JPS6234058A - Method for measuring reticulocyte by flow sight meter - Google Patents

Abstract

PURPOSE:To attain to enhance the measuring accuracy of a reticulocyte, by making the amount to be used of a reagent containing auramine O more excessive than the amount precipitating RNA in a blood specimen. CONSTITUTION:When a corpuscle measuring reagent containing auramine O is mixed with an anticoagulant whole blood specimen, the auramine dye vividly stains a corpuscle containing a reticulocyte and is bonded to RNA in a cell to precipitate RNA subjected to fluorescent staining. The specimen stained with the corpuscle measuring specimen used in an amount made more excessive than the amount precipitating RNA in the blood specimen is irradiated with light of a purple - blue wavelength region to measure fluorescence from the specimen. By this method, measuring accuracy can be enhanced even with respect to a corpuscle specimen low in the concn. of a corpuscle containing a reticulocyte or having a low reticulocyte ratio.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an optical blood measurement technique using flow cytometry (IJ), and particularly to a method for measuring immature (reticuloid) red blood cells, whose counting has recently become increasingly important.

Conventional technology Immature red blood cells in a blood sample are called reticulocytes (reticulocytes, also abbreviated as RET), and usually account for 0.7 to 2.2% of all red blood cells. It has become important in recent clinical tests because it supports various diagnoses such as acute internal bleeding, hemolytic anemia, and hypoplastic anemia by measuring .

Todoroki Hatsume New Methylene Blue was used to count the reticulocytes mentioned above.
A blood smear stained with a basic dye such as brilliant cresol blue was used, and the percentage of colored reticulocytes was counted while all the red blood cells were twisted under a microscope.

Staining with various dyes requires time and labor for pre-treatment of blood samples and visual calculation after staining, making it unsuitable for large numbers of tests.

Blood testing using flow cytometry, which was developed there, has become faster and more accurate, but problems still remain regarding reticulocyte counting.

Among the prior art, US Pat. No. 4,336,029, US Pat. No. 4,325,706, and US Pat. No. 4,284,412 propose the use of acridine orange for staining reticulocytes. This acridine orange is adsorbed to the RNA component in reticulocytes and emits red fluorescence, allowing them to be distinguished from mature red blood cells, which mainly emit green fluorescence, and to be counted. but,
In the case of acridine orange, the background fluorescence of the staining solution itself is strong, and it becomes background noise in the measurement of the fluorescence intensity derived from blood cells, which tends to cause errors. A highly sensitive fluorophotometer is required, (q) The degree of non-specific staining is strong and red fluorescence is generated in mature red blood cells, so it is difficult to prepare a staining solution composition with small sample volume error, (r)I Platelets Because red blood cells are strongly stained, if platelets and red blood cells pass simultaneously during measurement, the scattered light intensity of mature red blood cells and the red fluorescent light intensity of platelets will be measured simultaneously, and this will form a signal equivalent to reticulocytes, which can lead to errors. The cause of this problem was that the homogeneous acridine orange solution had a large environmental dependence and the fluorescence intensity was subject to change.

Furthermore, Japanese Patent Application Laid-Open No. 59-142465 discloses the use of Thioflavin T as a dye. this is,
After performing RNA staining of the blood sample, background fluorescence and nonspecific fluorescence of mature red blood cells are reduced by dilution and washing, and then RNA fluorescence of reticulocytes is detected using a flow cytometer.
However, as the non-specific fluorescence is reduced through the above dilution, the specific fluorescence due to the RNA-bound dye is also reduced, so a photometric device with high fluorescence sensitivity is required. However, there was still the problem that highly reproducible data could not be obtained after dilution of Gl because the fluorescence intensity decreased rapidly over time, and (G') staining took a relatively long time.

