JP3899845B2 - Method and apparatus for decolorizing stained specimen - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a decolorization method for decolorizing a biological tissue-stained specimen such as a blood smear to re-stain.
[0002]
[Prior art]
A representative staining of blood smears is Romanowsky staining. Wright staining, Giemsa staining, and Pappenheim double staining (May-Grunwald-Giemsa staining), which are widely used at present, are improved methods of Romanovsky staining.
[0003]
Examples of the dye used in the Romanovsky staining method or its improved method include methylene blue (basic dye), eosin (acidic dye), methylene blue eosinate (neutral dye), and oxides of methylene blue such as Azure B ( Generated in an alkaline methylene blue solution).
[0004]
Methylene blue binds to acidic substances in cells, for example, RNA in cells and dyes blue.
[0005]
Eodine binds to basic substances such as erythrocyte hemoglobin and eosinophil eosinophilic granules (eosinophil granules) and dyes red.
[0006]
Methylene blue eosin dissociates into methylene blue and eosin in an aqueous solution, functions as a pigment, and dyes special granules such as neutral granules in reddish brown or purple.
[0007]
Methylene blue oxide such as Azure B binds to nuclear DNA and cytosolic Azure granules and dyes purple-red (maroon).
[0008]
A blood smear that has undergone such staining cannot be re-stained if the staining fails, and another sample is stained and observed.
[0009]
[Problems to be solved by the invention]
The color of dyeing varies depending on the type of dyeing solution, the lot number, the temperature and humidity at the time of dyeing, and the desired dyeing result may not be obtained. In addition, a desired staining result cannot be obtained due to human error. In such a case, cell observation becomes difficult or impossible.
[0010]
However, once smeared smears could not be re-stained, if they were not stained properly, accurate observation would not be possible, and the opportunity for observation would be lost if they were valuable specimens that were difficult to collect. It was.
[0011]
The present invention has been made in view of such a conventional technique, and an object of the present invention is to decolorize a stained smear so that it can be stained again.
[0012]
[Means for Solving the Problems]
The present invention is a Romanowsky staining or bleaching method of the specimen stained with the improved method, including a first step of immersing the stained sample in methanol. As a result, a portion stained with a pigment that dissolves well in alcohol, such as Azure B or methylene blue, is decolorized.
[0013]
Furthermore, the method of the present invention, the above after the first step, including a second step of immersing the specimen in a mixture of phosphate buffer and methanol. As a result, a portion stained with a water-soluble pigment such as eosin is decolorized. Furthermore, the method of the present invention includes a step of immersing the specimen in methanol after the second step. Thereby, re-staining becomes easy.
[0014]
In the method of the present invention, the specimen is a biological tissue-stained specimen, and any one of the stained biological cell nucleus, blood cell nucleus, leukocyte nucleus DNA, platelet granule, or RNA has been decolored by the process of the first step. Or a step of confirming that the specimen portion stained with Azure B or methylene blue has been decolorized in the first step . Thereby, the process after the said 1st step can be performed reliably.
[0015]
In the method of the present invention, the specimen is a biological tissue-stained specimen, and the step of confirming that the eosinophil granule has been decolored by the processing of the second step, or the specimen portion stained with eosin Is confirmed to be decolorized by the process of the second step . Thereby, the process after the said 2nd step can be performed reliably.
[0016]
Further, in the method of the present invention, when the ratio of the phosphate buffer to methanol is in the range of 1: 3 to 1: 4, a suitable processing result can be obtained.
[0019]
The device of the present invention is a device for decolorizing a specimen stained by the Romanovsky staining method or an improved method thereof, and includes a first tank for storing methanol, and a first tank for storing a mixed solution of phosphate buffer and methanol. 2 tanks, a third tank containing methanol, and the specimen immersed in the first tank, then removed from the first tank and immersed in the second tank, the second tank and Eject from the bath, after immersion in the third tank, and the specimen conveyance means for retrieving from said third tank, the sample transport means and a control means for controlling the time for immersing the specimen in each tank .
