CN116413110A - Reticulocyte staining reagent set, box and reticulocyte staining method - Google Patents

Abstract

A reticulocyte staining reagent group, a test kit and a staining method include a staining reagent A and a stable reagent B; the stable reagent B is used after the staining reagent A is mixed with a sample for M seconds; the range of M is greater than or equal to 20 seconds; the staining reagent A includes dyeing agent A10, adjusting salt C10 and water; dyeing agent A10 is new methylene blue; the concentration of new methylene blue in dyeing reagent A is 0.8× ^10-4 g/ml～8.0× ^10-4 g/mL; adjusting Salt C10, used to provide a low osmotic pressure environment and a pH range of 7.0 to 8.0; Stabilizing Reagent B includes Dye Accelerating Stabilizer B10, Regulating Salt D10 and water; Dye Accelerating Stabilizer B10 in Stabilizing Reagent B includes aldehyde-containing substances . Using a unique two-step reagent formula and method, the rapid staining of reticulocytes is realized in a step-by-step manner. The staining can be done in 20 seconds, and the staining effect can be maintained for a long enough time to reach more than 120 minutes.

Description

Reticulocyte staining reagent set, box and reticulocyte staining method

technical field

The application belongs to the technical field of staining for cell bright field identification, and in particular relates to a reagent and method for staining reticulocytes in a cell suspension state, so as to realize the identification of reticulocytes under bright field.

Background technique

During the development of red blood cells in most mammals, including humans or dogs or cats, the RNA content has obvious regular changes, that is, it is relatively abundant in the primitive stage, then gradually decreases, and disappears or nearly disappears after the cells are fully mature. Reticulocytes (Ret) are cells in the transition stage between late immature red blood cells and mature red blood cells, slightly larger than mature red blood cells (8.0-9.5 μm in diameter), and the residual basophilic substance ribonucleic acid (RNA) in the cytoplasm Autonucleated red blood cells. RNA is a basophilic substance. After vital staining, it forms a granular or silk-like structure precipitate, so it is called reticulocytes. Reticulocytes still have the ability to synthesize hemoglobin after they are released from the bone marrow into the peripheral blood. After about 1 to 2 days, their nucleic acid substances disappear completely and transition into mature red blood cells.

For mammalian blood, mature erythrocytes in blood do not contain nuclear material and do not require staining, and can be seen after bright-field micromagnification. However, there is a small amount of nucleic acid substances in the reticulocytes. If the nucleic acid substances in them are not stained, the reticulocytes will be recognized as regular red blood cells. To be accurately identified, the nucleic acid substances in the reticulocytes need to be stained deeply enough to be identified. However, for the reticulocyte itself, it is a kind of immature red blood cell in the process of maturation; for the reticulocyte with more nucleic acid substances, it means that it has just developed from the late immature red blood cell, and the closer it is to the mature anucleated red blood cell The reticulocytes in the advanced state have less nucleic acid content in them, and are more difficult to be stained and identified. In the peripheral whole blood sample, the number of reticulocytes and the ratio of the number represent different diseases and the severity of a certain disease.

Reticulocytes, especially reticulocytes with less content of nucleic acid substances, are difficult to be stained by the staining methods in the prior art; that is to say, reticulocytes will be misidentified as normal red blood cells. Especially for the staining of reticulocytes based on suspension, it is difficult to distinguish between reticulocytes and ordinary red blood cells when the cell type is identified after bright field micro-magnification. For some pathological conditions, this cannot be accurately identified. Especially for some patients with anemia, the number of reticulocytes often indicates whether there is a certain disease or the degree of a certain disease; The key points of whether such diseases and their severity can be identified.

In the prior art, there are also some staining methods specifically for reticulocytes, such as the People's Republic of China health industry standard WS/T 346-2011 "Reference method for counting reticulocytes" provides a staining method for reticulocytes , in which phosphate is directly used as a buffer salt to provide an isotonic ion concentration environment, and new methylene blue dye is used for staining, and the blood sample and staining reagent are mixed at a ratio of 1:1 or 1:2 for staining The time is about 3 minutes to 5 minutes; the stained sample is then coated on a glass slide for observation after smearing. Due to the use of an isotonic ion concentration environment, the staining effect on reticulocytes rich in nucleic acid substances is better, but the time required for staining is relatively long, at least 3 minutes. As shown in Figure 2, it is a schematic diagram of the staining effect of reticulocytes after staining based on traditional liquid-based staining reagents. The staining takes at least 3 minutes, and the efficiency is not high.

