CN115615786A - Kit for staining megakaryocyte of bone marrow smear, staining method and application - Google Patents

Kit for staining megakaryocyte of bone marrow smear, staining method and application Download PDF

Info

Publication number
CN115615786A
CN115615786A CN202211271162.XA CN202211271162A CN115615786A CN 115615786 A CN115615786 A CN 115615786A CN 202211271162 A CN202211271162 A CN 202211271162A CN 115615786 A CN115615786 A CN 115615786A
Authority
CN
China
Prior art keywords
staining
fast red
reagent
agent
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202211271162.XA
Other languages
Chinese (zh)
Inventor
邢雅南
李三华
米贯勋
张林姿
牛银银
齐华
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Henan Celnovtebio Biotechnology Inc
Original Assignee
Henan Celnovtebio Biotechnology Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Henan Celnovtebio Biotechnology Inc filed Critical Henan Celnovtebio Biotechnology Inc
Priority to CN202211271162.XA priority Critical patent/CN115615786A/en
Publication of CN115615786A publication Critical patent/CN115615786A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • 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
    • 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
    • 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
    • G01N2001/305—Fixative compositions

Abstract

The invention relates to the technical field of staining of bone marrow smears, in particular to a kit for staining small megakaryocytes of a bone marrow smear, a staining method and application thereof. The kit for staining the small megakaryocytes of the bone marrow smear comprises a primary anti-reagent, a secondary anti-reagent and a staining agent, wherein the primary anti-reagent comprises at least one of a CD41 monoclonal antibody, a CD42b monoclonal antibody or a CD61 monoclonal antibody, the secondary anti-reagent comprises a secondary anti-polymer marked by alkaline phosphatase, and the staining agent comprises a fast red staining reagent and a hematoxylin counterstaining reagent. The kit is adopted to dye bone marrow cells, and the dyeing result shows that the kit provided by the invention has the advantages of strong dyeing specificity, accurate and clear signal expression, no background non-specific dyeing problem and convenient and rapid dyeing method.

