CN114252315A - Method for treating blood smear thick blood film for plasmodium microscopic examination - Google Patents
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- 239000008280 blood Substances 0.000 title claims abstract description 227
- 238000000034 method Methods 0.000 title claims abstract description 51
- 241000224016 Plasmodium Species 0.000 title claims abstract description 50
- 239000012528 membrane Substances 0.000 claims abstract description 50
- 238000001035 drying Methods 0.000 claims abstract description 36
- 239000011259 mixed solution Substances 0.000 claims abstract description 35
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims abstract description 25
- 210000003743 erythrocyte Anatomy 0.000 claims abstract description 24
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- 238000000386 microscopy Methods 0.000 claims abstract description 23
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- 238000010186 staining Methods 0.000 claims abstract description 17
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
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- 235000019441 ethanol Nutrition 0.000 claims description 10
- 239000012153 distilled water Substances 0.000 claims description 8
- 238000004043 dyeing Methods 0.000 claims description 7
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- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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Abstract
本发明属于疟原虫镜检的技术领域，特别涉及一种用于疟原虫镜检的血涂片厚血膜的处理方法，其具体步骤是：制备厚血膜和薄血膜；干燥；等待血膜干燥的同时配制25％乙醇(水)混合溶液；在载玻片上用上述混合溶液覆盖干燥的厚血膜；让25％乙醇(水)混合溶液作用厚血膜一定时间；干燥已完成厚血膜处理的血涂片；对干燥处理完成的血涂片进行染色处理；对染色后的血涂片进行镜检。该处理方法能完全有效破坏溶解厚血膜中的红细胞，同时也能够有效保留血膜中红细胞内的疟原虫物质、有核的白细胞和血小板等，并尽可能固定在原有厚血膜区域，保证后续的染色镜检和结果准确判断，为查找疟原虫提供了更加敏感和可靠的依据。
The invention belongs to the technical field of microscopic examination of malaria parasites, and particularly relates to a method for processing thick blood films of blood smears for microscopic examination of malaria parasites. The specific steps are: preparing thick blood films and thin blood films; drying; waiting for blood While the membrane is drying, prepare a 25% ethanol (water) mixed solution; cover the dried thick blood film with the above mixed solution on a glass slide; let the 25% ethanol (water) mixed solution act on the thick blood film for a certain period of time; drying has completed the thick blood film. Membrane-treated blood smears; stained blood smears after drying; microscopic examination of stained blood smears. The treatment method can completely and effectively destroy the red blood cells dissolved in the thick blood film, and at the same time, it can effectively retain the plasmodium, nucleated white blood cells and platelets in the red blood cells in the blood film, and fix them in the original thick blood film area as much as possible to ensure that Subsequent staining microscopy and accurate judgment of the results provide a more sensitive and reliable basis for finding malaria parasites.
Description
Technical Field
The invention belongs to the technical field of plasmodium microscopic examination, and particularly relates to a method for treating a blood smear thick blood film for plasmodium microscopic examination.
Background
Malaria is a parasitic disease caused by infection with human plasmodium and is distributed in a global epidemic, mainly concentrated in the tropics and subtropics. Malaria is the first of the world's lethal parasitic diseases, with approximately 100 to 300 ten thousand cases of death each year, most of which are children under the age of five. After the plasmodium invades the human body, the plasmodium firstly develops and breeds in liver cells and then invades into red blood cells to cause the rupture of the red blood cells, and the clinical manifestations of the plasmodium are that repeated intermittent chill, high fever and subsequent remission after profuse sweat appear.
