Novel super-living body S staining solution

Abstract

The invention discloses a novel super-living body S staining solution, which comprises a dye, water, an amino acid type surfactant, a buffer agent, a preservative, a stabilizer and an auxiliary agent. The preparation method comprises the following steps: S1, respectively preparing the cell nucleus staining component, the cytoplasm staining component and the fat staining component into aqueous solutions, and then uniformly mixing the aqueous solutions with the buffer agent; s2, adding an auxiliary agent, a preservative, a stabilizer and an amino acid type surfactant into the mixed solution obtained in the S1, defoaming to obtain the novel super-living body S staining solution, and sealing and storing at low temperature. By adopting the staining solution prepared by the method disclosed by the invention, the tangible substances in the urine stock solution can be dispersed more uniformly, the edge aggregation effect is effectively inhibited, the color contrast is more distinct and clear, the observation is convenient, and the sensitivity of microscopic examination of the urine stock solution is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of urine test reagents, and in particular relates to a novel super living S staining solution. Background Art

[0002] Urine fluid examination is to observe the formed elements in urine under a microscope, including red blood cells, white blood cells, epithelial cells, casts, crystals, parasites, etc. The quantity and morphological changes of these components are of great significance for the diagnosis of diseases, especially pediatric tubulointerstitial nephritis, renal amino aciduria, male urethral cancer, pregnancy complicated with acute pyelonephritis, pregnancy complicated with acute cystitis, renal artery occlusion, urogenital trichomoniasis, Henoch-Schonlein purpura, urinary tract infection in the elderly, etc.

[0003] The general steps for urine stock microscopy are: centrifuge the urine sample in a centrifuge using a sharp-tipped (spitz-type) centrifuge tube, remove the supernatant using an aspirator or pipette, and leave 0.2 mL of sediment. If the sediment volume exceeds 0.2 mL, the formed components of the urine will be diluted. Then use a pipette or other device to mix the precipitate thoroughly to achieve homogeneity while avoiding destroying the formed components of the urine. Take 15 μL of urine sediment and drop it on a slide for microscopic observation.

[0004] Urine stock examination can be further divided into unstained examination and stained examination. In principle, urine stock examination is performed without staining; however, staining can be used to confirm and identify urine sediment when necessary. Super Vital S staining solution is a chemical reagent used to stain various formed components in urine stock, aiming to improve microscopic identification and diagnostic accuracy. The specific ingredients of Super Vital S staining solution may vary by brand and manufacturer, but common ingredients include crystal violet, safranin (safranin O), methylene blue, eosin, etc. For example, Sternheimer-Malbin (SM) staining solution is composed of crystal violet and safranin, while some other staining solutions may contain methylene blue, eosin, etc. When the fat components in the urine stock solution include fat globules, fat casts, oval fat bodies, macrophages, fat granular cells, and fat casts, Sudan tri-staining is required for confirmation. The commonly used staining techniques at present are Stemheimer-Malbin (SM) staining and Stemheimer staining, which can meet the needs of formed element staining in most cases. However, in actual microscopy, it is found that formed elements in urine tend to gather along the edge of the coverslip, especially crystals are prone to overlap, affecting observation in the field of view. When the dilution factor is too high, it is easy to cause missed detection of tangible objects and reduce the detection sensitivity. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a novel super living body S dyeing solution, comprising a dye, water, an amino acid type surfactant, a buffer, a preservative, a stabilizer and an auxiliary agent.

[0006] Furthermore, the dye comprises a nucleus staining component, a cytoplasm staining component and a fat staining component, and the molar ratio of the nucleus staining component, the cytoplasm staining component and the fat staining component is (2~3):(2~3):(1~2).

[0007] Furthermore, the cell nucleus staining component is one or more of hematoxylin, methylene blue and Hoechst 33342, the cytoplasm staining component is one or more of eosin, 4',6-diamidino-2-phenylindole and mito tracker, and the fat staining component is Sudan III.

[0008] Furthermore, the buffer is MES Buffer or sodium barbital-hydrochloric acid, and the added mass of the buffer is 1% to 5% of the total mass of the staining solution. The role of adding the buffer is to adjust the pH value of the staining solution and control the pH of the staining solution within the range of 6 to 7.5.

