrations while unexpectedly (synergistically) improving leukocyte differentiation to the five subpopulations.

Thus, the present invention provides a reagent system that completely lyses erythrocytes without damaging (partially lysing) the white blood cells or adversely affecting the laser light scattering properties of leukocytes, particularly lymphocytes, during laser diffraction measurements.

The reagent we developed was tested on CELL-DYN 3000 and 3500 laser diffraction hematology analyzers. The initial technical phases of the measurement are identical to the Coulter (impedance) devices. After the automatic pre-dilution of the blood sample to be analyzed, the pre-diluted sample is divided into separate graduated cuvettes.

- determination of RBC and PLT parameters,

- photometric measurement of hemoglobin liberated from hemolysed red blood cells in the form of cyanomethemoglobin after the addition of a hemoglobin reagent,

- determination of leukocyte distribution parameters by laser diffraction after automatic addition of erythrocyte lysing / leukoprotective reagent and two-dimensional scatter plot of five subpopulations (LYM: lymphocyte, MONO: monocyte, EOS: eosinophil, BASO: basophil, NEU: neutrophil) , 90 ° scattering intensity vs. 10 ° scattering intensity, lobularity vs. granularity, etc.) in point groups.

The lysing / leukoprotective agent is a key component of the reagent system. From a measurement point of view, it is essential for the lysing reagent that the lysis reaction is carried out at an appropriate rate, i.e., moderated hemolysis completely resolving erythrocytes, but "sparing" the leukocytes, must occur within an optimum time (a few seconds incubation) and / measuring solution system must remain stable during the measuring time (5-8 seconds). In addition, the lysing reagent must also have leukoprotective properties to differentiate leukocytes into five subpopulations (lymphocyte, monocyte, eosinophil, neutrophil, basophil). The reagent system of the present invention further performs hydrodynamic focusing of the blood sample to be tested and prevents bubble formation in the tube system at high velocity fluid flow.

The most important factors determining the rate of lysis reaction (Ψ) are as follows: pH, osmotic pressure (σ), detergent strength (ξ), leukoprotective effect (π), temperature (t), blood concentration (c _blood ), which is written as a function: Ψ ( ρΗ, σ, ξ, π, c _blood , t).

When designing the reagent system, the task is to optimize the above multivariate function [Ψ (ρ, σ, ξ, π, c _blood , t)}. On a given measurement system (CELL-DYN 3000/3500 Hematology Analyzers), blood concentration ( _blood c) and measurement temperature (t) are given parameters, so the variables of the function to be optimized are reduced and modified as follows: Ψ ^ / ρΗ, σ, ξ, _p ).

The functional pH of the reagent is important in both respects. Namely, if the pH is relatively low (pH <4.5), the rate of erythrocyte membrane lysis reaction is significantly reduced, which significantly increases the time required for one assay, i.e. slows down the sample processing speed of the meter. On the other hand, the high pH of the reagent (pH> 9.0) causes rapid damage to leukocytes, leading to instability of evaluation. The ideal pH range was found to be between 7.0 and 9.0 and the optimum pH was 8.1 ± 0.4.

The role of the buffer system used and the osmotic pressure it generates are also essential for the proper functioning of the lysing reagent. Preferably, _the pKa of the buffer _is about the optimal pH (8.1 ± 0.4). Because a more concentrated buffer system osmotically slows down the lysis reaction, the ideal buffer is required to have a high buffer capacity and thus be able to buffer properly at low osmotic pressure (<90 mOs / kg). For our purpose, we found that 0.1-40 mM TRIS buffer is optimal (pK _a = 8, l [25 ° C]), but other organic buffer systems (barbiturate, ADA, MOPS, PIPES, HEPES, etc.) can be used. At a concentration of 1-40 mmol / l. Inorganic buffer systems (phosphate, borate, etc.) may also be used at concentrations of 0.1 to 40 mmol / L, however, leukocyte differentiation results in less stable results. The most suitable buffer system we found having an osmotic pressure of 5-80 mOs / kg (ideally 10-60 mOs / kg) - 0.1-40 mmol / l tris-hydroxymethylaminomethane and 0.05-15 mmol / l boric acid .