Currently, malaria is diagnosed primarily by epidemiological history, typical clinical manifestations, and laboratory examinations in which blood smears looking for plasmodium have a definitive diagnostic significance. The blood smear for searching the plasmodium has the advantages of simplicity, convenience, easy popularization and the like, and is also a diagnosis method recommended by WHO and the Chinese State Weijian Commission. The blood smear for searching the plasmodium is to use a small amount of blood of a detected person to make a thick and thin blood film, and then to perform dyeing microscopy to search the plasmodium after proper treatment. However, because of the high concentration and overlapping of blood cells in the thick blood membrane, the plasmodium is parasitic in the erythrocyte, and the plasmodium in the erythrocyte can not be observed by direct staining microscopy. Therefore, it is necessary to destroy the erythrocytes in the thick blood membrane to expose the plasmodium morphologies by sufficient extravasation, and then to find the plasmodium morphologies by microscopic examination through appropriate staining treatment. In conclusion, the key to the diagnosis of plasmodium infection by blood smear is the pretreatment of thick blood membrane staining, which is an important prerequisite for ensuring sensitivity and accuracy.
The current technical standard for searching plasmodium by blood smear staining microscopy is WS259-2015 document issued by the national health and health Committee of China, wherein appendix B has two treatments on thick blood films of blood smears:
the first method is that distilled water is directly dripped after drying for about 1 minute to destroy red blood cells (which is equivalent to hypotonic to destroy hemolysis), and then Swiss staining is carried out;
the second is direct microscopic examination with giemsa staining without treatment after drying.
However, the two methods have unsatisfactory effects in clinical practical application: the first method of directly dripping distilled water can completely destroy red blood cells, but all other cells on a thick blood film, including white blood cells, platelets, plasmodium and the like, are basically destroyed by water washing and can not be remained on a glass slide, so that the thick blood film is useless, and the subsequent staining microscopic examination and accurate result are seriously influenced. The second type of staining with Giemsa does not treat the thick blood membranes, but the thin blood membranes must be fixed with methanol (CH3-OH) for 30 minutes before staining. Although the method does not need to treat the thick blood membrane, the staining solution needs to be diluted before use, has more steps and long staining time, and still has the conditions of different degrees of thick blood membrane shedding and incomplete red blood cell destruction in the staining process, thereby finally influencing the detection timeliness and results.
In addition, researchers in China directly dye the thick blood membrane by adopting different dilutions of the Reye's dye solution, but due to the content difference of individual hemoglobin and red blood cells, the dyeing microscopic effect is unstable, and phenomena of thick blood membrane falling or incomplete hemolysis in different degrees can also occur.
The reason that the existing thick blood membrane treatment method is not ideal is probably that a simple aqueous solution only has the function of destroying blood cells by hypotonic, and the leucocyte, the blood platelet, the plasmodium and the like which are originally fixed on a glass slide are dissolved and suspended in water and lost along with the water while the blood cells are destroyed, so that all cells in the thick blood membrane are lost and shed.
Therefore, the development of a method for treating a blood smear thick blood membrane for plasmodium microscopy, which can completely and effectively destroy and dissolve red blood cells in the thick blood membrane, can effectively retain plasmodium substances, nucleated white blood cells, blood platelets and the like in the red blood cells in the blood membrane, can fix the substances, the nucleated white blood cells, the blood platelets and the like in the original thick blood membrane area as much as possible, and can ensure the subsequent staining microscopy and accurate result judgment, is in the forefront.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and particularly discloses a method for treating a blood smear thick blood membrane for plasmodium microscopy, which can completely and effectively destroy and dissolve red blood cells in the thick blood membrane, can effectively retain plasmodium substances, nucleated white blood cells, blood platelets and the like in the red blood cells in the blood membrane, is fixed in the original thick blood membrane area as far as possible, ensures subsequent staining microscopy and accurate judgment of results, and provides a more sensitive and reliable basis for searching plasmodium.
In order to achieve the technical purpose, the invention is realized according to the following technical scheme:
the invention relates to a method for treating blood smear thick blood film for plasmodium microscopy, which comprises the following specific steps:
(1) preparing a thick blood film and a thin blood film;
(2) drying;
(3) preparing a 25% ethanol (water) mixed solution while waiting for the blood membrane to be dry;
(4) covering the dried thick blood film on a glass slide by using the mixed solution;
(5) allowing 25% ethanol (water) mixed solution to act on thick blood membrane for a certain time;
(6) drying the blood smear which is subjected to the thick blood film treatment;
(7) dyeing the dried blood smear;
(8) and performing microscopic examination on the stained blood smear.