In the prior art, the patent application No. "CN2020112230298" titled "Cell Staining Reagent and Cell Staining Method" also provides a staining reagent for cell staining in the state of cell suspension. Although the staining reagent can achieve a better staining effect on white blood cells, the staining effect on reticulocytes in blood cells is not satisfactory; that is, the staining of reticulocytes cannot be completed. In the prior art, in the state of the blood cell suspension, when the blood cell suspension is stained with a conventionally formulated staining agent, the white blood cells are usually stained, but the reticulocytes have not been dyed, which is not enough for reticulation. Brightfield identification of erythrocytes.

Glossary:

Nucleic acid substances refer to the substances with genetic characteristics existing in cells, namely DNA or RNA or fragments thereof.

People's Republic of China health industry standard WS/T 346-2011 "Reference method for counting reticulocytes", see the following website for details:

"https://ebook.chinabuilding.com.cn/zbooklib/bookpdf/probation?SiteID=1&bookID=126960".

EDTA salt refers to ethylenediaminetetraacetic acid salt; ethylenediamine tetraacetic acid salt will combine with calcium in the blood to form a coordination compound, thereby preventing blood clotting. EDTA salt is routinely used as a blood anticoagulant.

Osmotic pressure: Separated by a semi-permeable membrane, one side is the water that dissolves enzymes, and the other side is the solution, and the water penetrates into the solution side through the semi-permeable membrane; the pressure added on the solution side to prevent the movement of water is called osmotic pressure. The motion of the water is stopped because this pressure is equal to the chemical potential energy of the water passing through the membrane. Osmolarity is expressed in units of osmolarity, which is usually expressed in milliosmoles of solute per kilogram of solvent.

The CAS numbers of some substances in this application are as follows:

(CASRegistryNumber or CASNumber, CASRn, CAS#), also known as CAS registration number or

The CAS registration number is a substance (compound, polymer material, biological sequence

(Biological sequences), mixtures or alloys) unique numerical identification number.

The water in this application is purified water. Purified water refers to the water that meets the purified water standard of "Chinese Pharmacopoeia (2015 Edition)". Water greater than 25 μS/cm (micro Siemens per centimeter).

Contents of the invention

In order to avoid the deficiency that reticulocytes cannot be stained and identified in the prior art, especially reticulocytes with low nucleic acid content cannot be fully stained, a method that can efficiently realize reticulocytes, especially those containing a small amount of nucleic acid, is proposed. A staining reagent kit for fully staining reticulocytes with a substance, a preparation method and a staining method. The technical solution of the present application to solve the above-mentioned technical problems is a reticulocyte staining reagent set, including staining reagent A and stabilizing reagent B; stabilizing reagent B is used after mixing the staining reagent A and the sample for M seconds; the range of M is greater than or equal to 20 seconds ; Dyeing reagent A includes dyeing agent A10, adjusting salt C10 and water; dyeing agent A10 is new methylene blue; the concentration of new methylene blue in dyeing reagent A is 0.8×10 ^-4 g/ml～8.0×10 ^-4 g/ mL; adjusting salt C10, used to provide a low osmotic pressure environment and a pH value range of 7.0 to 8.0 environment; stabilizing reagent B includes dyeing accelerator stabilizer B10, adjusting salt D10 and water; stabilizing reagent B includes dyeing accelerator stabilizer B10 containing Aldehydes. In the low osmotic environment of the dyeing reagent A, the osmotic pressure ranges from 140 mosmol/kg to 210 mosmol/kg.

In the staining reagent A for human or canine reticulocyte staining, the adjusting salt C10 includes disodium hydrogen phosphate and potassium dihydrogen phosphate; the osmotic pressure range is 140mosmol/kg～180mosmol/kg; In the stable reagent B for erythrocyte staining, the adjusting salt D10 includes disodium hydrogen phosphate and potassium dihydrogen phosphate; the osmotic pressure range is 180 mosmol/kg-240 mosmol/kg.