[0009] Furthermore, the auxiliary agent is cetylpyridinium chloride or cetylpyridinium bromide, and the mass of the auxiliary agent is 0.01% to 0.3% of the total mass of the new super living S staining solution. The auxiliary agent not only plays a color fixing role and increases the accuracy of detection, but also has an antiseptic effect, and the effect is very good.

[0010] Furthermore, the amino acid surfactant is sodium N-lauroyl aspartate, and the amount of the amino acid surfactant is 0.1% to 0.3% of the total mass of the dyeing solution. If the amount is too much, foam will be generated, and the foam that does not dissipate for a long time will affect the observation under the microscope. If the amount is too little, it cannot play a good role. When the feeding amount of sodium N-lauroyl glutamate is 0.05%, there is still a small amount of blurring around the crystal, and the formed components are unevenly dispersed and still tend to gather to the edge; when the feeding amount of sodium N-lauroyl glutamate is 0.5%, tiny bubbles can still be observed under the microscope, affecting the observation of formed components; when the feeding amount of sodium N-lauroyl glutamate is 0.1% and 0.3%, there is almost no blurring around the crystal, so the amount of surfactant added must be appropriate.

[0011] Furthermore, the preservative is methylisothiazolinone, and the added mass of the preservative is 0-0.3% of the total mass of the new super living S staining solution; the stabilizer is cyclodextrin, and the added mass of the stabilizer is 0-0.3% of the total mass of the staining solution.

[0012] Furthermore, the preparation method of the novel super living body S staining solution comprises the following steps: S1: preparing the cell nucleus staining component, the cytoplasm staining component and the fat staining component into aqueous solutions respectively, and then mixing them evenly with the buffer; S2: Add auxiliary agents, preservatives, stabilizers and amino acid surfactants to the mixed solution obtained in S1, defoam to obtain the novel super living S staining solution, and seal and store at low temperature.

[0013] Furthermore, the defoaming method in step S2 is vacuum defoaming, and the sealed low-temperature storage method is to fill with nitrogen, seal and store at 2~15°C, vacuum degassing to remove oxygen in the dyeing solution, and fill with nitrogen for protection, thereby extending the shelf life and increasing the service life.

[0014] At the same time, the present invention also provides a method for using the novel super living body S staining solution, which is characterized by comprising the following steps: S21: putting the urine sample into a centrifuge tube, centrifuging it, taking the urine stock solution, and mixing it with the Super Living S staining solution at a volume ratio of 1:(1-3); S22: Let the mixed solution in S21 stand for 1 to 10 minutes for sufficient staining, take an appropriate amount of the mixed solution and drop it on a glass slide, and place the glass slide on the detection equipment for observation.

[0015] The dyeing solution prepared by the method of the present application can make the tangible matter in the urine stock solution disperse more evenly, effectively inhibit the edge aggregation effect, make the color contrast more vivid and clear, facilitate observation, and improve the sensitivity of the urine stock solution microscopy. When using commercially available dyeing solutions to dye urine stock solution samples, the formed components of urine tend to gather along the edge of the cover glass, and crystals are prone to overlap, affecting observation in the field of view. When the dilution multiple is too high, it is easy to cause the missed detection of tangible matter and reduce the detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the detection diagram of Example 1.

[0017] Figure 2 This is the detection diagram of Example 2.

[0018] Figure 3 This is the detection diagram of Example 3.

[0019] Figure 4 This is the detection diagram of Example 4.

[0020] Figure 5 This is the detection diagram of Example 5.

[0021] Figure 6 This is the detection picture before staining of Example 6.

[0022] Figure 7 This is the detection picture after staining of Example 6.

[0023] Figure 8 This is the detection picture before staining of Example 7.

[0024] Fig. 9 This is the detection picture after staining of Example 7.

[0025] Fig.10 This is the detection picture of Example 8 before staining.

[0026] Fig.11 This is the detection picture after staining in Example 8.

[0027] Fig.12 This is the detection diagram of Comparative Example 1.

[0028] Fig.13 This is the detection diagram of Comparative Example 2. DETAILED DESCRIPTION

[0029] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0030] In Examples 1 to 3 of the present invention and Comparative Example 1, urine samples were collected from patient A.

[0031] In Examples 4 to 8 of the present invention, urine samples were collected from patients B, C, D, E, and F, respectively.

[0032] Some of the pictures in the embodiments and comparative examples have crystals and some have cells because the conditions of each patient are different. Only crystals prove that other components are normal. The appearance of crystals means that the patient may have stones and high uric acid. The appearance of different cells and different numbers of cells are related to the patient's own conditions. Through urine tests, it is determined which indicators of different patients are normal or abnormal, achieving a good diagnostic effect.

