CN116413110A - Reticulocyte staining reagent set, box and reticulocyte staining method - Google Patents
Reticulocyte staining reagent set, box and reticulocyte staining method Download PDFInfo
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N1/31—Apparatus therefor
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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Abstract
A reticulocyte staining reagent set, a reagent kit and a staining method comprise 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; 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. The unique double-step reagent formula and method are adopted, the reticulocyte is quickly dyed in a step-by-step mode, the reticulocyte can be successfully dyed in 20 seconds, and the dyeing effect can be maintained for more than 120 minutes long enough.
Description
Technical Field
The application belongs to the technical field of staining for cell bright field identification, and particularly relates to a reagent and a method for staining reticulocytes in a cell suspension state so as to realize the identification of the reticulocytes in the bright field.
Background
In the development of erythrocytes in most mammals, including humans or dogs or cats, the RNA content changes significantly regularly, i.e. from a relatively rich initial stage, and then gradually decreases until the cells disappear or nearly disappear after complete maturation. Reticulocytes (Ret) are transitional stage cells between late juvenile and mature red blood cells, slightly larger than mature red blood cells (8.0-9.5 μm in diameter), and the basophils ribonucleic acid (RNA) remaining in the cytoplasm are derived from nucleated red blood cells. RNA is a basophilic substance, and after living body dyeing, a precipitate with a dot-shaped or silk-screen structure is formed, so that the RNA is named as reticulocyte. Reticulocytes have the capacity to synthesize hemoglobin after release from the bone marrow to the peripheral blood, and after about 1-2 days, their nucleic acid material is depleted, transitioning to mature red blood cells.
For mammalian blood, the absence of nuclear material from mature red blood cells in the blood is also not required to be stained and can be seen after bright field microscopic magnification. But small amounts of nucleic acid material are present in the reticulocytes, which would be recognized as conventional red blood cells if they were not stained. To be accurately identified, nucleic acid materials in reticulocytes need to be stained deep enough to be identified. However, for reticulocytes themselves, this is a naive red blood cell that is approaching the maturation process; reticulocytes with a higher nucleic acid content, meaning that they have just evolved from late juvenile red blood cells, are more difficult to stain and to recognize the more mature, coreless red blood cell state they have. In peripheral whole blood samples, the respective numbers of reticulocytes and the ratio of the numbers thereof represent different diseases and the severity of a certain disease.
Reticulocytes, particularly reticulocytes having a reduced content of nucleic acid material, are difficult to stain by staining methods of the prior art; that means that reticulocytes will be misidentified as normal red blood cells. Especially, when the reticulocyte is stained based on suspension, and the cell type is identified after bright field microscopic amplification, the identification and the distinction between the reticulocyte and the normal red blood cell are difficult. This is not accurately identifiable for some pathological conditions. Especially for some anemic patients, the number of reticulocytes often means whether or not a certain disease is present or how much a certain disease is present; reticulocytes, and in particular, reticulocytes with a reduced amount of nucleic acid material, can be identified as a key point in whether such disease or severity can be identified.
In the prior art, there are also methods for staining reticulocytes, such as the standard WS/T346-2011 "reference method for reticulocyte count" in the sanitation industry of the people's republic of China, which provides a method for staining reticulocytes, wherein phosphate is directly used as buffer salt to provide an isotonic ion concentration environment, and a new methylene blue dye is used for staining, and a blood sample and a staining reagent are used as a reagent for 1:1 or 1:2 for a period of about 3 minutes to 5 minutes; the stained specimens were then smeared on a glass slide and observed. Because of the isotonic ion concentration environment, the reticulocyte dye has better effect on the reticulocyte with rich nucleic acid substance content, but the dye needs longer time which needs at least 3 minutes. As shown in FIG. 2, the effect of dyeing the reticulocytes based on the traditional liquid-based dyeing reagent is schematically shown, and the dyeing process requires at least 3 minutes and has low efficiency.
