JPS6179163A - Reagent for measuring blood cell - Google Patents

Abstract

PURPOSE:To make possible an exact measurement by kinds by constituting the reagent of a spheroidizing agent consisting of a cationic or a nonionic surface active agent expressed by the specific chemical formula, fixing agent and basic fluorescent dye. CONSTITUTION:The reagent is constituted with the cationic surface active agent expressed by formula I or the nonionic surface active agent expressed by formula II as the spheroidizing agent for spheroidizing red blood cells, the prescribed quantity of glutaraldehyde as the fixing agent for maintaining the state of the spherodized blood cells, the basic fluorescent dye for dyeing the blood cells, alkali metallic salt for making isotonic the entire part of the soln. and sodium hydroxide as a buffer agent. When such reagent is mixed with anti-clotting whole blood sample, the red blood cells and reticulocytes are efficiently spheroidized and dyed. The discrimination of the scattered light intensity of the red blood cell groups and platelet groups is thus made easy and the exact measurement of the cell groups of the whole blood by kinds is made possible.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a reagent for measuring blood cells, such as red blood cells, particularly reticulocytes, blood/J% plate, etc. suitable for use in optical blood measurement technology applying flow cytometry. .

Prior Art Prior art involves the use of acridine orange as a fluorescent dye to analyze whole blood samples, as shown, for example, in U.S. Pat. No. 4,536,029, issued June 22, 1982. There is a method that detects and processes the scattered light intensity and red fluorescent light intensity signal by a laser beam using a flow cytometry device to discriminate between red blood cells and platelets, and also to discriminate reticulocytes from red blood cell groups.

In the above flow cytometry device, as shown in Fig. 2, a whole blood sample stained with a staining solution containing acridine orange fluorescent dye flows through a sheath-shaped flow channel 3, forming approximately one optically stimulated area. Light from laser light #1 focused by lens 2 is irradiated onto the blood cells that pass by. Scattered light generated each time a blood cell passes is inputted to the analyzer 12 via a photodetector A5 and an amplifier 1. Further, the fluorescent light in the red region generated each time a blood cell passes through is condensed by a condenser lens 4 using an aberchirp Xc, Dike. After passing through a -7° filter 8, the signal is detected by a photodetector B9, amplified, and then input to an analyzer rIt, 12, where the intensity signal of only the wavelength components necessary for analysis is measured.

The measured scattered light intensity signal and red fluorescent light intensity signal are
The results are plotted in a distribution diagram as shown in the figure, and red blood cells and reticulocytes are distinguished by statistical processing by removing signals from platelet groups.

Problems to be Solved by the Invention Normal human red blood cells are disc-shaped with a concave center.
For this reason, it is inevitable that the scattered light intensity will vary depending on the angle of entry or passage in the optical stimulation area of the flow cytometry device. In other words, as shown in Figure 6, which depicts the spectrum of the scattered light intensity signal, red blood cells and platelets share a signal region that cannot be clearly distinguished from each other, and it is difficult to distinguish between the two using only information about the scattered light intensity. It is possible. Therefore, the two parameters of scattered light and fluorescence are used for discrimination, and as shown in Figure 3, the fluorescence intensity signal also shows that normal red blood cell groups and platelet and reticulocyte groups intersect with each other. Therefore, it is impossible to distinguish between each group. Even in the fluorescence intensity spectrum from which platelet groups are statistically removed using the scattered light intensity signal, as shown in Fig. 4, the mutual intersections hardly change, so that only the fluorescence intensity can be determined by K. It is not possible to accurately distinguish between red blood cells and red blood cells.

Reticulocytes are an indicator for determining the production capacity of red blood cells themselves and anemia, and their accurate measurement is a medically important factor, but as mentioned above, conventional flow cytometry is In the IJ-technology, errors were difficult to ignore even when both the scattered light and fluorescent light parameters were used. Therefore, in order to prevent variations in the intensity of scattered light due to changes in the passage angle of blood cells, attempts have been made to make red blood cells into spheres using an anionic surfactant such as sodium lauryl sulfate.

However, when measuring the reticulocyte group using a flow cytometry device, a staining solution containing basic fluorescent dyes such as acridine orange or vironin G is usually used to observe the difference in fluorescence intensity from the whole blood cell group. When an anionic surfactant is added to a staining solution containing a basic fluorescent dye that needs to be stained, this surfactant acts on the dye and blood cells in the staining solution and once stained. Not only does it cause the precipitation of dye in blood cells, but it also causes a state that inhibits the staining of blood cells themselves.For this reason, depending on the conventional staining solution, it is difficult to clearly stain the stained red blood cell group and reticulocyte group. It was not possible to make accurate measurements.