According to this configuration, by immersing the specimen in the first tank, a portion stained with a pigment that dissolves well in alcohol such as Azure B or methylene blue is decolorized, and by immersing the specimen in the second tank The portion stained with a water-soluble pigment such as eosin is decolorized.
In addition, the mixture of phosphate buffer and methanol had a phosphate buffer / methanol ratio in the range of 1: 3 to 1: 4. If it does in this way, after decoloring, it can dye | stain clearly and can suppress the morphological change of a blood cell.
[0020]
DETAILED DESCRIPTION OF THE INVENTION
An embodiment of the decoloring method of the present invention will be described. The blood smear stained by the Romanovsky staining method or its improved method is immersed in a tank containing methanol and vibrated up and down. If the methanol solution starts to change color, replace it with a new solution, repeat the same process of immersing the blood smear in this solution and vibrating it up and down. Here, for example, the methanol tank exchanges methanol every 5 minutes and is immersed in the methanol tank for a total of 20 minutes. The total immersion time depends on the elapsed time since the smear was stained, but if it is just after staining, it takes about 10 to 15 minutes as a whole, and about 30 minutes if one month has passed. By this treatment, for example, a specimen portion stained with a dye having a property that dissolves well in alcohol, such as Azure B or methylene blue, is decolorized. The methanol is preferably special grade methanol having a purity of 99.9%.
[0021]
When methanol is no longer colored, check for decolorization under a microscope. What is confirmed here is decolorization of blood cell nucleus, leukocyte nuclear DNA, platelet granules or RNA. For example, it is confirmed that the blood smear stained as shown in FIG. 1 becomes as shown in FIG. 2 by this processing. Here, most of the spheres shown in FIG. 1 are red blood cells. Red blood cells are entirely red in hemoglobin. A larger sphere around the center of FIG. 1 is an eosinophil (indicated by symbol A). The eosinophils are orange in eosinophil granules as a whole, and there are purple-red nuclei (indicated by symbol a) in them. Taking eosinophils as an example, as shown in FIG. 2, only the nuclei are decolored by this methanol decoloration treatment. FIG. 3 shows a specimen containing lymphocytes after methanol decolorization treatment. Lymphocytes are present around the center of FIG. 3, but the lymphocyte nucleus and RNA-containing cytoplasm are decolored, and the entire lymphocyte is invisible. Again, the spheres that remain unbleached are red blood cells.
[0022]
Next, the blood smear is put into a tank containing a mixed solution prepared by mixing phosphate buffer and methanol at a ratio of 1: 3 to 1: 4. The reason why the mixed solution is set to this ratio is that the mixture can be clearly stained and the morphological change of blood cells is suppressed. In this tank, the blood smear is vibrated up and down. Here, for example, it is immersed in the mixed solution tank for 5 minutes. If this immersion time is also adjusted according to the elapsed time after the smear is stained, decolorization can be performed efficiently. By this immersion treatment, for example, a specimen portion stained with a water-soluble pigment such as eosin is decolorized. After this treatment, decolorization is confirmed under a microscope. What is confirmed here is decolorization of erythrocyte hemoglobin, eosinophil granules and the like. In the example of FIG. 2 above, it is confirmed that the whole red blood cells and the whole eosinophils are decolorized, and all blood components are decolorized. Further, in the example of FIG. 3 above, it is confirmed that the remaining red blood cells are decolorized and all blood components are decolorized.
[0023]
Next, the smear is immersed in methanol for about 1 minute. By this process, the smear is fixed. The methanol is preferably a special grade methanol having a purity of 99.9% similar to that used for decolorization.
[0024]
Next, staining is again performed on the decolored smear. Since this dyeing may be a conventional dyeing that is usually performed, that is, dyeing by the Romanovsky dyeing method or its improved method, description thereof is omitted.
[0025]
Next, the decoloring apparatus according to the present invention will be described. FIG. 4 shows the overall configuration. As shown in this figure, a plurality of blood smears 2 are stored in the sample storage unit 1. The blood smear 2 is obtained by staining the blood smeared on the slide glass with the Romanovsky staining method or its improved method.
[0026]
A methanol tank 3 is provided adjacent to the specimen storage unit 1. The methanol tank 3 consists of four tanks, and methanol is accommodated in each of the tanks 3a to 3d. Here, methanol is the same as methanol used in the description of the above method.
