JPH0464588B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to an optical measurement technique for blood using flow cytometry, 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 (also abbreviated as reticulocytes.RET), and usually account for 0.7 to 2.2% of all red blood cells.Acute internal bleeding can be detected by measuring the reticulocyte count. In recent years, it has become important in clinical tests to support various diagnoses such as hemolytic anemia, poor production anemia, and others.

Initially, smeared blood stained with basic dyes such as new methylene blue and brilliant cresol blue was used to count the reticulocytes, and the percentage of colored reticulocytes was counted while observing all the red blood cells under a microscope. I was disappointed.

Staining with the above-mentioned various dyes requires time and labor for pretreatment of blood samples and visual calculation after staining, and is not suitable for cases where a large number of tests are to be performed.

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, U.S. Patent No. 4336029,
No. 4325706 and No. 4284412 propose the use of acridine orange for staining reticulocytes. This acridine orange is found in reticulocytes.
It is adsorbed to RNA components and emits red fluorescence, making it possible to distinguish it from mature red blood cells, which mainly emit green fluorescence, and to count them. However, in the case of acridine orange, (A) the background fluorescence of the staining solution itself is strong, which tends to cause background noise and errors in measuring the fluorescence intensity derived from blood cells, and (B) the fluorescence is in the red region of 630 nm or more. A highly sensitive fluorescence photometer is required. (C) The degree of non-specific staining is strong and red fluorescence is generated in mature red blood cells, making it difficult to adjust the composition of the staining solution with small errors between specimens. (D) Platelet Because it is strongly stained, if platelets and red blood cells pass through it simultaneously during measurement, the scattered light intensity of mature red blood cells and the red fluorescence intensity of platelets will be measured simultaneously, and this will constitute a signal level equivalent to reticulocytes, causing errors. (E) Acridine orange solution had problems such as being highly environmentally dependent and the fluorescence intensity easily changing.

Further, Japanese Patent Application Laid-Open No. 142465/1983 discloses the use of Thioflavin T as a dye. This involves performing RNA staining on a blood sample, reducing background fluorescence and non-specific fluorescence of mature red blood cells through dilution and washing, and then detecting and measuring the RNA fluorescence of reticulocytes with a flow cytometer. ,
(F) As the non-specific fluorescence is reduced through the above dilution, the specific fluorescence due to the RNA-bound dye is also reduced at the same time, so a measuring device with high fluorescence sensitivity is required. In addition, after (G) dilution, the fluorescence intensity decreases rapidly over time, making it difficult to obtain highly reproducible data.
There was a problem that dyeing took a relatively long time.

Furthermore, pyronine Y was also used as a dye, but after staining fixed red blood cells with RNA and washing to completely remove non-specific fluorescence and back fluorescence, the specific fluorescence caused by the RNA-binding dye was detected. It is something to be measured. According to this dye, (H) the pre-fixation of blood cells, staining, and washing steps require a large amount of time, several tens of times longer than other methods, so it is not suitable for processing multiple samples; (I) The fluorescence quantum yield of pyronine Y itself is extremely low, so it can only be measured by excitation with a high-power laser light source. (J) Pyronine Y is an RNA with a low degree of polymerization.
Since it only binds to DNA, specific fluorescence should be obtained.
There were problems such as low RNA staining efficiency.

Problems to be Solved by the Invention The above-mentioned problems are unavoidable when counting stained reticulocytes using conventional flow cytometry.
(A) to (E) for acridine orange, (F) to (G') for thioflavin T, (H) for pyronine Y
By selectively using a dye substance that can eliminate to the greatest extent all of the problems in (J), the fluorescence intensity of reticulocytes in the stained sample is significantly enhanced, while the background fluorescence of the sample solution is reduced. The problem to be solved by the present invention is to increase the S/N ratio in fluorescence measurement and to significantly improve the 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 for red blood cells in a whole blood sample, the RET ratio of mature red blood cells and reticulocytes, which are immature red blood cells and have a high amount of RNA, is adjusted to a certain degree. , which applies vivid and strong fluorescent staining to the reticulocyte group without being affected by the size of the red blood cell group, and significantly enhances the staining fluorescence intensity, making it possible to identify reticulocytes with high precision. To this end, the present invention provides a flow cytometry method characterized in that a reagent containing auramine O is used to fluorescently stain reticulocytes, and the amount of this reagent used is in excess of that used to precipitate RNA in sample blood. This is a method for measuring 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. .

Auramine O as a fluorescent dye or is used. Auramine O gives a clear hue to stained blood cells, and also has great coloring power itself.

When the amount of dye is small, the fluorescence intensity is low, and the scattered intensity distribution is not uniform, adding a small amount of a fixative that has a fluorescence-enhancing effect, such as glutaraldehyde, can equalize the fluorescence intensity and scattered light intensity of reticulocytes and mature red blood cells. is achieved. The effect of increasing the intensity of scattered light can be adjusted by adding Triton X-100 and maintained at an appropriate detection level. In this way, the background fluorescence of the solution is relatively suppressed,
Furthermore, it improves the unity of the scattered light intensity distribution of reticulocytes, and at the same time lowers the membrane viscosity and prevents adhesion of blood cells to each other, making it easier to differentiate and count them, thereby significantly improving the measurement accuracy of reticulocytes.

The content of auramine O may be 30 to 2500 μgr per ml of the reagent, and the content of alkali metal salts such as sodium chloride may be 5 to 20 mgr per ml, and the pH is preferably adjusted to 6-10. The isotonic buffered dye solution having the above-mentioned composition is a reagent for blood cell measurement, particularly reticulocyte measurement, to be mixed with a whole blood sample anticoagulated with EDTA, etc., and in the present invention, this reagent is used to
The method is characterized by using an excessive amount of reagent that sufficiently exceeds the amount of auramine O that is consumed by combining with RNA contained in blood cells in the specimen as a standard. The above reagent itself has been filed by the present applicant as Japanese Patent Application No. 12328/1983.

