JPS61165635A - Pretreatment of blood - Google Patents

Abstract

PURPOSE:To enable the accurate calculation of a reticulocite by preventing the generation of a dummy reticulocite, by immersing a stained blood specimen in an org. solvent for a predetermined time and subsequently coating the blood specimen with oil for an oil immersion lens. CONSTITUTION:The blood specimens 102 received in a cassette 101 are immersed in xylene for 1-10min. Next, the blood specimens are extruded from the cassette 101 one at a time by a lever 106 in a stage raising said blood specimens from xylene while a proper amount of oil 104 is dripped on the surface of each blood specimen to uniformly coat the surface thereof with oil for an oil immersion lens. By this operation, xylene is penetrated in the deformed part of one which was a dummy reticulocite at first and the contamination of the blood specimen is removed by oil and the greater part of dummy reticulocites can be eliminated when the oil for the oil immersion lens was dripped and erroneous discrimination can be prevented.

Description

[Detailed Description of the Invention] [Field of Application of the Invention] The present invention provides a blood pretreatment method for use in a reticulocyte count test in which red blood cells are distinguished from red blood cells using a microscopic image of a supravital-stained blood specimen and the reticulocyte ratio is calculated. Regarding.

[Background of the invention]

New methylene blue
In fresh blood specimens that have been stained with supravital dyes such as blue, reticulocytes are immature red blood cells in which purplish-blue rank substances and string-like substances (reticular bodies) are entangled in greenish-yellow red blood. be. Nucleated erythroblasts enucleate within the bone marrow and emerge into the peripheral blood, but in the process they inevitably pass through the reticulocyte stage. An increase or decrease in the reticulocyte ratio in peripheral blood indicates an increase or decrease in immature red blood cells, that is, an increase or decrease in erythropoietic ability. Therefore, calculation of reticulocytes is an important parameter for understanding erythropoiesis. This calculation of the reticulocyte ratio is performed using fresh blood specimens that have been stained with supravital staining.
It is performed using a high-magnification optical microscope using an oil immersion objective. In this case, wrinkle-like deformation may occur near the center of each red blood cell, depending on the drying conditions of the blood sample and the length of time elapsed before microscopy. When examining the red blood cells using the oil immersion method, the oil cannot penetrate into the wrinkled areas of the red blood cells, resulting in a microscopic image of the red blood cells that is very similar to a reticulocyte image. Such red blood cells are called pseudoreticulocytes.

The frequency of occurrence of this pseudoreticulocyte is, in rare cases, 1 red blood cell
In contrast, the frequency of IM red blood cells in normal people is very low at 3 to 11 per 1000 red blood cells, and the presence of pseudoreticulocytes is calculated. have a significant impact on the results. Particularly 1
For example, an automated device such as that proposed in Japanese Patent Publication No. 54-38758, etc., identifies mature red blood cells (hereinafter simply referred to as red blood cells) and reticulocytes by automatic identification processing of microscopic images of blood specimens, and calculates the reticulocyte percentage. It is extremely difficult to distinguish between the pseudo reticulocytes and true reticulocytes using a calculation device. Therefore, the occurrence of pseudo reticulocytes in blood specimens has been a major obstacle in automating reticulocyte calculation6. Prevent it from occurring in blood specimens.

The object of the present invention is to provide a blood pretreatment method that enables accurate reticulocyte count.

[Summary of the invention]

The blood pretreatment method of the present invention is characterized by immersing a supravital-stained blood specimen in an organic solvent for a predetermined period of time, and smearing oil for a microscope oil immersion lens on the blood specimen. According to this method, even if wrinkle-like deformation occurs in red blood cells, the oil for the oil immersion lens used during microscopy sufficiently penetrates into the wrinkle-like deformation, and the microscopic image shows the deformed part that can be mistaken for a reticular body. No image is generated. That is, the above-mentioned pseudo red blood cells do not appear in the microscopic image, and accurate reticulocyte count can be performed both by visual inspection and by automatic image processing.

[Embodiments of the invention]

FIG. 1 shows an example of a blood specimen pretreatment apparatus for carrying out the blood pretreatment method of the present invention. In FIG. 10, lot is a cassette case that stores a blood specimen 102;
has been stained supravital.