As a further improvement of the above technology, the process for preparing thick blood film and thin blood film in the step (1) is as follows: the blood volume of the thick blood film is 4-5 mu L, and the thick blood film is dripped at one end of the clean glass slide to prepare a circular thick blood film with the diameter of about 8 mm; then, 2. mu.L of whole blood or peripheral blood is dropped on the other end of the clean glass slide, and the push sheet is prepared into a thin blood film.
As a further improvement of the above technique, the drying process in the above step (2) is: naturally drying the prepared thick blood film and thin blood film at room temperature or drying the prepared thin blood film and thin blood film in a drying oven at 37 ℃ until no moisture exists on the surface of the thick blood film visible to naked eyes.
As a further improvement of the above technique, the preparation of the 25% ethanol (water) mixed solution in the step (3) is: : including a mixed solution of 1 part by volume of absolute ethyl alcohol and 3 parts by volume of distilled or deionized water, or a mixed solution of 1 part by volume of 75% ethyl alcohol and 2 parts by volume of distilled or deionized water.
As a further improvement of the above technique, the thick blood film process of covering the dried thick blood film on the slide glass with the above mixed solution of step (4) is specifically: the thick blood film and the thin blood film on the glass slide are separated by drawing lines with a crayon or a mark pen, the glass slide is horizontally placed, and the prepared 25 percent ethanol (water) mixed solution of 150 and 200 mu L is dripped on the dried thick blood film, wherein the dripping solution is in an amount which can completely cover the thick blood film and does not overflow and diffuse.
As a further improvement of the above technique, the step (5) of allowing the mixed solution of 25% ethanol (water) to act on the thick blood membrane for a certain time specifically comprises: according to the concentration of red blood cells or the depth of hemoglobin, a 25% ethanol (water) mixed solution is acted on a thick blood film for 2-3 minutes, and then a glass slide is erected to remove the solution acted on the thick blood film at one corner close to the thick blood film end.
As a further improvement of the above technique, the drying of the above step (6) has completed the thick blood film treatment of the blood smear: naturally drying the blood smear or drying the thick blood film in a drying oven at 37 ℃, and visually observing that the thick blood film area presents a gray white or gray yellow semitransparent state as a mark for successful treatment.
As a further improvement of the above technique, the step (7) is a method of staining a dried blood smear: the dried blood smears were stained by the Switzerland staining method or the Giemsa staining method according to the protocol of the staining solution.
As a further improvement of the above technology, the stained blood smear is subjected to a microscopic examination in the step (8) above, and the criteria for good thick blood film treatment are as follows: under oil microscope (1000 ×), 5-10 leukocytes can be seen in each visual field in the thick blood membrane area, and the morphology and search of plasmodium are referred to WS259-2015 file during microscopic examination.
Compared with the prior art, the invention has the beneficial effects that:
the method for treating the blood smear thick blood membrane for the plasmodium microscopic examination utilizes 25% ethanol (water) mixed solution to treat the thick blood membrane in the blood smear, can completely destroy the hypotonic hemolytic effect of red blood cells, expose plasmodium substances parasitized in the red blood cells, simultaneously has better fixing effect on other nucleated cells including white blood cells and blood platelets and exposed plasmodium substances (containing nucleic acid and protein) in each stage, can fix and adsorb the tangible substances to an original area of a carrier rupture disc under the action of the low-concentration ethanol, cannot be lost along with upper-layer solution, ensures the subsequent dyeing and microscopic examination results, and provides more sensitive, accurate and reliable basis for searching plasmodium.