When the above reagent for blood cell measurement containing auramine O is mixed with an anticoagulant whole blood sample, the auramine dye vividly stains all blood cells, including reticulocytes, and binds to RNA in the cells to precipitate it, resulting in fluorescent staining. . This state of staining with auramine O is caused by both fluorescence and scattered light being enhanced by the surfactant.
This lasts for more than a few hours, and combined with the effect of suppressing background fluorescence by aldehydes, these factors are combined to greatly improve the accuracy of reticulocyte discrimination and counting compared to conventional reagents. In the present invention, the precipitated RNA
In addition to fluorescent staining specifically adsorbed to chain molecules,
An excessive amount of auramine is used, which causes it to be non-specifically adsorbed to the multi-reticular substance containing RNA.
For blood samples with a RET ratio, the fluorescence intensity of reticulocytes is significantly enhanced, and compared to visual counting, there is almost no variation in measurement data, resulting in highly reliable diagnostic data. The stained reticulocytes are irradiated with violet or blue light using an excitation light source such as an argon ion laser, xenon or mercury arc lamp, helium-cadmium laser, etc., and the generated fluorescence of approximately 450 to 700 nm is detected using a flow cytometer. Discriminative counting is performed by detecting with . When measuring reticulocytes with a flow cytometer using this reagent, its composition is auramine O:
30-2000μgr/ml, NaCl: 8-13mgr/ml, glutaraldehyde: 0-500μgr/ml, Triton X
-100: 0 to 100μgr/ml, phosphoric acid, boric acid, barbital as buffer: 0.01 to 0.2M/,
It is preferable to bring the whole to 1 with the remaining water and adjust the pH to 7.0 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, the specific staining of reticulocytes competes with the non-specific staining of mature red blood cells and the solution itself, and the mature red blood cells themselves are stained. As a result, they overlap in the wavelength range to be measured, making it impossible to detect only reticulocytes. As shown in FIG. 3, 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 a specimen containing a large amount of reticulocytes, since the dye in the reagent was constant, the dye necessary for specific staining is insufficient, and the fluorescence intensity due to reticulocytes becomes weak as shown in FIG. 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 FIG. 3, making it increasingly difficult to distinguish specific adsorption. Such relative 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 is determined, and the light receiving filter and reticulocyte detection level are adjusted. It was necessary to check the reproducibility and accuracy in the determination.

In the pyronin Y and 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. For this reason, during actual measurements, background fluorescence due to the solution and mature red blood cells is reduced by dilution, but as shown in Figure 5, the blood cell signal, including the specific fluorescence based on the RNA itself, becomes extremely small, which interferes with photometry. leading to.

When auramine O is used as the dye, as shown in Figure 1a, the background 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 intensity from reticulocytes is relatively small. big. 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. Staining reveals that RNA, ribosomes, mitochondria,
As shown in Figure 1b, if a network substance consisting of aggregates such as Golgi bodies precipitates and there is an excess of dye,
It is adsorbed on the acidic sites of these substances and, therefore,
Since all the adsorbed dyes emit light C with a quantum yield as high as B adsorbed on RNA, the entire reticular material contributes to the discrimination of reticulocytes. That is, the reticular substance generates the fluorescence intensity of B and C in FIG. 1b, which far exceeds the intensity of A caused by the solution and mature red blood cells, as is clear from FIG. Thus, with a dye that uses auramine O in excess, this dye does not adversely affect the measurement of reticulocytes as with conventional methods, and on the contrary, it is possible to detect even reticulocytes that are very close to mature red blood cells and have little reticulum itself. In this case, there is no need to implement various fluorescence sensitivity improvement measures on the photometric device.

Example Auramine O: 1gr, sodium chloride: 9gr
Add 0.01M/phosphate buffer and water to make 1.
A yellow reagent with a pH of 7.20 was prepared. 10ml of this reagent
10μ of EDTA anticoagulated fresh blood was added, incubated at room temperature for 30 seconds, and then transferred to a fluorescence flow cytometer. The light source used was an argon ion laser that generated excitation light of approximately 500 nm, and fluorescence of 520 nm or more was received and detected. Forward scattered light - 0.1-26% RET with high reticulocyte content detected by backward fluorescence
Measurements were made on 30 samples. In this case, the correlation between measurement 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 O in a sufficient excess amount, the following very specific effects for the fluorescent flow meter are brought about: ( 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 connection,
Reticulocytes that gradually approach mature red blood cells from an immature state immediately after generation, and
Even substances with a small amount of reticular substances, 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 almost never missed.

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

(3) Fluorescence intensity is stable and does not fade for about 1 hour after staining, so measurement values are highly reproducible and reliable.

[Brief explanation of the drawing]

FIG. 1a is a graph showing the state of fluorescent staining of blood cells when using a reagent amount containing auramine O in an amount commensurate with the amount of reticulocyte RNA in the sample;
Figure 1b is a graph similar to that shown in a of staining using an excess amount of auramine O according to the method of the present invention;
The figure is a graph comparing the fluorescence intensity signals corresponding to Figure 1 a and b, respectively, Figure 3,...
Figure 4 is a graph similar to Figure 2 corresponding to Figure 3. Figure 5 is a graph similar to Figure 3 using the pyronine Y and thioflavin T method. It is.

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 violet to blue wavelength. 1. A method for measuring reticulocytes using a flow cytometer, characterized in that reticulocytes are measured by irradiating a region with light 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, 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, wherein the light source is a xenon arc lamp.