Means for Solving the Problems The present invention solves the above problems and provides a method for treating all blood cells, ie, reticulocytes, red blood cells, and white blood cells, in a whole blood sample. Accurate measurement of all these blood cell groups by type using a flow cytometry device, accurately reflecting the cell volume and staining sensitivity of each of the platelet groups and preserving the cell volume that contributes to staining and light scattering. This is what makes it possible. To this end, the present invention provides a cationic or nonionic surfactant as a spheronizing agent for spheroidizing red blood cells, a fixative for maintaining the shape of spheroidized blood cells, and a base for staining the blood cells. Provided is a reagent for blood cell measurement comprising an aqueous solution containing a fluorescent dye and a buffer for making the entire solution isotonic.

Among the above-mentioned spheronizing agents, those represented by the general formula are used as the cationic surfactants. Here, R8 is an alkyl group having 10 to 16 carbon atoms, Rt, Rs -R4 is -H
A lower alkyl group such as 1-CHs--Ct&, X may be a halogen such as chlorine or bromine.

In addition, nonionic surfactants include those represented by the general formula % (wherein R is an alkyl group having 8 to 18 carbon atoms, and n is 8 to 18) or those represented by the general formula (herein, R is an alkyl group having 8 to 18 carbon atoms, and n is 8 to 18). , Rt is an alkyl group having 8 to 12 carbon atoms, and n is 8 to 18).

The fixative is glutaraldehyde as a spheronizing agent to prevent the final hemolysis of red blood cells.

Formaldehyde, paraformaldehyde, etc. are used, and the dose thereof is preferably an amount that does not inhibit the spheroidization of blood cells by the spheronizing agent and is suitable for maintaining the shape of blood cells, that is, 0.3 to 6 grams per liter of reagent.

゛ Flavophosphine-R, coryphosphine-0, 4 are used as dyes for fluorescent staining of red blood cells including reticulocytes.
A basic fluorescent dye such as +4' diethylaminostyryl÷N-methylpyridinium iodide, acridine orange, or pyronine G is used.

Furthermore, in order to stabilize the spheroidized blood cells, an alkali metal salt such as sodium chloride is added to make the osmotic pressure of the staining solution reagent isotonic. At the same time, in order to efficiently stain all blood cells, sodium hydroxide or the like is added as a buffer to maintain the pH of the solution at 6 to 11.

The isotonic g:dark dye solution containing the above-described components serves as a blood cell measurement reagent for mixing with an anticoagulable whole blood sample.

When a blood cell measuring reagent prepared as described above is mixed with an anticoagulant whole blood sample, the cationic surfactant or nonionic surfactant in the composition efficiently spheroidizes red blood cells and reticulocytes, and It hardly inhibits the staining effect of fluorescent dyes on blood cells.

In addition, if an appropriate fixative containing aldehyde is used that does not inhibit the spheroidization of blood cells, the hemolytic effect of the spheronizing agent on the blood cells can be prevented and the morphology of the spheroidized blood cells can be maintained for a long period of time.
I Works to maintain stability. By using such a spheroidizing agent, the red blood cells are completely spheroidized, and the intensity of the scattered light changes depending on the angle of approach to the optical stimulation area in the flow cytometry device T or the difference between the light flux and the facing surface during passage. will be resolved. In other words, the intensity of scattered light from individual blood cells received by the photodetector accurately reflects the size and volume of each individual blood cell, and the distribution of the intensity of scattered light from platelets and red blood cells corresponds to the peak of both. In the conventional case (see Figure 6), it was difficult to clearly separate the erythrocyte peaks because they were relatively close to each other and the red blood cell peaks appeared only vaguely (see Figure 6).

In contrast, as shown in FIG. 5, they can be separated very clearly, and it is also possible to distinguish between red blood cell groups and platelet groups using the reagent of the present invention based only on the intensity of scattered light. Furthermore, as shown in FIG. 1, when the reagent according to the present invention is used, the discrimination area of the red blood cell group is reduced to a spherical shape, and there is a gap between the discrimination area of the reticulocyte group and the discrimination area of the normal red blood cell group. It should be understood that there is a significant difference in that they are now clearly distinguishable from each other, compared to the conventional situation shown in FIG. 7, where they are spread out and difficult to distinguish.

Example (1) Dissolve 5 mg of myristyltrimethylammonium bromide, 1 yr of glutaraldehyde, 1 yr of flavophosphine-R, and 9 r of phosphoric acid in 1 liter of water and hydroxylate it.
A reagent for blood cell measurement was prepared by adjusting the osmotic pressure to H8.0, adding sodium chloride, and adjusting the osmotic pressure to 280±10 mOzm/J.