Drawings
The invention is described in detail below with reference to the following figures and specific embodiments:
FIG. 1 is a schematic representation of thick blood membranes (circular area) and thin blood membranes for Plasmodium microscopy made according to conventional standards;
FIG. 2 is a schematic diagram of a proper amount of 25% ethanol (water) solution dropped on a dried thick blood membrane in the present invention;
FIG. 3 is a schematic diagram of a 25% ethanol (water) solution treated zone of Reye stained thick blood membrane in accordance with the present invention; (see the translucent cell region of grayish white or grayish yellow)
FIG. 4 is a schematic representation of the area of a treated blood smear of the present invention where the blood smear is stained by Giemsa staining;
FIG. 5 is a schematic diagram of the Swiss staining microscopic result of the thick blood membrane region treated by the method of the present invention (the background is clear, and the aggregated mauve white blood cells can be seen);
FIG. 6 is a schematic diagram of a Giemsa stained microscope (1000X) of the thick blood membrane region after treatment according to the method of the present invention. (purple blue leukocytes, with aggregates visible, and platelets with small blue dots).
Detailed Description
The invention relates to a method for treating blood smear thick blood film for plasmodium microscopy, which comprises the following specific steps:
(1) preparing a thick blood film and a thin blood film;
(2) drying;
(3) preparing a 25% ethanol (water) mixed solution while waiting for the blood membrane to be dry;
(4) covering the dried thick blood film on a glass slide by using the mixed solution;
(5) allowing 25% ethanol (water) mixed solution to act on thick blood membrane for a certain time;
(6) drying the blood smear which is subjected to the thick blood film treatment;
(7) dyeing the dried blood smear;
(8) and performing microscopic examination on the stained blood smear. The signs of good thick blood film treatment are: under oil microscope (1000 ×), more leukocytes (about 5-10) were visible per field in the thick blood membrane area, and the morphology and search of plasmodium on microscopy were referenced to WS259-2015 file.
Wherein the process for preparing the thick blood film and the thin blood film in the step (1) comprises the following steps: the blood volume of the thick blood film is 4-5 mu L, and the thick blood film is dripped at one end of the clean glass slide to prepare a circular thick blood film with the diameter of about 8 mm; then, 2. mu.L of whole blood or peripheral blood is dropped on the other end of the clean glass slide, and the push sheet is prepared into a thin blood film.
The drying process in the step (2) is as follows: naturally drying the prepared thick blood film and thin blood film at room temperature or drying the prepared thin blood film and thin blood film in a drying oven at 37 ℃ until no moisture exists on the surface of the thick blood film visible to naked eyes.
Preparing a 25% ethanol (water) mixed solution in the step (3): including a mixed solution of 1 part by volume of absolute ethyl alcohol and 3 parts by volume of distilled or deionized water, or a mixed solution of 1 part by volume of 75% ethyl alcohol and 2 parts by volume of distilled or deionized water.
The process of covering the dried thick blood film on the glass slide by the mixed solution in the step (4) is specifically as follows: the thick blood film and the thin blood film on the glass slide are separated by drawing lines with a crayon or a mark pen, the glass slide is horizontally placed, and the prepared 25 percent ethanol (water) mixed solution of 150 and 200 mu L is dripped on the dried thick blood film, wherein the dripping solution is in an amount which can completely cover the thick blood film and does not overflow and diffuse.
The step (5) of allowing the 25% ethanol (water) mixed solution to act on the thick blood membrane for a certain time specifically comprises the following steps: according to the concentration of red blood cells or the depth of hemoglobin, a 25% ethanol (water) mixed solution is acted on a thick blood film for 2-3 minutes, and then a glass slide is erected to remove the solution acted on the thick blood film at one corner close to the thick blood film end.
Drying the blood smear which is subjected to thick blood film treatment in the step (6): naturally drying the blood smear or drying the thick blood film in a drying oven at 37 ℃, and visually observing that the thick blood film area presents a gray white or gray yellow semitransparent state as a mark for successful treatment.
The method for staining the dried blood smear in the step (7) comprises the following steps: the dried blood smears were stained by the Switzerland staining method or the Giemsa staining method according to the protocol of the staining solution.
Performing microscopic examination on the stained blood smear in the step (8), wherein the standard for good thick blood film treatment is as follows: under oil microscope (1000 ×), 5-10 leukocytes can be seen in each visual field in the thick blood membrane area, and the morphology and search of plasmodium are referred to WS259-2015 file during microscopic examination.