In the staining reagent A for cats, the adjusting salt C10 includes disodium hydrogen phosphate and potassium dihydrogen phosphate; the osmotic pressure range is 170 mosmol/kg to 210 mosmol/kg; in the stable reagent B for cats, the adjusting salt D10 includes Disodium hydrogen phosphate and potassium dihydrogen phosphate; the osmotic pressure range is 160mosmol/kg～210mosmol/kg.

In dyeing reagent A, adjusting salt C10 includes disodium hydrogen phosphate and potassium dihydrogen phosphate; in stabilizing reagent B, adjusting salt D10 includes disodium hydrogen phosphate and potassium dihydrogen phosphate; neutralizing or stabilizing reagent B in dyeing reagent A, or Adjust the pH value with alkaline substances, including NaOH and/or KOH; or adjust the pH value with acidic substances, including hydrochloric acid.

Dyeing reagent A also includes anticoagulant dyeing accelerator A20; anticoagulant dyeing accelerator A20 includes EDTA salt; the concentration range of EDTA salt in dyeing reagent A is 2.0×10 ^-4 mol/L～2.0×10 ^-3 mol/L, The EDTA salt includes any one or more of EDTA·K ₂ , EDTA·K ₃ or EDTA·Na ₂ .

In the dyeing reagent A, the adjusting salt C10 includes NaCl; the concentration of NaCl in the dyeing reagent A is 2.2×10 ^-2 mol/L～3.5×10 ^-2 mol/L; or the adjusting salt C10 includes KCl; the concentration of KCl in the dyeing reagent A is 2.2×10 ^-2 mol/L～3.5× ^{10 -2} mol/L.

The pH value of staining reagent A used for staining human or canine reticulocytes is 7.0-7.6; in staining reagent A, the concentration of disodium hydrogen phosphate is 3.0×10 ^-2 mol/L～4.8×10 ^-2 mol/L , the concentration of potassium dihydrogen phosphate is 5.8×10 ^-3 mol/L～1.5×10 ^-2 mol/L; the concentration of disodium hydrogen phosphate is 2.5×10 in stable reagent B for human or canine reticulocyte staining ^-2 mol/L～6.0×10 ^-2 mol/L, the concentration of potassium dihydrogen phosphate is 2.4×10 ^-2 mol/L～5.4×10 ^-2 mol/L; the dyeing accelerator stabilizer B10 includes formaldehyde and amyl Dialdehyde; the concentration of formaldehyde is 1.5×10 ^-2 mol/L to 2.1×10 ^-2 mol/L; the concentration of glutaraldehyde is 6.0×10 ^-3 mol/L to 8.0×10 ^-3 mol/L.

The pH value of staining reagent A used for feline reticulocyte staining is 7.2-8.0; in staining reagent A, the concentration of disodium hydrogen phosphate is 4.3×10 ^-2 mol/L～6.0×10 ^-2 The concentration of potassium dihydrogen is 7.0×10 ^-3 ~ 1.4×10 ^{- 2} mol/L; the concentration of disodium hydrogen phosphate is 2.5×10 ^-2 mol/L ~ 6.0 in Stable Reagent B for cat reticulocyte staining ×10 ^-2 mol/L, the concentration of potassium dihydrogen phosphate is 2.4×10 ^-2 mol/L～5.4×10 ^-2 mol/L; the dyeing accelerator stabilizer B10 includes formaldehyde and glutaraldehyde; the formaldehyde concentration is 0.9 ×10 ^-2 mol/L～1.8×10 ^-2 mol/L; the concentration of glutaraldehyde is 4.0× ^{10 -3 mol} /L～7.5×10 ^-3 mol/L.

Every 500ml of Stable Reagent B includes 0.5mL of proClean 950 solution or preservative; every 500ml of Staining Reagent A includes 0.5mL of proClean 950 solution or preservative.

The technical solution of the present application to solve the above-mentioned technical problems may also be a reticulocyte staining kit, comprising: a first container and a second container; the first container includes the above-mentioned staining reagent A; the second container includes the above-mentioned stable reagent B; the volume ratio of dyeing reagent A in the first container to the volume of stable reagent B in the second container is 2:3.