In addition to the above parameters, the detergent strength of the lysing reagent substantially influences the rate of erythrocyte hemolysis. The detergent strength is determined by the hydrophilic / lipophilic character (HLB) and concentration of the detergent used. This component, by virtue of its cell membrane structure relaxant and solubilizing properties, is able to increase the lysis rate of the erythrocytamembrane and, by ideally modifying intermolecular interactions, enables flow turbulence to be avoided and hydrodynamic focusing of the laminar flow sample in the enclosure. By using them, maximum leukocyte signal / membrane noise ratio can be achieved by stabilizing the leukocyte signal and minimizing background caused by membrane fragments. It's optimal

EN 216 303 Β haemolysis rates of 0.001 to 4.0 g / l alkyl polyethylene glycol ether and / or 0.001 to 4.0 g / l alkoxy polyethylene glycol ether and / or 0.001 to 4.0 g / l aryl. polyethylene glycol ether and / or 0.001 to 4.0 g / l alkylaryl polyethylene glycol ether nonionic detergents. Polyethylene glycol (9,6) -octylphenyl ether (HLB = 13.5) and / or 0.001-4.0 g / l polyethylene glycol (7,8) -octyl are preferably used in a concentration of 0.001 to 4.0 g / l. -phenyl ether (HLB = 12.4) and / or 0.001 to 4.0 g / l polyethylene glycol (23)-lauryl ether (HLB = 16.9) and / or 0.001 to 4.0 g / l polyethylene glycol- sorbitan monolaurate (HLB = 16.7); and / or Pluronic F-108 (HLB = 27) at a concentration of 0.001 to 4.0 g / L, a polyethylene glycol-polypropylene glycol copolymer.

Although low osmotic pressure (<90 mOs / kg) buffer is capable of rapidly lysing erythrocytes, leukocytes, particularly lymphocytes, may be more or less degraded, which may result in a decrease and instability of the measured white blood cell count. This disadvantage can be avoided by using a leukoprotective reagent additive, which component selectively protects leukocytes during the lysis reaction. According to our studies the above-mentioned effect leukoprotektív comprising certain short-chain (C _10), low molecular weight (Mn <200) and a polar portion, however slightly lipophilic aliphatic and aromatic alcohols and glycol ethers have. During the haemolysis reaction, these diffusible, small molecules in the reagent system, by associating rapidly with lipophilic zones to the surface of cells or moderately diffused into their membrane structure, exert their protective effects by retaining polar, leucoprotective, membrane-permeable lipophilic zones of diffusing detergent molecules. This repulsive effect does not allow the displacement of the membrane-stabilizing leukoprotective molecules in the relatively short reaction and measurement time (up to 40 seconds) and the detergent molecules to loosen, then solvate and dissolve. At optimal concentrations of leukoprotective components, the membrane stabilizing effect described is differentially expressed: the membrane of erythrocytes without nuclei is soluble, but the membrane of leukocytes, especially the most vulnerable mononuclear lymphocytes due to their low lipoprotein content, is not damaged. The membrane protective and stabilizing effect of the leukoprotective component does not distort the cellular structure of the leukocytes, thereby not adversely affecting the laser light diffraction properties.

Analyzing the mechanism of action of the leukoprotective molecules, we found that the longer-chain, more lipophilic, leukoprotective molecules have higher protective efficacy and their concentration is concentration-dependent. At low concentrations, the protective effect is less pronounced: besides erythrocytes, some lymphocytes are lysed and membrane noise may become significant. At optimum concentrations, the substance protects lymphocytes and membrane noise is minimized, while at high concentrations, light scattering of leukocytes becomes unfavorable and lysis of erythrocytes is not complete.

The incomplete erythrocyte lysis may be caused by an increase in the osmotic pressure of the reagent or by the stabilization of the erythrocyte membrane as it exerts its leukoprotective effect primarily on lymphocytes.

Preferred leukoprotective components for use in our studies are: 0.5-20.0 g / l alkylene glycol monoalkyl ether and / or 0.5-20.0 g / l alkylene glycol monoaryl ether, and / or 0.5-20.0 g / l. g / l alkanol.