[0033] Example 1: Preparation of a super living S staining solution: Take 10 mL each of 6 mmol / L methylene blue, 6 mmol / L eosin, and 3 mmol / L Sudan III and mix them with 1 g of MES Buffer. The pH of the mixed solution is 6.5. Then add 0.01 g of cetylpyridinium chloride, 0.06 g of sodium N-lauroyl glutamate, 0.002 g of methylisothiazolinone, and 0.002 g of cyclodextrin. After mixing, vacuum defoam and fill with nitrogen. Then, seal and store at 2~15°C to obtain a super living S staining solution.

[0034] Place the urine sample in a centrifuge tube and centrifuge it. Then, mix 0.2 mL of the urine stock solution with 0.4 mL of a super living S staining solution and let it stand for 3 minutes to fully stain it. Place two drops of the mixed solution on a glass slide and place it on the detection device for observation. Figure 1 .

[0035] Compared with Comparative Example 1, the formed ingredients in the sample are more evenly dispersed in the droplets, the crystals do not overlap, there is no blooming around the crystals, and the color is brighter and more vivid.

[0036] Example 2: Preparation of a super living S staining solution: Take 10 mL of each of 6 mmol / L hematoxylin, 6 mmol / L 4',6-diamidino-2-phenylindole and 3 mmol / L sudantriol and mix them with 1 g of sodium barbital-hydrochloric acid, the pH of the mixed solution is 6.5, then add 0.012 g of cetylpyridinium bromide, 0.062 g of sodium N-lauroyl glutamate, 0.002 g of methylisothiazolinone and 0.002 g of cyclodextrin, defoam in vacuo after mixing, fill with nitrogen, and store in a sealed state at 2~10°C to obtain a super living S staining solution.

[0037] Place the urine sample in a centrifuge tube and centrifuge it. Then, mix 0.2 mL of the urine stock solution with 0.4 mL of a super living S staining solution and let it stand for 3 minutes to fully stain it. Place two drops of the mixed solution on a glass slide and place it on the detection device for observation. Figure 2 , the detection effect is good.

[0038] Example 3: Preparation of a super living S staining solution: Take 10 mL each of 6 mmol / L hematoxylin, 6 mmol / L 4',6-diamidino-2-phenylindole, and 3 mmol / L sudan tri and mix them with 1 g of sodium barbital-hydrochloric acid, the pH of the mixed solution is 6.5, then add 0.012 g of cetylpyridinium bromide and 0.062 g of sodium N-lauroyl glutamate, defoam under vacuum after mixing, fill with nitrogen, and store in a sealed state at 2~10℃ to obtain a super living S staining solution.

[0039] Place the urine sample in a centrifuge tube and centrifuge it. Then, mix 0.2 mL of the urine stock solution with 0.4 mL of a super living S staining solution and let it stand for 3 minutes to fully stain it. Place two drops of the mixed solution on a glass slide and place it on the detection device for observation. Figure 3 , there is micro-aggregation, which indicates that adding stabilizers and preservatives can also improve the detection effect.

[0040] Example 4: The urine of patient A was replaced with the urine of patient B. The other conditions were the same as those of Example 1. The test results are shown in Figure 4The formed components in the sample are dispersed more evenly in the droplets, and the crystals do not overlap, there is no blurring around the crystals, and the colors are brighter and more vivid.

[0041] Example 5: The urine of patient A was replaced with the urine of patient C. The other conditions were the same as those of Example 1. The test results are shown in Figure 5 The formed ingredients in the sample are dispersed more evenly, and the crystals do not overlap, and the color is brighter and more vivid.

[0042] Example 6: The urine of patient A was replaced with the urine of patient D. The other conditions were the same as in Example 1. The test results are shown in Figure 6 , Figure 7 , Figure 6 and Figure 7 Comparison of the images before and after staining shows that the cells are mainly epithelial cells, and the outline is clearer after staining, including whether there is overlap and whether there are other cells, which can be distinguished at a glance.