The technical solution of the present application to solve the above-mentioned technical problems can also be a method for staining reticulocytes, including step 10: taking a standard sample and mixing it with staining reagent A to form a staining mixture 1; step 20: waiting for staining for M seconds; M range It is greater than or equal to 20 seconds, and the time can also be other numbers from 20 seconds to 120 seconds; Step 30: Mix the dyeing mixture 1 with the stable reagent B to form the dyeing mixture 2; take the volume of the standard sample and the volume of the dyeing reagent A The ratio ranges from 5:400 to 20:400; the volume ratio of the staining reagent A to the stabilizing reagent B is 2:3; the staining reagent A is the aforementioned staining reagent A; the stabilizing reagent B is the aforementioned stabilizing reagent B.

One of the technical effects: using a unique two-step reagent formula and method, the rapid staining of reticulocytes is realized in a step-by-step manner, and the staining effect can be maintained for a long enough time to reach more than 120 minutes; low-osmotic and weakly alkaline staining reagents A and hypotonic weakly alkaline stable reagent B are added at specific time intervals, which provides just the right staining time window for the staining of nucleic acid substances in reticulocytes. The low-osmotic weak alkaline environment and the dye binding method based on the mutual attraction of positive and negative charges enable the dye to quickly enter the reticulocytes within M seconds to complete the staining of nucleic acid substances; and reach a state close to equilibrium after M seconds The value range of M is greater than or equal to 20 seconds, that is, sufficient staining of reticulocytes can be completed in 20 seconds. After the sample is mixed with the staining reagent for 24 seconds, after adding the stabilizing reagent B in this state, the aldehyde-containing substance in the dye-promoting stabilizer B10 is used to chemically cross-link the protein on the surface of the cell membrane, so that the cell membrane is fixed and it is easier to maintain the cell The excellent staining effect and cell morphology make the staining effect last for a long time, and the staining effect can be maintained even after 120 minutes; it is convenient for subsequent microscopic magnification imaging in suspension state and identification of reticulocytes after imaging. It solves the problem of dyeing in a suspension state. Based on the principle of entropy increase, the dye is easily diluted in a suspension state and it is difficult to maintain a stable dyeing state. There is no surfactant in the reagent, the cell membrane is not perforated by the surfactant, and it is easier to maintain the shape; and there is no permeabilizing agent in the reagent; the cell membrane is not perforated by the permeabilizing agent, and it is easier to maintain the shape in a hypotonic state.

The second technical effect: the hypotonic and weakly alkaline dyeing reagent A provides reticulocytes with an atmosphere that is very suitable for being dyed; weakly alkaline, especially an environment with a pH value of 7-8, will make the reticulocytes Phosphate groups exhibit negatively charged characteristics, so they can be combined with positively charged basic dyes. This dye binding method based on the mutual attraction of positive and negative charges makes the nucleic acid substances in reticulocytes complete dyeing and at the same time reunite The effect is easier to see and identify, and it is especially suitable for staining cells with only a small part of the nucleus material such as reticulocytes; the acid-base characteristics of basic dyes are also just combined with acidic phosphate groups to form a stable combination to ensure staining The effect can last longer. The above two aspects make it possible to have a good dyeing and binding effect on low-concentration nucleic acid substances, thereby showing sufficient dyeing effects on reticulocytes in various states. The problem in the prior art that reticulocytes with low content of nucleic acid substances cannot be dyed or dyed insufficiently is avoided. Provided is a staining reagent and method capable of fully staining reticulocytes with low nucleic acid content; such staining can fully stain reticulocytes in different states, thereby improving the recognition rate of reticulocytes, It can provide a more accurate basis for the number of reticulocytes for the early diagnosis of some diseases, and is the key path for the identification of the next generation of reticulocytes.

The third technical effect: both the hypotonic and weakly alkaline dyeing reagent A and the hypotonic weakly alkaline stable reagent B provide a hypotonic environment for cell staining, which makes the cell membrane slightly swell, and accordingly the nucleic acid substances inside the cell membrane and the cell membrane The boundaries between the dyes are easier to distinguish; the dyes based on the mutual attraction of the positive and negative charges and the phosphate groups are also more easily highlighted, so that the expression of the staining is more sufficient.