In the prior art, a staining reagent for staining cells in a state of a cell suspension is also provided in a patent application having the name "CN2020112230298" entitled "cell staining reagent and cell staining method". Although the dyeing reagent can realize better dyeing effect of white blood cells, the dyeing effect of reticulocytes in blood cells is not ideal; that is, staining of reticulocytes cannot be completed. In the prior art, when the blood cell suspension is dyed by adopting a dyeing agent with a conventional formula, white blood cells are usually dyed, but reticulocytes are not dyed yet, and are insufficient for bright field identification of the reticulocytes.
Noun interpretation:
nucleic acid material refers to a material having genetic properties, i.e., DNA or RNA, or fragments thereof, that is present within a cell.
The standard WS/T346-2011 "reference method for reticulocyte count" of the health industry of the people's republic of China is described in detail in the following websites:
“https://ebook.chinabuilding.com.cn/zbooklib/bookpdf/probationSiteID＝1&bookID＝126960”。
EDTA salt refers to ethylenediamine tetraacetate; ethylenediamine tetraacetate may bind to calcium in blood to form complex compounds, thereby inhibiting blood coagulation. EDTA salts are routinely used as blood anticoagulants.
Osmotic pressure: separating a semipermeable membrane, wherein one side is water for dissolving enzyme, the other side is solution, and the water permeates to one side of the solution through the semipermeable membrane; the pressure applied on the solution side to prevent the movement of water is called osmotic pressure. The movement of water is stopped by the pressure equaling the chemical potential of the water passing through the membrane. Osmotic pressure is expressed in osmolality as a unit, which is typically expressed in milliosmoles of solute per kilogram of solvent.
The CAS numbers of the partial substances in this application are as follows:
(CASRegistryNumber or CASNumber, CASRn, CAS #), also known as CAS registry number or CAS number
CAS registry number is a substance (compound, polymer material, biological sequence)
(biologicals), mixtures or alloys).
The water in this application is purified water. Purified water is water meeting the standard of purified water in Chinese pharmacopoeia (2015 edition), and the main parameters are as follows: the water with the conductivity of not more than 25 mu S/cm (micro Siemens per cm) is detected by an off-line conductivity meter at the temperature of 25 ℃ when the mark loading amount is not more than 10ml (milliliter).
Disclosure of Invention
In order to avoid the defect that reticulocytes cannot be dyed and identified in the prior art, particularly the reticulocytes with low nucleic acid content cannot be fully dyed, a dyeing reagent kit capable of efficiently realizing the full dyeing of the reticulocytes, particularly the reticulocytes with a small amount of nucleic acid substances, and a preparation method and a dyeing method thereof are provided. The technical scheme for solving the technical problems is that the reticulocyte staining reagent set 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; 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. In the low-permeability environment of the dyeing reagent A, the osmotic pressure molar concentration ranges from 140mosmol/kg to 210mosmol/kg.
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.
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.
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.
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.
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。
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。
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。
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.
The technical scheme of this application solution above-mentioned technical problem still can be a reticulocyte staining kit, includes: a first container and a second container; the first container comprises the dyeing reagent A; the stabilizing reagent B is contained 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.
The technical solution for solving the above technical problems may also be a reticulocyte staining method, including, 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 ranges from 20 seconds or more, and the time may be other numbers in the range of 20 seconds to 120 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 dyeing reagent A is the dyeing reagent A; the stabilizing reagent B is the stabilizing reagent B.