By mixing 500 parts of the above reagent with 1 part of anticoagulant whole blood sample, blood cell spheroidization and fluorescent staining could be performed completely and in a short time. (2) Hexadecyltrimethylammonium chloride 7-5 rng ” *Glutaraldehyde 0.3qy.

Dissolve acridine orange l Q mgr, phosphorus 01yγ in 1 liter of water and add aqueous dispersion of sodium to P)I7.0
Adjust to 280±I Q r with sodium chloride.
The osmotic pressure was adjusted to 7 kg of Osm and used as a reagent. To 1 liter! Dissolve and adjust the pH to 85 with sodium hydroxide, and add sodium chloride to adjust the osmotic pressure to 280±1Q rJLOsm.
/kg of reagent was prepared.

(4) Polyoxyethylene nonylphenol ether (Nonion NS-210, manufactured by NOF) 3 Q mg
r, Ho/I/A aldehyde 3! 1-, 4+
Dissolve 4' dimethylaminostyryl + 30 mg of N-methylpyridinium iodide and 1 yr of phosphoric acid in 1 liter of water, adjust the pH to 10.0 with sodium hydroxide, and chlorinate) Add IJium to make the osmotic pressure 280 ± 1.
9 reagents were prepared in Qmozm/.

(Triton-X 100) 50 m, 'l'
, Glutaraldide 0.3yy; yLabophosphy 7-R40F'LP.

Dissolve phosphorus D1yr in 1 liter of water, add sodium hydroxide to adjust the pH to 8.5, and salt it)
A reagent with an osmotic pressure of 280±1° m05 m/yu was prepared by adding Jum.

Effects of the Invention According to the configuration described above, when the reagent of the present invention is used in a blood sample to be fed to a flow cytometry device, first, red blood cell groups and platelets can be distinguished very clearly only by the intensity of scattered light. (see figure), these individual counts are now possible using only the scattered light factor. In addition, since cationic surfactants and nonionic surfactants are used as spheronizing agents, staining of blood cells with basic fluorescent dyes is carried out completely efficiently, and reticulocytes are also spheroidized under these favorable staining conditions. As a result, it has become possible to distinguish them from normal red blood cells through accurate and simple analysis, and the accuracy of diagnosis through blood tests has been significantly improved.

[Brief explanation of drawings]

Figure 1 is a scattering light intensity-fluorescence intensity distribution diagram measured by a flow cytometer VC using the reagent of the present invention;
The figure shows the basic configuration of a flow cytometry device, Figure 3 shows the fluorescence intensity distribution of stained blood cells before platelets are separated, and Figure 4 shows the same distribution of platelets as Figure 3. After separation, Figure 5 is a diagram depicting the scattered light intensity distribution using the reagent of the present invention.The scattered light intensity of the red blood cell group has a sharp peak, making it possible to distinguish it from the platelet group. Fig. 6 shows the scattered light intensity distribution diagram by the conventional reagent, and comparing it with Fig. 5 shows that the difference between the two is clear [7, Fig. 7 shows the scattered light intensity distribution by the conventional reagent - A fluorescence intensity distribution map is shown. Patent Applicant: Toa Medical Electronics Co., Ltd. (5 others) Jyo/I2] 2 tails, 3 figures, 41'2] Sou, 5 Concave accounts, sales destinations, filtrates, scattered light, etc.

Claims (1)

[Scope of Claims] 1. A blood cell measuring reagent comprising an aqueous solution containing a spheroidizing agent made of a cationic or nonionic surfactant, a fixing agent, and a basic fluorescent dye. 2. The above-mentioned spheroidizing agent has a general formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (However, R_1: an alkyl group having 10 to 16 carbon atoms, R
_2, R_3, R_4: -H or -CH_3, -C
The reagent for blood cell measurement according to claim 1, which is a cationic surfactant represented by a lower alkyl group such as _2H_5, X: halogen. 3. The spheronizing agent has the general formula R_1-[OCH_2CH_2]-_nOH (however,
R_1: alkyl group having 8 to 18 carbon atoms, n=8 to 18)
The reagent for blood cell measurement according to claim 1, which is a nonionic surfactant represented by: 4. The above-mentioned spheroidizing agent has a general formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (However, R_2: an alkyl group having 8 to 12 carbon atoms, n =
The reagent for blood cell measurement according to claim 1, which is a nonionic surfactant represented by 8 to 18).