The fourth technical effect: the aldehyde-containing substance in the dye-promoting stabilizer B10 will react with the protein on the surface of the cell membrane to form a colloidal layer, thereby stabilizing and fixing the cell shape, which is convenient for subsequent cell shape recognition of various cells.

The fifth technical effect: ethylenediaminetetraacetic acid salt; usually, ethylenediamine tetraacetic acid salt will combine with calcium in the blood to form a coordination compound, thereby preventing blood coagulation, so it is a conventional anticoagulant. However, in the formula of the present application, the anticoagulant and stain accelerator A20 does not play the role of anticoagulation, but plays the role of promoting dyeing of nucleic acid substances in reticulocytes.

Description of drawings

Fig. 1 is a schematic diagram of the steps of the reticulocyte staining method in the present application;

Figure 2 is a schematic diagram of the staining effect of reticulocytes after staining based on traditional liquid-based staining reagents;

Fig. 3 is a schematic table of components based on multiple embodiments and comparative examples of the reticulocyte staining reagent group;

Fig. 4 is to include two parts of the picture; The upper part of the picture in Fig. 4 is according to the reticulocyte staining method among the present application, after embodiment 1, embodiment 2, embodiment 3 are applied to canine blood cell staining for 20 seconds, to After staining, the sample is microscopically enlarged to observe a collection of pictures of some of the reticulocytes. In the figure, it can be seen that the reticulocytes have been colored; the lower part of the picture in Figure 4 is Example 1, Example 2, and Example 3 After 120 minutes of staining of canine blood cells, a collection of pictures of some of the reticulocytes observed by microscopically zooming in on the stained sample shows that the staining effect of the reticulocytes can continue to be maintained;

Fig. 5 is to comprise two parts picture among; Fig. 5 upper part picture is according to the reticulocyte staining method among the present application, after embodiment 11, embodiment 12, embodiment 13 are applied in cat blood cell staining 20 seconds, to After staining, the sample is microscopically enlarged to observe a collection of pictures of some of the reticulocytes. In the figure, it can be seen that the reticulocytes have been colored; the lower part of the picture in Figure 5 is Example 11, Example 12, and Example 13 After 120 minutes of cat blood cell staining, a collection of pictures of some of the reticulocytes observed by microscopically zooming in on the stained sample shows that the staining effect of the reticulocytes can continue to be maintained.

Detailed ways

The content of the present application will be described in further detail below in conjunction with the accompanying drawings. It should be noted that the following are descriptions of preferred embodiments of the present invention, which do not constitute any limitation to the present invention. The description of the preferred embodiment of the invention is provided as an illustration only of the general principles of the invention. The numbers such as "first", "second" and "A" and "B" involved in the present invention are only for the convenience of description, and do not represent the sequential relationship in time or space.

As shown in Figure 3, in the schematic table of components based on multiple embodiments and comparative examples of the reticulocyte staining reagent group, the embodiment of the reticulocyte staining reagent group includes staining reagent A and stabilizing reagent B; stabilizing reagent B is used after dyeing reagent A is mixed with the sample for M seconds; the range of M is greater than or equal to 20 seconds; dyeing reagent A includes dyeing agent A10, adjusting salt C10 and water; dyeing agent A10 is new methylene blue; new methylene blue in dyeing reagent A The concentration of methylene blue is 0.8×10 ^-4 g/ml～8.0×10 ^-4 g/mL; the adjustment salt C10 is used to provide a low osmotic pressure environment and a pH value range of 7.0 to 8.0 environment; Stabilizing Reagent B includes dyeing accelerator stabilizer B10, adjusting salt D10 and water; the dyeing-accelerating stabilizer B10 in the stabilizing reagent B includes aldehyde-containing substances. In the low osmotic environment of the dyeing reagent A, the osmotic pressure ranges from 140 mosmol/kg to 210 mosmol/kg.

In the staining reagent A for human or canine reticulocyte staining, the adjusting salt C10 includes disodium hydrogen phosphate and potassium dihydrogen phosphate; the osmotic pressure range is 140mosmol/kg～180mosmol/kg; In the stable reagent B for erythrocyte staining, the adjusting salt D10 includes disodium hydrogen phosphate and potassium dihydrogen phosphate; the osmotic pressure range is 180 mosmol/kg-240 mosmol/kg.