Furthermore, pyronine Y has also been used as a dye, but fixed red blood cells are stained with RNA and washed to completely remove non-specific fluorescence and background fluorescence, and then specific fluorescence with an RNA-binding dye is applied. It measures light. According to this dye, (HJ) each step of pre-fixation of blood cells, staining, and washing takes a long time and is several tens of times longer than other methods, making it unsuitable for processing multiple samples. (J) Pyronine Y has an extremely low fluorescence quantum yield and can only be measured by excitation with a high-power laser light source.
, R should obtain specific fluorescence because it binds only to DNA.
There were problems such as low efficiency of NA staining.

As mentioned above, it is unavoidable to count the reticulocytes stained with the conventionally used 70-Sidemed Ij-. In case (H), the fluorescence intensity of the reticulocytes in the stained sample is significantly enhanced by selectively using a dye substance that can be removed to the extent that it covers all the problems in the sample solution, while the background of the sample solution is The problem to be solved by the present invention is to reduce fluorescence and increase the S/N ratio in fluorescence measurement, thereby significantly improving measurement accuracy of reticulocytes, especially in the low concentration or low RET ratio range.

Means for Solving the Problems The present invention solves the above problems and distinguishes between mature red blood cells and immature red blood cells in a whole blood sample.
A vivid and strong fluorescent staining is applied to the reticulocyte group without being affected by the RET ratio or the red blood cell concentration, etc., and the intensity of the dyed fluorescence is significantly increased. To this end, the present invention uses a reagent containing auramine O to fluorescently stain reticulocytes, and describes the use of this reagent to identify RNA in sample blood. A method for measuring blood cells by flow cytometry is realized, which is characterized in that an excessive amount is used to precipitate the blood cells. In order to stabilize the blood cell shape during optical measurement, an alkali metal salt such as sodium chloride can be added to make the staining solution reagent isotonic, and a buffer can also be added to increase the efficiency of staining blood cells. .

As auramine O as a fluorescent dye, NH, HNO3 or NH-HIJj is used. Auramine 0 gives a vivid hue to stained blood cells, and its coloring power is also high.

If the amount of dye is small, the fluorescence intensity is low, and the scattered light intensity distribution is uneven, use a fixative that has a fluorescence increasing effect, such as glutaraldehyde, and adjust the increasing effect by adding Triton X-100. It is possible to maintain an appropriate detection level.

In this way, the background fluorescence of the solution is relatively suppressed, and the scattered light intensity distribution of reticulocytes is improved, and at the same time, the membrane viscosity is lowered to prevent blood cells from adhering to each other, thereby improving their differential counting. This makes it easier to measure reticulocytes and significantly improves the accuracy of reticulocyte measurement.

Auramine 0 is 30-2500 μ9r per 1 mg of reagent.
The content of alkali metal salts such as sodium chloride may be 5 to 20 mgr per rttl, and the pH is 6-1.
It is preferable to adjust it to 0. The isotonic buffered dye solution having the above-mentioned composition is used as a reagent for blood cell measurement, particularly reticulocyte measurement, to be mixed with a whole blood sample anticoagulated with EDTA or the like. The method is characterized by using an excess amount of the reagent that sufficiently exceeds the amount of auramine 00 that is consumed by combining with RNA contained in cells. The above reagent itself is disclosed in Japanese Patent Application No. 60-12312 by the present applicant.
It has been filed as No. 8.

When the above reagent for blood cell measurement containing auramine 0 is mixed with an anticoagulant whole blood sample, the auramine dye vividly stains all the reticulocytes and blood cells, and binds to RNA in the cells to precipitate it, causing it to fluoresce. dye. This dyed state caused by auramine O lasts for more than an hour because both fluorescence and scattered light are enhanced by the surfactant, but the background fluorescence is suppressed by the aldehydes, and these effects are combined and are The accuracy of differential counting of reticulocytes is significantly improved compared to the reagent described above. In the present invention, in addition to fluorescent staining that is specifically adsorbed to the precipitated RNA chain molecules, an excessive amount of auramine is used in which auramine is non-specifically adsorbed to other reticular substances containing RNA, and therefore, reticulocytes are Even when the blood cell concentration is low or the blood sample has a low RET ratio, the fluorescence intensity of reticulocytes is significantly enhanced, and the variation in measurement data is almost eliminated compared to visual counting, giving high reliability of diagnostic data. Become. The stained reticulocytes are
Approximately 450 to 70
Discriminative counting is performed by detecting fluorescent light of about 0 nm with a flow cytometer. When measuring reticulocytes with a flow cytometer using this reagent, its composition is OramiyO: 30-20001'? ,'/me, Na(
J: 8-13"+9'/rne, Glutaraldehyde: O-500 PI'/ml.