As shown in fig. 1, fig. 1 is a schematic diagram of a hemogram and a blood film for plasmodium microscopy, which are manufactured according to the conventional standard, wherein the hemogram is a darker circular area on the left side of each group, and the blood film is a lighter area on the right side of each group.
As shown in fig. 2-6, 10 cases of preliminary experiments were performed according to the method provided by the present invention, all of which achieve the expected effect, and the method has been already popularized and tried in the clinical laboratory of the sixth people hospital in the southern sand area of guangzhou city, and the operator has good feedback.
Compared with the method for treating the blood smear thick blood membrane for the plasmodium microscopic examination, which directly treats the thick blood membrane with water, directly dyes the thick blood membrane with giemsa and treats the thick blood membrane with other ethanol solutions with different concentrations, the method obviously improves the comprehensive performance on two key indexes of hemolysis effect and thick blood membrane flaking rate, and the method is shown in the following table.
| Method | Number of examples | Hemolytic effect | Rate of exfoliation of thick blood film | Comprehensive evaluation |
| The method of the |
10 | Complete hemolysis | 0(0/10) | Is suitable for |
| Direct water treatment | 5 | Complete hemolysis | 80％(4/5) | Not applicable to |
| Direct Giemsa staining | 5 | Complete hemolysis | 60％(3/5) | Not applicable to |
| 10% ethanol solution | 5 | Complete hemolysis | 60％(3/5) | Not applicable to |
| 50% ethanol solution | 5 | Small part of hemolysis | 0(0/5) | Not applicable to |
| 75% ethanol solution | 5 | Non-hemolytic disease | 0(0/5) | Not applicable to |
Watch 1
The processing method of the blood smear thick blood membrane for the plasmodium microscopic examination provided by the invention treats the thick blood membrane through the low-concentration ethanol solution, namely treats the thick blood membrane through the 25% ethanol (water) solution, thereby ensuring that the hypotonic hemolysis (the erythrocyte is swollen and broken in the hypotonic environment) of the solution enables plasmodium substances in the erythrocyte to overflow and expose, simultaneously, the ethanol in the solution as a polar molecule has the functions of fixing cells and proteins, and can quickly fix nucleated cells such as leucocytes and blood platelets and plasmodium substances on a carrier fragment without falling off along with the water solution. Therefore, the effects of dissolving red blood cells and fixing other visible cells are achieved, the cell forms except the red blood cells are retained to the maximum extent, and the subsequent dyeing microscopic examination effect is ensured.
The present invention is not limited to the above-described embodiments, and various changes and modifications of the present invention are also intended to be included within the scope of the claims and equivalent technologies of the present invention, unless they depart from the spirit and scope of the present invention.
Claims (10)
1. A method for processing blood smear thick blood film for plasmodium microscopy comprises the following specific steps:
(1) preparing a thick blood film and a thin blood film;
(2) drying;
(3) preparing a 25% ethanol (water) mixed solution while waiting for the blood membrane to be dry;
(4) covering the dried thick blood film on a glass slide by using the mixed solution;
(5) allowing 25% ethanol (water) mixed solution to act on thick blood membrane for a certain time;
(6) drying the blood smear which is subjected to the thick blood film treatment;
(7) dyeing the dried blood smear;
(8) and performing microscopic examination on the stained blood smear.
2. The method for processing blood smear thick blood film for plasmodium microscopy according to claim 1, wherein: the process for preparing the thick blood film and the thin blood film in the step (1) comprises the following steps: the blood volume of the thick blood film is 4-5 mu L, and the thick blood film is dripped at one end of the clean glass slide to prepare a circular thick blood film with the diameter of about 8 mm; then, 2. mu.L of whole blood or peripheral blood is dropped on the other end of the clean glass slide, and the push sheet is prepared into a thin blood film.
