JPH01301166A - Standard liquid for calibrating blood cell counter - Google Patents

Abstract

PURPOSE:To impart high dispersion stability to particles and to obtain high reliability in both the measurement of the number of platelets and the number of red blood cells and the measurement of a hematocrit value by dispersing the 1st particles and 2nd particles respectively having specific average grain sizes into an aq. electrolyte soln. CONSTITUTION:The 1st particles which are substantially spherical have a sulfonic acid group and/or sulfonate group on the surface and have 1.9-3mum average grain size and <=5% coefft. of fluctuation. The range of the average grain size is approximately equal to the size of the platelets of a normal man. The 2nd particles which are substantially spherical have the number of the ratio at which the number is 10-40 times the number of the 1st particles. Said particle have the sulfonic acid group on the surface and have 4.5-5.5mum average grain size and <=5% coefft. of fluctuation. The 2nd particles correspond to the red blood cells and the range of the average grain size thereof is smaller than the size of the red blood cells of the normal man. These 1st, 2nd particles are dispersed in the aq. electrolyte soln.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a standard solution for calibrating a hemocytometer.

[Conventional technology]

In general, it is important to measure the number of red blood cells, platelets, or hematocrit (1) of blood in order to know the state of a disease. A blood cell counter is usually used for such measurements, but this blood cell counter needs to be calibrated with high accuracy. In order to fli8B this, conventionally, a standard solution for calibrating a hemocytometer (hereinafter simply referred to as "standard solution") made of a dispersion of polymer particles has been used.

[Problem that the invention seeks to solve]

However, with conventional standard solutions, (1) it is necessary to add surfactants, etc. to the standard solution because the dispersion stability of the particles is low, and this causes the standard solution to foam violently, causing erroneous measurements. (2) The number of particles in the standard solution is said to correspond to the normal number of red blood cells, so there is no problem in measuring the number of red blood cells, but the hematocrit value is different from that of red blood cells. If a standard solution containing substantially spherical particles of the same size (6 to 9 sides) is used, the measurement will be large, so
There is a problem in that high reliability cannot be obtained for one hematocrit shift.

The present invention has been made in the technical background as described above, and its purpose is to provide particles with high dispersion stability and to be suitable for both platelet count, red blood cell count measurement, and hematocrit value measurement. Our goal is to provide highly reliable standard solutions.

[Means for solving problems]

The object of the present invention is to have a sulfonic acid group and/or a sulfonic acid group (hereinafter simply referred to as "sulfonic acid group") on the surface, an average particle size of 1.9 to 3 mm, and a coefficient of variation of 5%. a substantially spherical first particle that is
Substantially spherical particles with sulfonic acid groups on the surface, an average particle size of 4.5 to 5.5 spines, and a coefficient of variation of 5% or less, in a proportion of 10 to 40 times the number of particles. This is achieved by a standard solution characterized in that the second particles are dispersed in an aqueous electrolyte solution.

The present invention will be specifically explained below.

The standard solution of the present invention is one in which first particles and second particles each having a specific average particle size are dispersed in an electrolyte aqueous solution at a specific ratio.

The first particles have a sulfonic acid group on the surface and are substantially spherical with an average particle diameter of 1.9 to 3 mm, preferably 2 to 2.92 IJ11. This average particle size range is within the size and tract of normal human platelets.

Further, the second particles have a sulfonic acid group on the surface, have an average particle size within the range of 4.5 to 5.5 mm, and are substantially spherical like the first particles. It is. This second particle corresponds to red blood cells, but its average particle size range is smaller than the size of normal human red blood cells (6-9 μs), and this makes the hematocrit value highly reliable. can be obtained. If the average particle size of the second particles is less than 4.5 particles, the hematocrit value will be small, and if the average particle size exceeds 5.5, the hematocrit value will be large. It can no longer be used as a standard for measuring values.

In the present invention, "substantially spherical" for the first particles and the second particles (hereinafter referred to as "dispersed particles") means
When the longest diameters of the front view, elevation view, and side view images of the particles are projected using the orthogonal projection method are x and ySz, the values of y X x of the particles are all 0. It refers to a state in which the value is in the range of .5 to 2, and is preferably perfectly spherical.

The coefficient of variation of the dispersed particles, that is, the ratio of the standard deviation to the average particle diameter, must all be 5% or less, and more preferably 3% or less.

The dispersed particles in the present invention need to have sulfonic acid groups on their respective surfaces. Such dispersed particles having a sulfonic acid group are usually made of a thermoplastic polymer obtained from a vinyl monomer, a thermosetting polymer such as a phenol resin, and can be obtained by the following method.