Description

Kit for staining megakaryocyte of bone marrow smear, staining method and application
Technical Field
The invention relates to the technical field of staining of bone marrow smears, in particular to a kit for staining small megakaryocytes of a bone marrow smear, a staining method and application thereof.
Background
Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal diseases of the myeloid lineage that originate from hematopoietic stem cells and is characterized by abnormal development of myeloid cells, manifested by ineffective hematopoiesis, refractory cytopenia, and high risk of transformation into Acute Myeloid Leukemia (AML). MDS is mainly characterized by three pathogenic states, granulocytic, erythrocytic and megakaryocytic, while megakaryocytic disease is mainly characterized by a marked increase in microscopic megakaryocytes, especially lymphoid small megakaryocytes.
The micro megakaryocytes are small-sized megakaryocytes with abnormal morphology, are found in various hematopathy, and are one of characteristics of myelodysplastic hematopoiesis. At present, pathological megakaryocytes have been listed as one of the diagnostic indicators of MDS. Because of small volume and various forms, the megakaryocytes are difficult to identify and identify clinically by morphological routine Rui-Geimsa staining, are easy to be confused with lymphocytes, immature erythrocytes and the like, and have high omission factor. Therefore, the special staining method or the staining method of megakaryocyte immunohistochemistry is used for staining the small megakaryocytes, and the small megakaryocytes are positive, thereby having important significance for diagnosing and typing myelodysplastic syndromes.
Myelodysplastic syndrome is explicitly mentioned in chinese diagnostic and treatment guidelines (2019 edition): patients suspected of being MDS should undergo Gomori silver staining and in situ Immunohistochemistry (IHC), and commonly used detection markers include CD34, MPO, GPA, CD61, CD42, CD68, CD20 and CD3. The cytomorphological examination result indicates that the megakaryocyte is pathologically hematopoietic, and a certain proportion of small and micro megakaryocytes are found by immunocytochemical staining detection. The 2010 international MDS blood pathology working meeting suggests that 1-2 megakaryocyte markers including CD42b, CD61, CD31 and CD41 are selected from bone marrow paraffin sections.
Currently, bone marrow smear staining methods adopted in the market include an SP method, an SAP staining method, an APAAP staining method, an avidin-biotin-peroxidase technique, and the like, and all of these methods have certain defects, such as complicated steps, the need of multi-step antibody incubation, long operation time, the problem of binding with endogenous biotin, and the increase of non-specific background staining while enhancing positive signals.
CN104865377A provides a kit for staining of bone marrow smear micromegakaryocytes marked with a combination of CD41 and CD61 antibodies. The method has the following defects: (1) the two antibodies, namely CD41 and CD61, are marked in a cocktail way to carry out immunohistochemical staining, so that the steps are complicated, and the sensitivity and the specificity are difficult to obtain; (2) the prepared bone marrow blood smear sample is not fixed, the inherent form and structure of cells are difficult to maintain better, and the antigen is likely to disperse or inactivate; (3) the bone marrow blood smear sample is subjected to antigen thermal repairing by using the antigen repairing liquid, on one hand, the fixed bone marrow blood smear sample is not used, the cell morphology is extremely easy to damage by high-temperature thermal repairing, and the cell is detached, on the other hand, the heating and cooling time is long, heating equipment is required, and the process is complicated; (4) by adopting a DAB color development system marked by horseradish peroxidase, endogenous peroxidase cannot be completely eliminated by 3% hydrogen peroxide sealing treatment, so that the final dyeing result has non-specific staining of neutrophils, and the accurate interpretation of the result is influenced.
Disclosure of Invention
In view of this, the technical problem to be solved by the present invention is to provide a staining kit for bone marrow smear megakaryocytes, a staining method and applications thereof, and the present invention provides the staining kit and the staining method which have the advantages of high detection sensitivity, good specificity and simple operation, and simultaneously avoid the background non-specific staining problem.
The invention provides a staining kit for bone marrow smear megakaryocytes, which comprises: a primary antibody reagent, a secondary antibody reagent, and a staining agent;
the primary anti-agent comprises an antibody and an antibody diluent, wherein the antibody is selected from at least one of a CD41 monoclonal antibody, a CD42b monoclonal antibody or a CD61 monoclonal antibody;
the secondary antibody reagent comprises a secondary antibody polymer marked by alkaline phosphatase;
the staining agent comprises a fast red color developing agent and a hematoxylin counterstaining agent.
The CD41 monoclonal antibody, the CD42b monoclonal antibody or the CD61 monoclonal antibody is beneficial to the identification of megakaryocytes, can obviously improve the detection rate of the tiny megakaryocytes by matching with an Alkaline Phosphatase (AP) labeled secondary antibody reagent, a fast red staining reagent and a hematoxylin counterstaining reagent, and has important clinical value for early diagnosis and differential diagnosis of MDS.