One of the technical effects is as follows: the unique double-step reagent formula and method are adopted, the quick dyeing of reticulocytes is realized in a step-by-step mode, and the dyeing effect can be maintained for a long time to reach more than 120 minutes; the hypotonic weak alkaline staining reagent A and the hypotonic weak alkaline stabilizing reagent B are added at specific time intervals, so that a proper staining time window is provided for staining nucleic acid substances in reticulocytes. The hypotonic weak alkaline environment and the dye combination mode based on the mutual attraction of positive and negative charges enable the dye to quickly enter the reticulocytes to complete the dyeing of nucleic acid substances within M seconds; and reaches a near equilibrium state after M seconds; the range of M is 20 seconds or more, namely 20 seconds or more, so that the reticulocyte can be fully dyed. After the sample is mixed with the staining reagent for M seconds, and the stabilizing reagent B is added in the state, aldehyde-containing substances in the dyeing promoting stabilizer B10 are used for chemically crosslinking proteins on the surface of a cell membrane, so that the cell membrane is fixed, the staining effect and the cell morphology of the cell are easier to keep, the staining effect can be kept for a long time, and the staining effect can be kept even after 120 minutes; and the subsequent microscopic amplification imaging under the suspension state is convenient for identifying the imaged reticulocytes. Solves the problems that the dye is easy to dilute and dilute in the suspension state based on the entropy increasing principle, and the stable dyeing state is difficult to maintain. The reagent has no surfactant, and the cell membrane is not perforated by the surfactant, so that the shape is easier to maintain; and the reagent is also free of permeabilizing agent; the cell membrane is not perforated by the permeabilizing agent and is also easier to maintain in its morphology in the hypotonic state.
The second technical effect is: in the hypotonic weak alkaline staining reagent A, an atmosphere very suitable for being stained is provided for reticulocytes; the weak alkalinity, especially the pH value of 7-8, can make the phosphoric acid group in the reticulocyte present the characteristic of negatively charged, therefore can combine with positively charged basic dye, this dye combination mode based on the mutual attraction of positive and negative charges, make the nucleic acid substance in the reticulocyte complete the dyeing and have the agglomeration effect at the same time, it is easier to be seen and recognized, especially suitable for the cell dyeing of only a small part of nuclear substance such as the reticulocyte; the acid-base characteristic of the basic dye is just combined with the phosphoric acid group with acidity to form stable combination, so that the dyeing effect can be kept for a longer time. The above two aspects lead to good dyeing and combining effects on nucleic acid substances with low concentration, thereby being capable of showing sufficient dyeing effects on reticulocytes in various states. The problems that reticulocytes with low nucleic acid substance content cannot be dyed or are insufficiently dyed in the prior art are avoided. Provides a staining reagent and a method which can sufficiently stain reticulocytes with low nucleic acid content; the dyeing can fully dye the reticulocytes in different states, so that the recognition rate of the reticulocytes can be improved, a more accurate reticulocyte quantity basis can be provided for early diagnosis of some diseases, and the method is a key path for recognition of next-generation reticulocytes.
Third technical effect: the hypotonic weak alkaline staining reagent A and the hypotonic weak alkaline stabilizing reagent B both provide hypotonic environment for cell staining, so that cell membranes slightly expand, and correspondingly, the boundaries between nucleic acid substances inside the cell membranes and the cell membranes are easier to distinguish; the groups after the dye and the phosphate groups are combined based on the mutual attraction of positive and negative charges are also more easily highlighted, so that the expression of dyeing is more sufficient.
Fourth, technical effects: the aldehyde-containing substances in the dyeing promoter B10 can react with proteins on the surface of a cell membrane to form a colloid aggregation layer, so that the cell morphology is stabilized and fixed, and the cell morphology recognition of various subsequent cells is facilitated.
Fifth technical effect: ethylenediamine tetraacetic acid salt; generally, edetate salts bind to calcium ions in the blood to form complex compounds, thereby inhibiting blood coagulation and thus are conventional anticoagulants. In the formulation of the present application, however, the anticoagulation promoting agent A20 does not play an anticoagulation role, but rather plays a role in promoting the staining of nucleic acid substances in reticulocytes.