(1) A vinyl monomer having one sulfonic acid group represented by the formula -3O3X (X represents a monovalent cation such as a lithium ion, potassium ion, sodium ion, or ammonium ion), such as sodium styrene sulfonate, acrylamide - A method for obtaining dispersed particles by polymerizing or copolymerizing N-propanesulfonic acid or the like.

(2) To synthesize polymer particles by polymerization or copolymerization and then introduce sulfonic acid groups onto the surface of the polymer particles, for example, the chloromethyl groups on the particle surface derived from chloromethylstyrene used as a vinyl monomer are replaced with bisulfite) Alternatively, the benzene ring on the particle surface derived from the aromatic vinyl monomer used as the vinyl monomer or the phenol used as the monomer of the phenol resin may be reacted with concentrated sulfuric acid, fuming sulfuric acid, sulfuric anhydride, chlorosulfonic acid, etc. By reacting, −
3O, H is introduced, and if necessary, -3O3H is converted to monovalent ions such as lithium ions, potassium ions, sodium ions, ammonium ions, etc. using lithium hydroxide, potassium hydroxide, sodium hydroxide, ammonium hydroxide, etc. A method of converting into a salt by cations and obtaining dispersed particles.

The monomer copolymerizable with the vinyl monomer having a sulfonic acid group in the method (1) above or the vinyl monomer for synthesizing polymer particles in the method (2) above is preferably styrene, vinyltoluene, α
- Aromatic vinyl monomers such as methylstyrene, divinylbenzene, chloromethylstyrene, and chlorostyrene can be mentioned, and these monomers are not limited to one type, but two or more types can also be used. Further, the aromatic vinyl monomer may be copolymerized with other vinyl monomers, and examples of other vinyl monomers include conjugated dienes such as butadiene, isoprene, and chloroprene; methyl (meth)acrylate, ethyl (meth)acrylate, and ethyl (meth)acrylate; (meth)acrylic acid esters such as acrylate and hydroxy(meth)acrylate; halogenated vinyl monomers such as vinylidene chloride; (meth)acrylonitrile, vinyl acetate, and (meth)acrylic acid.

Note that methods for polymerizing or copolymerizing vinyl monomers in method (1) above, and methods for synthesizing polymer particles by polymerization or copolymerization in method (2) above include emulsion polymerization, seed polymerization, and suspension polymerization. , swelling polymerization, precipitation polymerization, etc. In particular, seed polymerization,
Precipitation polymerization or swelling polymerization is preferred.

The density of sulfonic acid groups on the surface of these dispersed particles is usually 0.001 to 1 per person, preferably 0.01
~0.5 piece/to2. If this density is less than 0.001 particles/person2, the dispersion stability of the dispersed particles becomes insufficient and aggregation tends to occur in the electrolyte aqueous solution, while on the other hand, 1 particle/person2
If it exceeds this range, the hydrophilicity of the dispersed particles becomes too large, causing swelling or cracking of the particles themselves, which is not preferable.

In the present invention, examples of the electrolyte for the electrolyte aqueous solution in which the dispersed particles are dispersed include sodium chloride, potassium chloride, sodium phosphate, disodium phosphate,
Monosodium phosphate, potassium phosphate, dipotassium phosphate, monopotassium phosphate, sodium citrate, sodium benzenesulfonate, salicylate, thorium, sodium carbonate, sodium bicarbonate, sodium borate,
Hydrochloric acid, sulfuric acid, sodium hydroxide, ammonium chloride, etc. can be used, and not only one of them, but also two
More than one species can also be used. The electrolyte concentration in these electrolyte aqueous solutions is preferably 0.1 to 20% by weight.

Further, as specific examples of these electrolyte aqueous solutions, r l
5OTON-UJ (manufactured by Coulter), physiological saline, and the like.

The number of dispersed particles dispersed in the electrolyte aqueous solution varies depending on the dilution ratio of the hemocytometer used, but usually the number of the first particles is 4XIO' to 3XIO'/mi', and the number of the second particles is 4X10 The number of particles is within the range of 12×10 particles/rnl, and the ratio of the number of first particles to the number of second particles is 1:10 to 40, preferably 1:15 to 20. The measurement value by the hemocytometer was 2 to 3 XIO@particles/mi for the first particle! , the second particle is preferably expressed as a numerical value in the range of 4 to 5×10s particles/rnl.

The standard solution of the present invention can also be used as a standard solution for measuring hemoglobin concentration in blood by coloring the electrolyte aqueous solution and making its absorption spectrum similar to that of cyanmethemoglobin.