[0027]
A liquid mixture tank 4 is provided adjacent to the methanol tank 3. The mixed solution tank 4 is a tank that contains a mixed solution of a phosphate buffer and a methanol solution. This mixed liquid is the same as the mixed liquid used in the description of the above method.
[0028]
A methanol tank 5 is provided adjacent to the mixed liquid tank 4. The methanol stored in the methanol tank 5 is the same as that stored in the methanol tank 3.
[0029]
Furthermore, this apparatus includes a transport unit 6 including an arm 6a that is movably held in the upper part of the sample storage unit 1, the methanol tank 3, the mixed liquid tank 4, and the methanol tank 5. The transport unit 6 is controlled by the control unit 7, and by moving the arm 6a horizontally and vertically, the blood smear sample 2 is taken out from the sample storage unit 1, and thereafter the methanol tanks 3a to 3d, the mixed liquid tank 4, Immersion and removal are performed in the order of the methanol tank 5.
[0030]
In this apparatus, the methanol tank 3 is the first tank, the mixed liquid tank 4 is the second tank, the transport unit 6 is the first sample transport unit, and the control unit 7 is the first control unit. is there.
[0031]
The operation of the apparatus thus configured will be described. The transport unit 6 controlled by the control unit 7 moves the arm 6a to first take out a plurality of blood smears 2 from the specimen storage unit 1 and sequentially immerse and take them out from the methanol tanks 3a to 3d. . Here, the time immersed in the methanol tanks 3a to 3d is 5 minutes each, and is 20 minutes as a whole. By this treatment, the specimen portion stained with Azure B or methylene blue is decolorized. That is, blood cell nucleus, leukocyte nuclear DNA, platelet granule or RNA is decolorized.
[0032]
Next, the transport unit 6 moves the arm 6 a to take out the blood smear 2 by immersing it in the mixed solution tank 4 for 5 minutes. By this treatment, the portion stained with eosin or the like is decolored. That is, hemoglobin of erythrocytes, eosinophil granules and the like are decolorized.
[0033]
Next, the transport unit 6 moves the arm 6a to take out the blood smear 2 by immersing it in the methanol tank 5 for a predetermined time (for example, about 1 minute). This process fixes the blood components of the blood smear 2 and prepares for subsequent staining.
[0034]
In addition, in said operation | movement, if the conveyance part 6 is equipped with the function to vibrate the arm 6a up and down while the blood smear sample 2 is immersed in each tank, decoloring will be performed more effectively.
[0035]
Further, when the next blood smear 2 is continuously decolored, it is necessary to replace the previously used methanol or mixed solution in each tank with a new one before the decoloring process. For this replacement, a discharge means for discharging the liquid to each tank and a supply means for supplying the corresponding liquid to each tank are provided, and each tank is discharged and supplied every time the entire decolorization process is completed. If the control is performed, the liquid can be automatically replaced.
[0036]
When such a discharge means, a supply means, and a control means for controlling these are provided, only one methanol tank for the methanol decoloring process is required.
[0037]
An example of such an apparatus is shown in FIG. In this figure, a plurality of blood smears 2 are stored in the sample storage part 11. A methanol tank 13 is provided adjacent to the specimen storage unit 11.
[0038]
A liquid mixture tank 14 is provided adjacent to the methanol tank 13. A methanol tank 15 is provided adjacent to the mixed liquid tank 14.
[0039]
The apparatus further includes a transport unit 20 including an arm 20a that is movably held in the upper part of the specimen storage unit 11, the methanol tank 13, the mixed solution tank 14, and the methanol tank 15. The transport unit 20 is controlled by the control unit 21 and moves the arm 20a horizontally and vertically to take out the blood smear sample 2 from the sample storage unit 11. Thereafter, the methanol tank 13, the mixed liquid tank 14, and the methanol tank 15 are removed. In this order, storage and removal are performed.