In the staining reagent A for cats, the adjusting salt C10 includes disodium hydrogen phosphate and potassium dihydrogen phosphate; the osmotic pressure range is 170 mosmol/kg to 210 mosmol/kg; in the stable reagent B for cats, the adjusting salt D10 includes Disodium hydrogen phosphate and potassium dihydrogen phosphate; the osmotic pressure range is 160mosmol/kg～210mosmol/kg.

In dyeing reagent A, adjusting salt C10 includes disodium hydrogen phosphate and potassium dihydrogen phosphate; in stabilizing reagent B, adjusting salt D10 includes disodium hydrogen phosphate and potassium dihydrogen phosphate; neutralizing or stabilizing reagent B in dyeing reagent A, or Adjust the pH value with alkaline substances, including NaOH and/or KOH; or adjust the pH value with acidic substances, including hydrochloric acid.

Dyeing reagent A also includes anticoagulant dyeing accelerator A20; anticoagulant dyeing accelerator A20 includes EDTA salt; the concentration range of EDTA salt in dyeing reagent A is 2.0×10 ^-4 mol/L～2.0×10 ^-3 mol/L, The EDTA salt includes any one or more of EDTA·K ₂ , EDTA·K ₃ or EDTA·Na ₂ .

In the dyeing reagent A, the adjusting salt C10 includes NaCl; the concentration of NaCl in the dyeing reagent A is 2.5×10 ^-2 mol/L～3.5×10 ^-2 mol/L; or the adjusting salt C10 includes KCl; the concentration of KCl in the dyeing reagent A is 2.5×10 ^-2 mol/L～3.5× ^{10 -2} mol/L.

The pH value of staining reagent A used for staining human or canine reticulocytes is 7.0-7.6; in staining reagent A, the concentration of disodium hydrogen phosphate is 3.0×10 ^-2 mol/L～4.8×10 ^-2 mol/L , the concentration of potassium dihydrogen phosphate is 5.8×10 ^-3 mol/L～1.5×10 ^-2 mol/L; the concentration of disodium hydrogen phosphate is 2.5×10 in stable reagent B for human or canine reticulocyte staining ^-2 mol/L～6.0×10 ^-2 mol/L, the concentration of potassium dihydrogen phosphate is 2.4×10 ^-2 mol/L～5.4×10 ^-2 mol/L; the dyeing accelerator stabilizer B10 includes formaldehyde and amyl Dialdehyde; formaldehyde concentration is 1.5×10 ^-2 mol/L to 2.1×10 ^-2 mol/L; glutaraldehyde concentration is 6.0×10 ^-3 mol/L to 8.0×10 ^-3 mol/L.

The pH value of staining reagent A used for feline reticulocyte staining is 7.2-8.0; in staining reagent A, the concentration of disodium hydrogen phosphate is 4.3×10 ^-2 mol/L～6.0×10 ^-2 The concentration of potassium dihydrogen is 7.0×10 ^-3 ~ 1.4×10 ^{- 2} mol/L; the concentration of disodium hydrogen phosphate is 2.5×10 ^-2 mol/L ~ 6.0 in Stable Reagent B for cat reticulocyte staining ×10 ^-2 mol/L, the concentration of potassium dihydrogen phosphate is 2.4×10 ^-2 mol/L～5.4×10 ^-2 mol/L; the dyeing accelerator stabilizer B10 includes formaldehyde and glutaraldehyde; the formaldehyde concentration is 0.9 ×10 ^-2 mol/L～1.8×10 ^-2 mol/L; the concentration of glutaraldehyde is 4.0× ^{10 -3 mol} /L～7.5×10 ^-3 mol/L.

Every 500ml of Stable Reagent B includes 0.5mL of proClean 950 solution or preservative; every 500ml of Staining Reagent A includes 0.5mL of proClean 950 solution or preservative.

In an embodiment of a reticulocyte staining kit not shown in some drawings, comprising: a first container and a second container; the first container includes a staining reagent A; the second container includes a stabilizing reagent B ; The volume ratio of the dyeing reagent A in the first container to the volume of the stabilizing reagent B in the second container is 2:3.