For such coloring, the aqueous medium is colored with a combination of two or more acidic dyes and/or direct dyes, and similar to the light absorption characteristics of cyanmethemoglobin, a wavelength of 540 nm is obtained.
has a maximum absorption at 540 nm and its absorption spectrum is 540 nm.
What is necessary is to have absorption characteristics that are flat in the vicinity.

The dyes used here are acidic dyes and/or direct dyes, and other dyes, such as basic dyes and oil-based dyes, are used to dye the standard solution container, measurement cell, dispersed particles, serum proteins attached to the measurement cell, etc. It also stains the skin, causing errors in measurements.

Specific examples of acidic dyes that are preferably used include, for example, Anradred 8, Anradred 26, Anradred 27, Anradred 35, Anradred 37, Anradred 49, Anradred 52, Anradred 131, Anradred 134, Anradred 138, Anradred 14.
5, 224, 249, 254, 274, Acid Violet 49, 51, Food Red No. 2, No. 3,
Examples include No. 102, No. 104, No. 105, and No. 106. Specific examples of direct dyes include Direct Red 9, Direct Red 9, Direct Red 3L, Direct Violet 51, and the like. Among these, preferable ones are Undradred 8 and Undradred 26.
, 27, 35, 37, 52, 145, 22
4, 254 and Direct Red 224.

These dyes have a maximum absorption in the range of 500 to 550 nm, but when two of them are used in combination, it is possible to obtain
It is possible to obtain flat absorption characteristics over a wavelength range of more than 100%. The difference between the maximum absorbance and the minimum absorbance of this flat portion is usually 0.02 or less, preferably 0.01 or less.

Specific examples of dye combinations suitable for obtaining such properties include [Acid Red 35/Food Red No. 2/Food Red No. 106 J1 [Acid Red 35/Acid Red 254], [Acid Red 37/Direct Red]. [Acid Red 224], [Acid Red 27/Acid Red 35/Acid Red 52], etc.
Particularly preferred dye combinations are [Acid Red 37/Direct Red 224] and [Acid Red 27/
Assisted Dred 35/Assisted Dred 52].

The amount of these dyes used is usually 1p in the standard solution.
The amount is within the range of 10% by weight from pm, preferably from 2 ppm to 5% by weight, and is appropriately determined depending on the dilution ratio used in the hemocytometer and the absorbance required as a standard solution.

In addition, the standard solution of the present invention may contain an anti-staining agent, if necessary.
Additives such as antibacterial and antifungal agents can be added.

Examples of the stain inhibitor include alcohols such as ethyl cellosolve; organic acid salts such as benzoic acid, sodium tartrate, and lactic acid; hydrophilic silicone compounds, and the like.

These more completely prevent staining of serum proteins adhering to the standard solution container, particles dispersed on the measurement surface, and measurement cells.
Used to achieve stable absorbance measurements.

Examples of antibacterial/antifungal agents include IJumdiazide, sodium benzoate, and sodium salicylate.

〔Example〕

Examples of the present invention will be described in detail below, but the present invention is not limited thereto.

Example 1 Particle Δ Weight ratio of styrene to methyl methacrylate is 98:2
10 g of substantially spherical particles (average particle size 2.08 μs, coefficient of variation 2.5%) made of styrene-methyl methacrylate copolymer were added to 100 ml of concentrated sulfuric acid, and the sulfonation reaction was carried out at 60°C with stirring. The reaction was carried out for 1 hour, neutralized with reaction condensate sodium hydroxide, and thoroughly washed with water to obtain substantially spherical particles A having sulfonic acid groups on the surface. The average particle size of this particle A is 2.09 gm, and the coefficient of variation is 2.5%.
The density of sulfonic acid groups determined by conductivity titration was 0.22/person2.

Particle B The weight ratio of styrene and methyl methacrylate is 98:2
Sulfonic acid groups were added to the surface in the same manner as Particle A, except that substantially spherical particles (average particle diameter 5.16 μs, coefficient of variation 1.4%) made of styrene-methyl methacrylate copolymer were used. Substantially spherical particles B were obtained. The average particle diameter of this particle B is 5.30 μs, and the coefficient of variation is 1.5
%, and the density of sulfonic acid groups determined by conductivity titration was 0.23 pieces/2.

Preparation of standard solution In an electrolyte aqueous solution containing about 0.9% by weight of potassium chloride, 5.5XIQ'/rn! of the above particles were added. ,, the ratio of I
Standard solution 1 was obtained by adding and dispersing the solution (11 g).