However, it is particularly preferred to use the following compounds in the following concentrations: 1.0-10.0 mL / L ethylene glycol monobutyl ether and / or 0.1-5.0 mL / L ethylene glycol monophenyl ether, and / or , 0-8.0 ml / l butanol, and / or 1.2-12.2 ml / l isopropanol.

From a measurement point of view, it is very important that the refractive index of the lysing reagent should be the same as the refractive index of the diluent: it should be between 1.332 and 1.335, the best result being obtained with a refractive index of 1.334.

To ensure storage stability of the lysing reagent, an antioxidant to prevent auto-oxidation degradation of the detergent is also added. Preferred antioxidants are 0.001 to 2.0 g / l of Na-3,3'-thiodipropionate and / or 0.001 to 2.0 g / l of butylhydroxytoluene and / or 0.001 to 2.0 g / l of ascorbic acid, and / or in a concentration of 0.001 to 2.0 g / l of p-methoxyphenol.

In addition to the above-mentioned components, the lysing reagent also contains a preservative that inhibits bacterial and fungal growth. The ideal preservative is nonionic and low in activity, thus reducing its osmolality effect. Ideal preservative grains have been found in ethylene glycol monophenyl ether at a concentration of 0.001-5.0 g / l, which allows storage at room temperature (25 ° C) for at least 12 months, but preservative grains of 0.001-3.0 g / l Na- 2-mercaptopyridine 1-oxide and / or 0.001-3.0 g / l gentamicin sulfate and / or 0.0013.0 g / l neomycin sulfate and / or 0.001-3.0 g / l 1 procaine hydrochloride and / or 0.001-3.0 g / l8-oxyquinoline and / or 0.001-3.0 g / l8-oxyquinoline sulfate and / or 0.001-1.0 g / l mertiolate .

The following general formulas are characterized by specific examples:

Lysing / Leukoprotective / Flow Orientation Reagent

Example 1 For a liter of deionized water: 21.0 g of tris-hydroxymethylaminomethane, 1.5 g of citric acid, 6.5 g of Tergitol Type 15-S-12, 45.0 ml of ethylene glycol monophenyl ether, 650.0 ml of n-butanol, 3.8 g of butylhydroxytoluene, 0.5 g of Na-2-mercaptopyridine-1-oxide are made up to 100 liters with deionized water. After complete dissolution, the pH is adjusted to 8.20 with 4M NaOH or 6M HCl and then passed through a 0.22 µm membrane filter into a suitable container.

Example 2 To be added to a liter of deionized water: 20.0 g of tris-hydroxymethylaminomethane, 10.0 g of boric acid, 4.8 g of polyethylene glycol (23) lauryl ether, 50.0 ml of ethylene glycol monophenyl ether 800.0 ethylene glycol monobutyl ether, 4.0 g butyl

Make up to 100 liters with hydroxytoluene, deionized water. After complete dissolution, the pH was adjusted to 8.10 with 4M NaOH or 6M HCl and then passed through a 0.22 µm membrane filter into a suitable container.

Example 3 To be added to a liter of deionized water: 22.0 g of tris-hydroxymethylaminomethane, 4.0 g of acetic acid, 9.5 g of polyethylene glycol sorbitan monolaurate, 55.0 ml of ethylene glycol monophenyl ether, 800.0 ml isopropanol, 4.0 g of butylhydroxytoluene, is made up to 100 liters with deionized water. After complete dissolution, the pH is adjusted to 8.10 with 4M NaOH or 6M HCl and then passed through a 0.22 µm membrane filter into a suitable container.