In the embodiment of a kind of reticulocyte staining method as shown in Figure 1, comprise step 10: get standard sample and mix with staining reagent A, form staining mixture 1; Step 20: wait for staining M seconds; M range is 20 seconds to 120 seconds; Step 30: Mix the dyeing mixture 1 with the stable reagent B to form the dyeing mixture 2; the ratio of the volume of the standard sample to the volume of the dyeing reagent A is in the range of 5:400 to 20:400; the dyeing reagent A The volume ratio of volume to stabilized reagent B is 2:3.

As shown in Figure 4, the upper part of the picture in Figure 4 is based on the reticulocyte staining method in this application, after applying Example 1, Example 2, and Example 3 to dog blood cells for 20 seconds, the stained sample A collection of pictures of part of the reticulocytes observed under microscopic magnification. It can be seen in the figure that the reticulocytes have been colored; After 120 minutes of blood cell staining, a collection of pictures of some of the reticulocytes observed by microscopically zooming in on the stained sample shows that the staining effect of the reticulocytes can continue to be maintained.

As shown in Figure 5, the upper part of the picture in Figure 5 is based on the reticulocyte staining method in this application, after applying Example 11, Example 12, and Example 13 to cat blood cells for 20 seconds, the sample after staining A collection of pictures of part of the reticulocytes observed under microscopic magnification. It can be seen in the figure that the reticulocytes have been colored; After 120 minutes of blood cell staining, a collection of pictures of some of the reticulocytes observed by microscopically zooming in on the stained sample shows that the staining effect of the reticulocytes can continue to be maintained.

While the invention has been illustrated and described in terms of preferred embodiments and several alternatives, the invention is not to be limited by what has been specifically described in this specification. Other alternative or equivalent components may also be used to practice the invention.

Claims (11)

1. A reticulocyte staining reagent set is characterized in that,

comprises a staining reagent A and a stabilizing reagent B;

the stabilizing reagent B is used after the staining reagent A is mixed with the sample for M seconds; m is greater than or equal to 20 seconds;

the dyeing reagent A comprises a dyeing agent A10, a regulating salt C10 and water;

the coloring agent A10 is new methylene blue; the concentration of the new methylene blue in the dyeing reagent A is 0.8X10 ^-4 g/ml～8.0×10 ^-4 g/mL；

Adjusting salt C10 to provide low osmotic pressure environment and pH value range 7.0-8.0 environment; in the low osmotic pressure environment of the dyeing reagent A, the osmotic pressure molar concentration ranges from 140mosmol/kg to 210mosmol/kg;

the stabilizing agent B comprises a dyeing promoting stabilizing agent B10, a regulating salt D10 and water;

the dyeing promoter B10 in the stabilizing reagent B comprises aldehyde-containing substances.

2. A reagent set for staining reticulocytes according to claim 1, wherein,

in staining reagent a for staining human or canine reticulocytes, the regulating salt C10 includes disodium hydrogen phosphate and potassium dihydrogen phosphate; osmotic pressure ranges from 140mosmol/kg to 180mosmol/kg;

in stabilizing reagent B for human or canine reticulocyte staining, the modulating salt D10 comprises disodium hydrogen phosphate and potassium dihydrogen phosphate; the osmotic pressure range is 180mosmol/kg to 240mosmol/kg.

3. A reagent set for staining reticulocytes according to claim 1, wherein,

in the staining reagent A for cats, the regulating salt C10 comprises disodium hydrogen phosphate and potassium dihydrogen phosphate; osmotic pressure range is 170 mosmol/kg-210 mosmol/kg;

in stabilizing agent B for cats, the modulating salt D10 comprises disodium hydrogen phosphate and potassium dihydrogen phosphate; the osmotic pressure ranges from 160mosmol/kg to 210mosmol/kg.

4. A reagent set for staining reticulocytes according to claim 1, wherein,

in the staining reagent a, the regulating salt C10 includes disodium hydrogen phosphate and potassium dihydrogen phosphate;

in stabilizing reagent B, the regulating salt D10 comprises disodium hydrogen phosphate and potassium dihydrogen phosphate;

the dyeing reagent A is neutralized or stabilized in the reagent B, or the pH value is adjusted by alkaline substances, wherein the alkaline substances comprise NaOH and/or KOH; or adjusting pH value with acidic material including hydrochloric acid.