[0043] Example 7: The urine of patient A was replaced with the urine of patient E. The other conditions were the same as those of Example 1. The test results are shown in Figure 8 , Fig. 9 , Figure 8 and Fig. 9 Comparison of the images before and after staining shows that they are mainly white blood cells, but there will be confusion before staining, and it is difficult to judge whether there are red blood cells or other cells. After staining, the nucleus, cytoplasm, size, and color of the formed elements have obvious contrast before and after staining, clear contours, and greatly increased recognition, which is convenient for identification, thereby increasing the positive detection rate.

[0044] Example 8: The urine of patient A was replaced with the urine of patient F. The other conditions were the same as those of Example 1. The test results are shown in Fig.10 , Fig.11 , Fig.10 and Fig.11 Comparison of the images before and after staining shows that for crystals with special shapes, it is easier to identify them after staining.

[0045] Comparative Example 1: Preparation of a super living S staining solution: Take 10 mL each of 6 mmol / L hematoxylin, 6 mmol / L 4',6-diamidino-2-phenylindole, and 3 mmol / L Sudan III and mix them with 1 g of sodium barbital-hydrochloric acid. The pH of the mixed solution is 6.5. Then add 0.062 g of sodium N-lauroyl glutamate. After mixing, vacuum defoam and fill with nitrogen. Then, seal and store at 2~10℃ to obtain a super living S staining solution.

[0046] Place the urine sample in a centrifuge tube and centrifuge it. Then, mix 0.2 mL of the urine stock solution with 0.4 mL of a super living S staining solution and let it stand for 3 minutes to fully stain it. Place two drops of the mixed solution on a glass slide and place it on the detection device for observation. Fig.12 It can be observed that the formed elements gather along the edge of the coverslip, and some crystals overlap, and there is a halo phenomenon around the crystals, indicating that the addition of additives has a significant effect on improving the detection effect.

[0047] Comparative Example 2: In Example 1, 0.06 g of sodium N-lauroyl glutamate component was removed, and the rest was the same as in Example 1. The test results are shown in Fig.13 The formed elements gathered along the edge of the coverslip, some crystals overlapped, and there was a halo phenomenon around the crystals.

Claims

1. A novel super living S staining solution, characterized in that: Including dyes, water, amino acid surfactants, buffers, preservatives, stabilizers and additives; The dye comprises a cell nucleus staining component, a cytoplasm staining component and a fat staining component, and the molar ratio of the cell nucleus staining component, the cytoplasm staining component and the fat staining component is (2-3):(2-3):(1-2); The cell nucleus staining component is one or more of hematoxylin, methylene blue and Hoechst 33342, the cytoplasm staining component is one or more of eosin, 4',6-diamidino-2-phenylindole and mito tracker, and the fat staining component is Sudan III; The buffer is MES Buffer or sodium barbital-hydrochloric acid, and the added mass of the buffer is 1% to 5% of the total mass of the staining solution; The auxiliary agent is cetylpyridinium chloride or cetylpyridinium bromide, and the mass of the auxiliary agent is 0.01% to 0.3% of the total mass of the new super living S staining solution; The amino acid surfactant is sodium N-lauroyl aspartate, and the amount of the amino acid surfactant used is 0.1% to 0.3% of the total mass of the dyeing solution; The preservative is methylisothiazolinone, and the added mass of the preservative is 0-0.3% of the total mass of the new super living S staining solution. The stabilizer is cyclodextrin, and the added mass of the stabilizer is 0-0.3% of the total mass of the staining solution.

2. The method for preparing the novel super living body S staining solution according to claim 1, characterized in that: The following steps are involved: S1: preparing the cell nucleus staining component, the cytoplasm staining component and the fat staining component into aqueous solutions respectively, and then mixing them evenly with the buffer; S2: Add auxiliary agents, preservatives, stabilizers and amino acid surfactants to the mixed solution obtained in S1, defoam to obtain the novel super living S staining solution, and seal and store at low temperature.

3. The method for preparing the novel super living body S staining solution according to claim 2, characterized in that: The defoaming method in step S2 is vacuum defoaming, and the sealed low-temperature storage method is to fill with nitrogen and then seal and store at 2~15°C.

4. The method for using the novel super living body S staining solution according to claim 1, characterized in that: The steps include: S21: putting the urine sample into a centrifuge tube, centrifuging it, taking the urine stock solution, and mixing it with the Super Living S staining solution at a volume ratio of 1:(1-3); S22: Let the mixed solution in S21 stand for 1 to 10 minutes for sufficient staining, take an appropriate amount of the mixed solution and drop it on a glass slide, and place the glass slide on the detection equipment for observation.

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