3. The method for processing blood smear thick blood film for plasmodium microscopy according to claim 1, wherein: the drying process in the step (2) is as follows: naturally drying the prepared thick blood film and thin blood film at room temperature or drying the prepared thin blood film and thin blood film in a drying oven at 37 ℃ until no moisture exists on the surface of the thick blood film visible to naked eyes.
4. The method for processing blood smear thick blood film for plasmodium microscopy according to claim 1, wherein: the preparation of the 25% ethanol (water) mixed solution in the step (3) is as follows: comprising a mixed solution of 1 part by volume of absolute ethyl alcohol and 3 parts by volume of distilled or deionized water.
5. The method for processing blood smear thick blood film for plasmodium microscopy according to claim 1, wherein: the preparation of the 25% ethanol (water) mixed solution in the step (3) is as follows: a mixed solution of 1 part by volume of 75% ethanol and 2 parts by volume of distilled or deionized water.
6. The method for processing blood smear thick blood film for plasmodium microscopy according to claim 1, wherein: the process of covering the dried thick blood film on the glass slide by the mixed solution in the step (4) is specifically as follows: the thick blood film and the thin blood film on the glass slide are separated by drawing lines with a crayon or a mark pen, the glass slide is horizontally placed, and the prepared 25 percent ethanol (water) mixed solution of 150 and 200 mu L is dripped on the dried thick blood film, wherein the dripping solution is in an amount which can completely cover the thick blood film and does not overflow and diffuse.
7. The method for processing blood smear thick blood film for plasmodium microscopy according to claim 1, wherein: the step (5) of allowing the 25% ethanol (water) mixed solution to act on the thick blood membrane for a certain time specifically comprises the following steps: according to the concentration of red blood cells or the depth of hemoglobin, a 25% ethanol (water) mixed solution is acted on a thick blood film for 2-3 minutes, and then a glass slide is erected to remove the solution acted on the thick blood film at one corner close to the thick blood film end.
8. The method for processing blood smear thick blood film for plasmodium microscopy according to claim 1, wherein: drying the blood smear which is subjected to thick blood film treatment in the step (6): naturally drying the blood smear or drying the thick blood film in a drying oven at 37 ℃, and visually observing that the thick blood film area presents a gray white or gray yellow semitransparent state as a mark for successful treatment.
9. The method for processing blood smear thick blood film for plasmodium microscopy according to claim 1, wherein: the method for staining the dried blood smear in the step (7) comprises the following steps: the dried blood smears were stained by the Switzerland staining method or the Giemsa staining method according to the protocol of the staining solution.
10. The method for processing blood smear thick blood film for plasmodium microscopy according to claim 1, wherein: performing microscopic examination on the stained blood smear in the step (8), wherein the standard for good thick blood film treatment is as follows: under oil lens (1000 ×), 5-10 leukocytes per field of view were visible in the thick blood membrane area.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024138651A1 (en) * | 2022-12-30 | 2024-07-04 | 深圳迈瑞生物医疗电子股份有限公司 | Sample treatment device |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1985005446A1 (en) * | 1984-05-15 | 1985-12-05 | Merck Patent Gmbh | Single dye replacement for romanowsky plural dye variable compositions in human biopsy specimen constituent identifications |
| US20070059763A1 (en) * | 2004-08-03 | 2007-03-15 | Kazunori Okano | Cellomics system |
-
2021
- 2021-12-20 CN CN202111561994.0A patent/CN114252315A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1985005446A1 (en) * | 1984-05-15 | 1985-12-05 | Merck Patent Gmbh | Single dye replacement for romanowsky plural dye variable compositions in human biopsy specimen constituent identifications |
| US20070059763A1 (en) * | 2004-08-03 | 2007-03-15 | Kazunori Okano | Cellomics system |
Non-Patent Citations (1)
| Title |
|---|
| 何群力 等: "医学微生物学与人体寄生虫学实验技术", 第四军医大学出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024138651A1 (en) * | 2022-12-30 | 2024-07-04 | 深圳迈瑞生物医疗电子股份有限公司 | Sample treatment device |
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