Toridor X -100:0~1001"9'/rrtl
, phosphoric acid, boric acid, barbital as buffer: 0.
01-0.2M/l, adjust the total to 11 with remaining water and pH 7,
It is suitable to adjust it to 0 to 9.0.

Effects In the conventional staining method using acridine orange, although it is desirable to measure all the fluorescence emitted by reticulocytes, which is shown in the shaded area in Figure 3, there is a difference between specific staining of reticulocytes and non-specific staining of mature red blood cells and the solution itself. The staining competes and mature red blood cells themselves are stained, which overlaps in the wavelength range to be measured, making it impossible to detect only reticulocytes. As can be seen in FIG. 3 (1), it can be seen that the wavelength range in which fluorescence photometry can be performed without being affected by background fluorescence from mature red blood cells is extremely small. On the other hand, in samples containing many reticulocytes,
Since the dye in the reagent was constant, there was a shortage of dye necessary for specific staining, and the intensity of fluorescence by reticulocytes became weak as shown in FIG. 3(n). In addition, in the case of a blood cell sample with a small number of red blood cells and an excess of dye, and the dye is easily adsorbed, the signal intensity of both mature red blood cells and reticulocytes increases as shown in Figure 3 (fil), making it increasingly difficult to distinguish specific adsorption. Become.

Such dynamic changes in signal strength are compared in FIG.

Therefore, in the acridine orange method, the number of blood cells to be detected in the sample is adjusted to some extent in advance, and then the dye concentration that maximizes specific adsorption and minimizes non-specific adsorption (C) is determined, and the light-receiving filter and net are adjusted. It was necessary to confirm reproducibility and accuracy in determining the red blood cell detection level.

In the pyronine Y1 thioflavin T method, when RNA is stained to the maximum, the fluorescence intensity ratio between mature red blood cells is small and it is difficult to distinguish blood cell signals from solution fluorescence, as shown in FIG. 5 (1). For this reason, during actual measurements, the background fluorescence caused by the solution and mature red blood cells is reduced by dilution, but as shown in Figure 5 (n), the blood cell signal, including the specific fluorescence caused by the RNA itself, is extremely small. This may cause problems with photometry.

When auramine O is used as the dye, as shown in Figure 1(a), the back fluorescence from the solution and the fluorescence intensity from the dye adsorbed to relatively low viscosity mature red blood cells are small, and the fluorescence from reticulocytes is small. The strength is relatively high. Therefore, even in a specimen containing a large amount of reticulocytes, reticulocytes can be sufficiently distinguished from mature red blood cells with an amount of reagent containing enough dye to completely stain RNA. Upon staining, reticular substances consisting of aggregates of RNA, liposomes, midochondria, Golgi bodies, etc. are deposited and precipitated within the reticulocytes. The entire reticulum contributes to the discrimination of reticulocytes since all the adsorbed dye emits light (q) with a quantum yield as high as that of the RNA adsorbed (B). In other words, the reticular substance is shown in Figure 1(b).
As is clear from FIG. 2, the fluorescence intensity of B%C is generated, which far exceeds that of A produced by solution and mature red blood cells. Thus,
This staining uses excessive amounts of auramine ○, which does not have a negative effect on reticulocyte measurements as in conventional methods.
On the contrary, it is possible to detect even reticulocytes that are very close to mature red blood cells and have little reticulum itself, and in this case there is no need to implement various measures to improve the fluorescence sensitivity of the photometric device.