Regarding the standard solution 1 that is above the measurement level, on the day of preparation of the standard solution 1 and after it has been allowed to stand at room temperature for 3 months, use a hemocytometer rPc 601J (manufactured by Elma) and the same rPc 601J (manufactured by Elma)
Measurement was performed using PC604J (manufactured by Emaru Co., Ltd.), and the displayed platelet count, red blood cell count, and hematocrit (Ht> value (by W+ntrobe method) were as shown in Table 1.

Table 1 From the results in Table 1, it is clear that even after 3 months, this standard solution 1 had the same properties as at the time of preparation and had excellent dispersion stability. Further, in this standard solution 1, the particles were sufficiently dispersed by gentle shaking, and no variation was observed in the measurement results even in the five measurements at each measurement.

Comparative Example 1 Substantially spherical particles C (coefficient of variation 3.0%, sulfonic acid group Density of 0.20
The particles C were dispersed at a ratio of 8 XIO' particles/d and the particles A used in Example 1 were dispersed at a ratio of 4.4 When the obtained comparison standard solution 1 was measured using a hemocytometer rPc 604J, the number of red blood cells displayed was 4.05.
Although the number of red blood cells/ml is not particularly problematic, the displayed hematocrit value is 48.8%, and the number of red blood cells is 4.
Hematocrit value approximately 34 calculated from Xl09 pieces/gloss
%.

Comparative Example 2 Particle D The weight ratio of styrene and n-butyl acrylate is 70:
30, substantially spherical particles (average particle size 2, 88 ua, coefficient of variation 1.5%) of 100 m! The mixture was added to 2N IJ hydroxide and hydrolyzed at 80° C. for 8 hours with stirring, and thoroughly washed with water to obtain substantially spherical particles having carboxyl groups on the surface. The average particle size of the particles was 2.85 gm, the coefficient of variation was 1.9%, and the density of carboxyl groups determined by conductivity titration was 0.35 pieces/person2.

Particle E Substantially spherical particles (average particle diameter 5.25 μs, coefficient of variation 2.3%) made of styrene-n-butyl acrylate copolymer with a weight ratio of styrene and n-butyl acrylate of 70 to 30. Substantially spherical particles E having carboxyl groups on the surface were obtained in the same manner as in the particle preparation except that particles E were used. The average particle diameter of the particles E was 5.28JLII+, the coefficient of variation was 2.3%, and the density of carboxyl groups determined by conductometric titration was 0.56/person2.

Preparation of standard solution The above particles were added to the same electrolyte aqueous solution as in Example 1 in 5.
5X10' pieces/m! ! , the above particles E are 1 x 105 pieces/-
A comparative standard solution 2 was obtained by adding and dispersing the particles at a ratio of (Particle D: Particle E = 1:1 B).

Regarding this comparative standard solution 2, in the same manner as in Example 1,
Measurements were performed on the day of preparation and after one month of standing at room temperature, and the displayed platelet counts and red blood cell counts were as shown in Table 2.

From the results shown in Table 2, it is clear that the displayed measurement value of Comparative Standard Solution 2 decreased after one month, and the storage stability was low.

Example 2 Acid Red 35 and Acid Red 254 were added and dissolved in the standard solution 1 obtained in Example 1 at a ratio of 1:1 so that the concentration was 3 ppm.

The maximum absorption of this colored standard solution 1 is at a wavelength of 515 to 545 nm.
The absorbance in this range is flat at 0.2 ± 0.003, and this colored standard solution 1 does not stain plastic containers, glass or quartz cells, and it does not stain glass to which human serum albumin has been adsorbed. It also did not stain.

〔Effect of the invention〕

In the standard solution of the present invention, all of the dispersed particles have sulfonic acid groups on their surfaces, so the particles have high dispersion stability and storage stability without adding surfactants. Because it is large and contains a specific proportion of two types of substantially spherical particles, each with a specific average particle size, it is fully reliable for both red blood cell and platelet measurements and hematocrit measurements using a hemocytometer. Results with high quality will be displayed.

Claims (1)

[Claims] 1) Substantially spherical first particles having a sulfonic acid group and/or a sulfonic acid group on the surface, an average particle size of 1.9 to 3 μm, and a coefficient of variation of 5% or less, and this first particle The particles have a number of sulfonic acid groups and/or sulfonic acid groups on the surface in a ratio of 10 to 40 times the number of particles with an average particle size of 4.
A standard solution for calibrating a hemocytometer, characterized in that substantially spherical second particles having a diameter of 5 to 5.5 μm and a coefficient of variation of 5% or less are dispersed in an aqueous electrolyte solution.