Drawings
FIG. 1 is a schematic illustration of the steps of the reticulocyte staining method of the present application;
FIG. 2 is a schematic diagram of the effect of reticulocyte staining after staining based on a conventional liquid-based staining reagent;
FIG. 3 is a schematic table of the components of various examples and comparative examples based on reticulocyte staining reagent sets;
FIG. 4 is a two-part picture included therein; the upper part of the pictures in fig. 4 is a picture set of a part of reticulocytes observed by microscopic magnification of a sample after staining for 20 seconds by applying example 1, example 2, and example 3 to canine blood cells according to the reticulocyte staining method in the present application, in which it is seen that the reticulocytes have been stained; the lower part of the pictures in fig. 4 are the picture sets of the reticulocytes of the parts of the samples after 120 minutes of staining of the canine blood cells, wherein the staining effect of the reticulocytes can be continuously maintained;
FIG. 5 is a two-part picture included therein; the upper part of the pictures in fig. 5 is a picture set of a part of reticulocytes observed by microscopic magnification of a stained sample after 20 seconds of staining of cat blood cells according to the reticulocyte staining method in the present application, in which it is seen that the reticulocytes have been colored; the lower part of the pictures in fig. 5 is a picture set of reticulocytes of a part of the samples after 120 minutes of staining of the blood cells of the cats, which is observed by microscopic magnification after the staining of the blood cells of the cats, and the staining effect of the reticulocytes can be maintained.
Detailed Description
The present application is described in further detail below in conjunction with the various figures. The following description of the preferred embodiments of the present invention is not intended to limit the present invention. The description of the preferred embodiments of the present invention is merely illustrative of the general principles of the invention. The numbers "first", "second" and "a", "B" in the present invention are for convenience of description and do not represent a time or space sequential relationship.
As shown in fig. 3, in the schematic component tables based on the various examples and comparative examples of the reticulocyte staining reagent set, examples of the reticulocyte staining reagent set include 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; 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. In the low-permeability environment of the dyeing reagent A, the osmotic pressure molar concentration ranges from 140mosmol/kg to 210mosmol/kg.
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.
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.
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.
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.
In staining reagent a, the conditioning salt C10 comprises NaCl; naCl concentration in staining reagent A was 2.5X10 -2 mol/L～3.5×10 -2 mol/L; or the regulating salt C10 comprises KCl; KCl concentration in staining reagent A was 2.5X10 -2 mol/L～3.5×10 - 2 mol/L。
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。
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 The mol/L of the catalyst is equal to the mol/L, potassium dihydrogen phosphate concentration was 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。
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.
In an embodiment of a reticulocyte staining kit not shown in some of the figures, the kit comprises: a first container and a second container; including a staining reagent a in a first container; including stabilizing reagent B 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.
In one embodiment of a method of staining reticulocytes as shown in FIG. 1, the method includes the steps of 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 ranges from 20 seconds to 120 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 staining reagent A to the stabilizing reagent B is 2:3.
As shown in fig. 4, the upper part of the picture in fig. 4 is a picture set of a part of reticulocytes observed by microscopic magnification of a sample after staining for 20 seconds by applying example 1, example 2, and example 3 to canine blood cells according to the reticulocyte staining method in the present application, and it can be seen that the reticulocytes have been stained; the lower part of the pictures in fig. 4 are the picture sets of the reticulocytes of the parts of the samples after 120 minutes of staining of the canine blood cells, which are observed by microscopic magnification after the staining of the canine blood cells, in example 1, example 2 and example 3, and the staining effect of the reticulocytes can be maintained.
As shown in fig. 5, the upper part of the picture in fig. 5 is a picture set of a part of reticulocytes observed by microscopic magnification of a sample after staining for 20 seconds by applying example 11, example 12, and example 13 to cat blood cells according to the reticulocyte staining method in the present application, and it can be seen that the reticulocytes have been stained; the lower part of the pictures in fig. 5 is a picture set of reticulocytes of a part of the samples after 120 minutes of staining of the blood cells of the cats, which is observed by microscopic magnification after the staining of the blood cells of the cats, and the staining effect of the reticulocytes can be maintained.
While the invention has been illustrated and described in terms of a preferred embodiment and several alternatives, the invention is not limited by the specific description in this specification. Other alternative or equivalent components may also be used in the practice of the present 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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