Example Auramine O: 1.9r, sodium chloride: 9.9r, o, oIM/l phosphate buffer and water were added to make 1g and the pH was adjusted to 1g.
A yellow reagent of 7,20 was prepared. This reagent 10rrt
1 opz of EDTA anticoagulated fresh blood was added to the tube, incubated for 30 seconds at room temperature, and then transferred to a fluorescence flow cytometer. An argon ion laser that generates excitation light of about 500 nm was used as a light source, and fluorescent light of 520 nm or more was received and detected. 30. of 0.1 to 26% RET with a high content ratio of reticulocytes detected by forward scattered light-backward fluorescence.
i measurements were made for the t* body. In this case, the correlation between the measured data and visual counting data was extremely high even for low RET samples.

Effects As mentioned above, when a blood sample is dyed with a fluorescent staining reagent containing Auramine 0 in a sufficient excess amount, the following effects are very specific to the fluorescent flow meter: (1 ) The difference in fluorescence intensity signals between mature red blood cells and reticulocytes is large, and the accuracy of signal discrimination between both and solution fluorescence is further improved. In this regard, reticulocytes that have gradually approached mature red blood cells from an immature state immediately after generation, and even blood cells with little reticulum such as RNA, can be distinguished from mature red blood cells, platelets, etc. with high accuracy. . Therefore, in a sample solution with a low RET ratio, reticulocytes are hardly missed.

(2) Staining time is extremely short. The acridine orange method stains for 5 minutes, but when RET exceeds 2 to 3%, it takes 10 minutes or more to complete staining. Staining with excess auramine ○ can be completed in 30 seconds even for specimens with RET=13%.

(3) The fluorescence intensity is stable and does not easily fade for about 1 hour after staining, so the reproducibility and reliability of measured values is high.

[Brief explanation of drawings]

Figure 1 (a) is a graph showing the state of fluorescent staining of blood cells when using a reagent amount containing auramine○ in an amount that is commensurate with the amount of reticulocyte RNA in the sample. A graph similar to [a) showing staining using an excess amount of auramine O by the invention method, Figure 2 is the same as Figure 1 t&), t
b) Fluorescence intensity signals corresponding to 1. Graph comparing II, Figure 3 (D, (II), (Il [)) is a graph similar to Figure 1 using the acridine orange method, Figure 4 is a graph similar to Figure 2 corresponding to Figure 3. , Figure 5 (1
1, (ID is pyronine Y, third by thioflavin T method)
This is a graph similar to the one shown in the figure. Patent Applicant Toa Medical Electronics Co., Ltd. (5 others)) Snoring η--Kichicho and n~ 450 500 550 60゜-Kayako Rokucho (nyn) 1 N Fig. 3 500 580 600 (77
7.? N-V direction fu 11v'-Kaze Tsuyakito-Quasi-Ya Atmosphere 1

Claims (1)

[Claims] 1) Using a fluorescent staining reagent for blood cells containing auramine O, a sample stained with an amount of the reagent in excess of the amount required to precipitate RNA in the sample blood cells is treated with a purple to blue color. 1. A method for measuring reticulocytes using a flow cytometer, characterized in that reticulocytes are measured by irradiating light in a wavelength range from a light source and measuring fluorescence from the sample. 2) A method for measuring reticulocytes using a flow cytometer, wherein the light source is an argon-ion laser. 3) A method for measuring reticulocytes using a flow cytometer according to claim 1, wherein the light source is a helium-cadmium laser. 4) A method for measuring reticulocytes using a flow cytometer according to claim 1, wherein the light source is a mercury arc lamp. 5) A method for measuring reticulocytes using a flow cytometer according to claim 1, wherein the light source is a xenon arc lamp.