Example 4 To be measured for 1 liter of deionized water: 26.0 g tris-hydroxymethylaminomethane, 13.0 boric acid, 4.5 g polyethylene glycol (9,6) octylphenyl ether, 400.0 ml ethylene glycol monobutyl ether, 330.0 mL of n-butanol, 3.5 g of butyl hydroxytoluene, 1.5 g of gentamicin sulfate, make up to 100 L with deionized water. After complete dissolution, the pH is adjusted to 8.00 with 4 M NaOH or 6 M HCl and filtered through a 0.22 µm membrane filter into a suitable container. 25

Synergistic coefficient

Surprisingly, an unexpected (synergistic) interaction between the detergent and the leukoprotective component used in the reagent was observed, when the above-mentioned ingredients were used at optimum concentrations, the differentiation of leukocytes into five subpopulations was stabilized and the erythrocyte solubilized by diminished red blood cells.

The following experimental results prove this:

Measurements were performed on CELL-DYN 3000 and 3500 hematology analyzers. The reference reagent used for the device is the factory-supplied preparation.

Preparation of lysing "stock solution": To 800 ml deionized water, add the following components with constant stirring: 0.20 g tris-hydroxymethylaminomethane, 0.10 g boric acid, 0.50 ml ethylene glycol monophenyl ether, 04 g butylhydroxytoluene. After complete dissolution, the solution is made up to 1 liter with deionized water under constant stirring and filtered through a 0.22 µm pore size membrane into a polyethylene container.

-Reference Reagent: CELL-DYN REAGENT, (ABBOTT), SHEATH, Product No. 99 311-01 The following components were added to the stock solution and the resulting solutions were labeled with:

- Reagent A: Stock solution

Reagent B: Stock solution + 0.04 # g of polyethylene glycol (23) lauryl ether

- reagent C: stock solution + 0 · 0 ml ethylene glycol monobutyl ether,

Reagent D: Stock solution + 0.048 polyethylene glycol (2 3) lauryl ether + 8.0 ml ethylene glycol monobutyl ether.

The leukocyte distribution stabilizing effect of the formulations was characterized by the reproducibility of the measurements, by the coefficient of variation (CV%) of the measured leukocyte subpopulations (LYM: lymphocyte, MONO: monocyte, EOS: eosinophil, BASO: basophil, NEU: neutrophil). Higher distribution stability has a lower% variance. For 6 blood samples (2 abnormal WBC, 2 normal WBC, 2 abnormally high WBC) 5 consecutive measurements were made with each reagent. (The rest of the reagent system consisted of factory-supplied diluent, hemoglobin reagent, cleaning detergent solution.)

The results are as follows:

Reagent used CV (LYM)% CV (MONO)% CV (EOS)% CV (BAS)% CV (NEU)% Reference 2.2 2 (2 3.2 3.1 2.4 Reagent A 10.5 8.8 9.7 9.2 8.1 Breagens 8.5 7.9 8.5 8.9 6.2 Creagens 6.9 6.1 6.9 5.5 5.1 Dreagens 2.1 2.1 3.0 2.9 2.2

The lowest variance, and thus the highest distribution stability, was found for the reagent D of the present invention.

Advantages of the invention:

Compared to the reagent compositions of the present invention, it was found that prior formulations did not allow for hydrodynamic focusing of the samples and differentiation of leukocytes by laser diffraction into five subpopulations in the pH range used by the older formulations. The additives we use allow for more accurate, stable and reproducible measurements.

Claims (12)