5. A reagent set for staining reticulocytes according to claim 1, wherein,

the dyeing reagent A also comprises an anticoagulation accelerant A20;

anticoagulation promoting agent a20 includes EDTA salt; EDTA salt concentration range in staining reagent A is 2.0X10 ^-4 mol/L～2.0×10 ^-3 mol/L, EDTA salts include EDTA salts including EDTA.K ₂ ，EDTA·K ₃ Or EDTA-Na ₂ Any one or more of the following.

6. A reagent set for staining reticulocytes according to claim 1, wherein,

in staining reagent a, the conditioning salt C10 comprises NaCl; naCl concentration in staining reagent A was 2.2X10 ^-2 mol/L～3.5×10 ^-2 mol/L; or the regulating salt C10 comprises KCl; KCl concentration in staining reagent A was 2.2X10 ^-2 mol/L～3.5×10 ^-2 mol/L。

7. A reagent set for staining reticulocytes according to claim 2, wherein,

the pH value of the staining reagent A for staining human or canine reticulocytes is 7.0-7.6; in the staining reagent A, the concentration of disodium hydrogen phosphate is 3.0X10 ^-2 mol/L～4.8×10 ^-2 mol/L, potassium dihydrogen phosphate concentration of 5.8X10 ^-3 mol/L～1.5×10 ^-2 mol/L；

In the stabilizing reagent B for staining human or canine reticulocytes, disodium hydrogen phosphate concentration is 2.5X10 ^-2 mol/L～6.0×10 ^-2 mol/L, potassium dihydrogen phosphate concentration of 2.4X10 ^-2 mol/L～5.4×10 ^-2 mol/L；

The dyeing promoter B10 comprises formaldehyde and glutaraldehyde; formaldehyde concentration of 1.5X10 ^-2 mol/L～2.1×10 ^-2 mol/L; glutaraldehyde concentration of 6.0X10 ^-3 mol/L～8.0×10 ^-3 mol/L。

8. A reagent set for staining reticulocytes according to claim 3, wherein,

the pH value of the staining reagent A for staining the reticulocytes of cats is 7.2-8.0; in the staining reagent A, the concentration of disodium hydrogen phosphate is 4.3X10 ^-2 mol/L～6.0×10 ^-2 mol/L, potassium dihydrogen phosphate concentration of 7.0X10 ^-3 ～1.4×10 ^-2 mol/L; in the stabilizing reagent B for cat reticulocyte staining, the concentration of disodium hydrogen phosphate is 2.5X10 ^-2 mol/L～6.0×10 ^{- 2} mol/L, potassium dihydrogen phosphate concentration of 2.4X10 ^-2 mol/L～5.4×10 ^-2 mol/L；

The dyeing promoter B10 comprises formaldehyde and glutaraldehyde; formaldehyde concentration of 0.9X10 ^-2 mol/L～1.8×10 ^-2 mol/L; glutaraldehyde concentration of 4.0X10 ^-3 mol/L～7.5×10 ^-3 mol/L。

9. The reticulocyte staining reagent set according to claim 1, wherein

Each 500mL of stabilizing agent B comprises 0.5mL of proClean 950 solution, namely preservative;

each 500mL of staining reagent A included 0.5mL of a proClean 950 solution, i.e., preservative.

10. A reticulocyte staining kit is characterized in that,

comprising the following steps: a first container and a second container; in a first container, comprising a staining reagent a according to any of claims 1 to 9; comprising the stabilising agent B of any one of claims 1 to 9 in a second container; the volume ratio of the staining reagent A in the first container to the stabilizing reagent B in the second container is 2:3.

11. A method for staining reticulocytes, which is characterized by comprising the steps of,

step 10: mixing a standard sample with a staining reagent A to form a staining mixed solution 1;

step 20: waiting for dyeing for M seconds; m is greater than or equal to 20 seconds;

step 30: uniformly mixing the dyeing mixed solution 1 with the stabilizing reagent B to form a dyeing mixed solution 2;

the ratio of standard sample volume to staining reagent a volume was in the range of 5:400 to 20:400;

the volume ratio of the dyeing reagent A to the stabilizing reagent B is 2:3;

the staining reagent a is any one of the staining reagents a of claims 1 to 9;

the stabilizing agent B is a stabilizing agent B according to any one of claims 1 to 9.

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