[0040]
The apparatus further includes a methanol supply means 16 for supplying methanol to the methanol tank 13, a mixed liquid supply means 17 for supplying the mixed liquid to the mixed liquid tank 14, and a methanol supply means 18 for supplying methanol to the methanol tank 15. , A discharge means 19 for selectively discharging the liquid stored in each tank is provided. In addition to controlling the transport unit 20, the control unit 21 controls the methanol supply unit 16, the mixed solution supply unit 17, the methanol supply unit 18, and the discharge unit 19.
[0041]
In this apparatus, the methanol tank 13 is a first tank, the liquid mixture tank 14 is a second tank, the transport unit 20 is a second sample transport unit, and the control unit 21 is a second control unit. is there.
[0042]
Next, the operation of the apparatus configured as described above will be described. First, the control unit 21 controls the methanol supply unit 16, the mixed solution supply unit 17, and the methanol supply unit 18, so that methanol is stored in the methanol tank 13, the mixed solution is stored in the mixed solution tank 14, and methanol is stored in the methanol tank 15. Supply. Here, methanol and the mixed solution are the same as those used in the apparatus shown in FIG.
[0043]
Next, the transport unit 20 moves the arm 20 a to take out the blood smear 2 from the sample storage unit 11 and immerse it in the methanol tank 13 for a predetermined time. The control unit 21 controls the discharge unit 19 to discharge the used methanol stored in the methanol tank 13, and then controls the methanol supply unit 16 to supply new methanol to the methanol tank 13. For this reason, the blood smear 2 is immersed again in methanol. The control unit 21 performs this immersion for a predetermined time (for example, 5 minutes), then controls the discharge means 19 to discharge the used methanol from the methanol tank 13, and then controls the methanol supply means 16 to Fresh methanol is supplied to the methanol tank 13. As described above, the control unit 21 immerses the blood smear sample 2 in the methanol tank 13 and discharges the used methanol a predetermined number of times (for example, four times). By this treatment, the specimen portion stained with Azure B or methylene blue is decolorized.
[0044]
Next, the control unit 21 moves the arm 20a of the transport unit 20 to take out the blood smear 2 from the methanol tank 13, soak the blood smear 2 in the mixed liquid tank 14 for a predetermined time (for example, 5 minutes), and then Take out. By this treatment, the specimen portion stained with eosin or the like is decolored.
[0045]
Next, the control part 21 moves the arm 20a of the conveyance part 20, and immerses the blood smear sample 2 in the methanol tank 15 for a predetermined time (for example, about 1 minute) and takes it out. This process fixes the blood components of the blood smear 2 and prepares for subsequent staining. Then, the control unit 21 moves the arm 20a of the transport unit 20 to store the decolored blood smear sample 2 in a decolored sample storage unit (not shown).
[0046]
Next, the control unit 21 controls the discharge unit 19 to discharge the used liquid stored in each tank, and moves the arm 20a of the transport unit 20 to the upper part of the sample storage unit 11 to stop it. . Then, the control unit 21 returns to the initial process of supplying the liquid corresponding to each tank, and causes each unit to repeat the above operation. This operation is performed until the blood smear 2 disappears in the specimen container 11.
[0047]
In the above operation, if the transport unit 20 has a function of vibrating the arm 20a up and down while the blood smear 2 is immersed in each tank, decolorization is performed more effectively.
[0048]
As described above, if one methanol tank is used for the methanol decoloring process, the apparatus can be miniaturized.
[0049]
Note that the above is an example of an apparatus used when performing all of the methanol decoloring process, the mixed liquid decoloring process, and the fixing process for fixing the decolored specimen.
[0050]
On the other hand, when only the methanol decoloring process is sufficient, as the apparatus, for example, in the apparatus of FIG. 4, a process after performing the methanol decoloring process using the methanol tank 3 under the control of the control unit 7, That is, a configuration in which the mixed liquid decoloring process and the methanol fixing process are omitted may be employed. For example, in the apparatus of FIG. 5, the control unit 21 may be configured to omit the process after the methanol decoloring process using the methanol tank 13, that is, the mixed liquid decoloring process and the methanol fixing process. .