PATENT CLAIMS 1. Flow cytometric lysing / leukoprotective / current50-targeting reagent for differentiation of leukocytes into five subpopulations of the laser diffusion principle containing 0.1-40.0 mmol / l organic and / or inorganic buffer, 0.5-20.0 g / l leukoprotective component, 0.001 to 4.0 g / l of a nonionic detergent, and optionally 0.001 to 2.0 g / l 55 antioxidants and / or 0.001 to 5.0 g / l of preservative, and deionized water in an amount of 100% by weight, characterized in that the leukoprotective component is C ₁ -C ₁ aliphatic and / or aromatic alcohols, and / or aliphatic and / or aromatic glycol cholesters and alkyl5 as nonionic detergent EN 216 303 Β polyethylene glycol ether and / or alkoxy polyethylene glycol ether and / or aryl polyethylene glycol ether and / or alkylaryl polyethylene glycol ether. 2. The reagent of claim 1, wherein the organic buffer is 0.1-40.0 mmol / L TRIS buffer and / or 0.1-40.0 mmol / L PIPES, and / or 0.1 -40.0 mmol / L HEPES and / or 0.1-40.0 mmol / L barbital buffer and / or 0.0515.0 mmol / L borate buffer and / or 0.05-15, 0 mM phosphate buffer and / or 0.05-15.0 mM citrate buffer and / or 0.05-15.0 mM acetate buffer. 3. A reagent solution according to any one of claims 1 to 4, characterized in that it contains 0.1-40.0 mmol / l tris-hydroxymethylaminomethane and 0.05-15.0 mmol / l boric acid. 4. A reagent solution according to any one of claims 1 to 4, characterized in that its osmotic pressure is between 5 and 80 mOs / kg and the pH is between 7.0 and 9.0. 5. A reagent solution according to any one of claims 1 to 3, characterized in that it has an osmotic pressure of 10-60 mOs / kg, a pH of 7.7-8.5 and a refractive index of 1.332-1.335. 6. A reagent solution according to any one of claims 1 to 3, characterized in that the leukoprotective component is 0.5-20.0 g / l alkylene glycol monoalkyl ether and / or 0.5-20.0 g / l alkylene glycol monoaryl ether, and / or Containing 5-20.0 g / l alcohol. 7. A reagent solution according to any one of claims 1 to 3, characterized in that the leukoprotective ethylene glycol monoalkyl ether is 0.5-20.0 g / l ethylene glycol monoethyl ether and / or 0.5-20.0 g / l ethylene glycol monopropyl ether, and / or or 0.5-20.0 g / l ethylene glycol monobutyl ether and / or 0.5-20.0 g / l propylene glycol monoethyl ether and / or 0.5-20.0 g / l propylene glycol monopropyl ether and / or 0.5-20.0 g / l propylene glycol monobutyl ether. 8. A reagent solution according to any one of claims 1 to 5, characterized in that the leukoprotective alkylene glycol monoaryl ether is 0.5-20.0 g / l ethylene glycol monophenyl ether and / or 0.5-20.0 g / l propylene glycol monophenyl ether. 9. A reagent solution according to any one of claims 1 to 5, characterized in that the leukoprotective alkanol is 0.5-20.0 g / l ethanol and / or 0.5-20.0 g / l npropanol and / or 0.5-20.0 g / l 1 isopropanol and / or 0.5-20.0 g / l butanol and / or 0.5-20.0 g / l isobutanol. 10. A reagent solution according to any one of claims 1 to 6, characterized in that the nonionic detergent is present in a concentration of 0.001 to 4.0 g / l polyethylene glycol (9.6) octylphenyl ether and / or 0.001 to 4.0 g / l polyethylene glycol (7.8) -. octylphenyl ether and / or 0.001 to 4.0 g / l polyethylene glycol (23) lauryl ether and / or 0.001 to 4.0 g / l polyethylene glycol sorbitan monolaurate and / or 0.001 to 4 g / l, 0 g / l polyethylene glycol sorbitan monopalmitate and / or 0.0014.0 g / l Pluronic F-108 polyethylene glycol-polypropylene glycol copolymer. 11. A reagent solution according to any one of claims 1 to 3, characterized in that the antioxidant is 0.001-2.0 g / l butylhydroxytoluene and / or 0.0012.0 g / l Na-3,3'-thiodipropionate, and / or 0.001-2 , 0 g / l of ascorbic acid and / or 0.001 to 2.0 g / l of p-methoxyphenol. The reagent solution according to any one of claims 1 to 11, characterized in that the preservative is 0.001 to 3.0 g / l of Na-2-mercaptopyridine 1-oxide and / or 0.001 to 5.0 g / l of ethylene glycol. monophenyl ether and / or 0.001 to 3.0 g / l gentamicin sulfate and / or 0.0013.0 g / l neomycin sulfate and / or 0.001 to 3.0 g / l procaine hydrochloride, and / or from 0.001 to 3.0 g / l of 8-oxoquinoline and / or from 0.001 to 3.0 g / l of 8-oxoquinoline sulfate and / or from 0.001 to 1.0 g / l of mertiolate.