103 is an organic solvent (xylene in this example), 104
is a microscope oil immersion lens oil (for example, US R, P, CAR
GILE LABORATORrES.

TYPEA, manufactured by INC., Inc., 105 is a tube that guides the oil 104 to the blood specimen 102, and 106 is the blood specimen 10.
Reference numeral 2 is a lever that moves the blood sample 102 to the oil dripping position, reference numeral 1.07 is a driving portion of the lever 106, and reference numeral 108 is a mechanism portion that returns the blood specimen 102 to the cassette position after the oil is dripped. Blood specimen 102 stored in cassette 101 is first immersed in xylene solution 103 for 1 to 10 minutes. The soaking time is adjusted depending on the drying conditions and elapsed time after dyeing. However, if immersed for more than 10 minutes, stained red blood cells will begin to decolorize, so it is not desirable to soak for too long.

Next, in the step of pulling up the blood specimen from the xylene solution, the lever 106 is used to pull the blood specimen one by one into the cassette.
01 and oil 104 on the surface of this blood specimen.

An appropriate amount of oil for an oil immersion lens is dripped from the tube 105 to evenly spread the oil for an oil immersion lens on the surface of the blood specimen. As for the smearing, it goes without saying that a method of pushing out the oil through a slit-like hole or a method of smearing the oil with a side hand may be used. At this time, it is desirable to drop the oil as soon as possible before the xylene 103 mentioned above completely evaporates from the surface of the blood sample. After dropping, the blood specimen is reset into the cassette 101. Next, the cassette 101 is moved upward by one blood specimen, and the above operation is repeated. The lowest blood sample 1.02 of the cassette 101 contains xylene solution 1.
Since this will be the longest period of immersion in 03, it is desirable that this time not exceed 10 minutes.

By carrying out the above operations, the xylene penetrated into the wrinkled deformed parts of what were originally pseudoreticulocytes, the stains from the blood sample due to oil etc. were removed, and the oil for oil immersion lenses was dropped. At this time, this oil can sufficiently penetrate into the wrinkle-like deformed areas and eliminate most of the pseudoreticulocytes. If pseudo reticulocytes remain in the blood specimen, repeating the above procedure once more will completely remove the pseudo reticulocytes. In the second operation, the above-mentioned operation may be repeated while the oil for the oil immersion lens that was performed in the first time is still smeared.

Blood specimens smeared with oil for oil immersion lenses in this way can be examined under a microscope immediately, and since the oil itself does not evaporate, the wrinkle-like deformation will disappear even if left for a long time. Since it is maintained in a stable condition, you can take a microscopic examination after some time.

In the above explanation, we have explained the case where xylene is used as an organic solvent; .

An organic solvent can be used that has the same effects as xylene, such as not causing denaturation, dissolution, corrosion, etc., and (1) volatilization and being easily removable.

According to such a blood pretreatment method, even for blood specimens in which the frequency of pseudoreticulocytes is 100 or more per 1000 red blood cells, the frequency of pseudoreticulocytes can be reduced to 1 per 1000 red blood cells. It was confirmed that it is possible to significantly reduce the amount to less than

〔Effect of the invention〕

As described above, according to the present invention, it is possible to extremely reduce the incidence of pseudoreticulocytes in a blood specimen, thereby preventing misidentification of red blood cells and reticulocytes in a reticulocyte counting test.

[Brief explanation of the drawing]

FIG. 1 shows an example of a processing apparatus that implements the processing method of the present invention. 102...Blood specimen, 103...8 organic agents. 104... Oil agent for oil immersion lenses Patent attorney Katsuo Ogawa 29. −． . Ha

Claims (1)

[Scope of Claims] 1. A method in which a blood specimen is subjected to supravital staining and a microscopic image is obtained by an oil immersion method to identify red blood cells and reticulocytes, in which the stained blood specimen is immersed in an organic solvent for a predetermined period of time. , and then smearing oil for an oil immersion lens on the blood specimen. 2. The blood pretreatment method according to claim 1, characterized in that xylene is used as the organic solvent.