[0051]
In addition, when only such methanol decoloring process is required, a dedicated apparatus may be used. As the dedicated apparatus, for example, in the apparatus of FIG. 4, the liquid mixture tank 4 and the methanol tank 5 are not provided, but the transport unit 6 transports the blood smear 2 from the specimen storage unit 1 to the methanol tank 3. It may be performed until the immersion treatment.
[0052]
In the apparatus shown in FIGS. 4 and 5, a detection means for detecting the staining concentration of the blood smear is provided, and the immersion time in the decoloring process (methanol decoloring process and / or mixed liquid decoloring process) is determined depending on the concentration. decide. That is, a long time is determined when it is dark, and a short time is determined when it is light. Then, the blood smear may be immersed for the determined time.
[0053]
In the above, although the sample was a blood smear sample, it may be a smear sample stained by the Romanovsky staining method or its improved method, and is not limited to a blood sample. For example, a specimen including a general living cell nucleus may be used. Living cell nuclei are decolorized by methanol decolorization.
[0054]
【The invention's effect】
According to the present invention, it is possible to re-stain a smear that has failed to be stained. For this reason, even if it is a valuable specimen that is difficult to collect, there is an effect that a single observation opportunity can be obtained many times. In some examinations, a plurality of smears may be dyed and observed by different staining methods. In such a case, according to the present invention, since one smear can be used repeatedly, the amount of specimen collected from the patient can be reduced.
[Brief description of the drawings]
FIG. 1 shows an example of a stained blood smear.
FIG. 2 is a diagram showing that eosinophil nuclei have been decolorized by the methanol decolorization treatment of the method of the present invention.
FIG. 3 is a view showing that the entire lymphocyte has been decolored by the methanol decolorization treatment of the method of the present invention.
FIG. 4 is a configuration diagram showing an example of the device of the present invention.
FIG. 5 is a configuration diagram showing another example of the device of the present invention.
[Explanation of symbols]
1, 11 Specimen storage unit 3, 5, 13, 15 Methanol tank 4, 414 Mixed solution tank 6, 20 Transport unit 7, 21 Control unit

Claims (8)

A method for decolorizing a specimen stained by the Romanovsky staining method or its improved method,
A first step of immersing the stained specimen in methanol ;
After this first step, a second step of immersing the specimen in a mixture of phosphate buffer and methanol;
A method for decoloring a stained specimen, comprising a third step of immersing the specimen in methanol after the second step . The method for decoloring a stained specimen according to claim 1, wherein the mixture of the phosphate buffer and methanol has a ratio of phosphate buffer to methanol in the range of 1: 3 to 1: 4. A method for decoloring a stained specimen according to claim 1 or 2,
The specimen is a biological tissue stained specimen,
Decolorization of a stained specimen, comprising the step of confirming that any one of stained biological cell nucleus, blood cell nucleus, leukocyte nucleus DNA, platelet granule, or RNA is decolored by the process of the first step Method. A method for decoloring a stained specimen according to claim 1 or 2,
A method for decoloring a stained specimen, comprising the step of confirming that a specimen portion stained with Azure B or methylene blue has been decolorized in the first step. A method for decoloring a stained specimen according to claim 1 or 2 ,
The specimen is a biological tissue stained specimen,
A method for decoloring a stained specimen, comprising the step of confirming that the eosinophil granule has been decolored by the process of the second step. A method for decoloring a stained specimen according to claim 1 or 2 ,
A method for decoloring a stained specimen, comprising the step of confirming that a specimen portion stained with eosin has been decolored by the process of the second step. A device for decolorizing a specimen stained by the Romanovsky staining method or its improved method,
A first tank containing methanol;
A second tank containing a mixture of phosphate buffer and methanol;
A third tank containing methanol;
After immersing the specimen in the first tank, taken out from the first tank, after immersion in the second bath, and Eject from said second tank, after immersion in the third tank , Sample transport means for taking out from the third tank,
A depigmenting device for stained specimens, characterized in that the specimen transport means has control means for controlling the time for immersing the specimen in each tank . 8. The staining specimen decoloring apparatus according to claim 7, wherein the mixture of the phosphate buffer and methanol has a ratio of phosphate buffer to methanol in the range of 1: 3 to 1: 4.

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