In the embodiment of the invention, the reagent composition in the kit is as follows:
Figure BDA0003893772720000021
in the present invention, the antibody diluent comprises water, tris-HCl, bovine serum albumin, sodium chloride, a nonionic surfactant, a fruit green pigment and a bacteriostatic agent. Compared with other antibody diluents, the antibody diluent provided by the invention provides a more stable working environment for the antibody, and can ensure the stability and the specificity of the antibody for a longer time, so that a more accurate technical effect is obtained.
In some embodiments, the nonionic surfactant is Tween-20 and the bacteriostatic agent is ProClin950. Compared with other reagents, tween-20 and ProClin950 are respectively used as a surfactant and a bacteriostatic agent to be applied to an antibody diluent, have good compatibility with the antibody, and can be more effectively matched with the antibody and other reagents to finish detection, so that more accurate and more sensitive technical effects are obtained.
Further preferably, the antibody diluent consists of water and 6g/L Tris-HCl, 15g/L bovine serum albumin, 8.5g/L sodium chloride, 0.1vol% Tween-20, 0.01wt% physalin and 0.15wt% ProClin950, and the pH of the antibody diluent is 7.60 +/-0.05. Experiments have shown that at this concentration and pH, the antibody diluent is most compatible with the antibody.
In the invention, the secondary antibody reagent comprises water, alkaline phosphatase-labeled secondary antibody polymer, tris, a protein protective agent, sodium chloride, tween-20, a bacteriostatic agent, an enzyme protective agent and a stabilizing agent IV. Compared with other reagents, the secondary antibody reagent can be matched with the primary antibody reagent and other reagents more specifically and stably, so that more accurate and sensitive technical effects can be obtained.
In some embodiments, the protein protectant is bovine serum albumin, the enzyme protectant is a metal ion, the enzyme protectant includes one or more of a mixture of zinc ions, magnesium ions, and calcium ions, the stabilizer iv is glycerol, and the bacteriostatic agent is ProClin950. Compared with other reagents, bovine serum albumin, glycerol and ProClin950 are respectively applied to the secondary antibody reagent as a protein protective agent, a stabilizing agent and a bacteriostatic agent, have good compatibility with the antibody, can more effectively cooperate with the antibody and other reagents to complete detection, and further obtain more accurate and sensitive technical effects.
Further preferably, the secondary antibody reagent is prepared from water and 8-12 mug/mL alkaline phosphatase labeled anti-rabbit polymer, 6g/L Tris, 10g/L bovine serum albumin, 8.5g/L sodium chloride, 3g/L Tween-20, 0.1wt% ProClin950, 0.1-1 mM enzyme protective agent and 40vol% glycerol, the enzyme protective agent is metal ions, the enzyme protective agent comprises one or more of zinc ions, magnesium ions and calcium ions, and the pH value of the secondary antibody reagent is 7.20 +/-0.05. Experiments show that at this concentration and pH, the specific binding of the secondary antibody reagent to the primary antibody reagent is optimal.
Still further preferably, the zinc ions are derived from zinc chloride and the magnesium ions are derived from magnesium chloride.
In the present invention, the fast red staining reagent comprises: fast red color developing agent, fast red activating agent and fast red buffer solution;
the fast red color developing agent comprises an aromatic primary amine compound and inorganic strong acid, wherein the aromatic primary amine compound is any one of basic fuchsin, new fuchsin and 3-amino-4-methoxybenzamide, and the inorganic strong acid is any one of hydrochloric acid, hydrobromic acid, fluoboric acid, sulfuric acid and nitric acid;
the fast red activating agent is a nitro reagent;
the fast red buffer solution comprises naphthol phosphate, tris, sodium chloride, a magnesium ion activator, an endogenous alkaline phosphatase inhibitor, a scale remover and a bacteriostatic agent.
Compared with other reagents, the fast red color developing agent provided by the invention is used together with the primary antibody reagent, the secondary antibody reagent and other reagents, so that the fast red color developing agent can be more effectively matched with antibodies to realize dyeing; the fast red activator and the fast red buffer provide a stable staining environment for the primary anti-reagent, the secondary anti-reagent, and the stain. The matching dyeing among the components obtains the technical effects of more accuracy and sensitivity and small background dyeing problem.
In some specific embodiments, in the fast red developer, the aromatic primary amine compound is 3-amino-4-methoxybenzamide, and the inorganic strong acid is hydrochloric acid;
the fast red activating agent is sodium nitrite, and compared with other reagents, the activating effect of the fast red developing agent participated by the sodium nitrite is more obvious;
in the fast red buffer solution, the magnesium ion activator is MgCl 2 ·6H 2 O, the endogenous alkaline phosphatase inhibitor is levamisole hydrochloride, the scale remover is Tween-20, the bacteriostatic agent is ProClin950, and compared with other reagents, mgCl 2 ·6H 2 The O, the levamisole hydrochloride, the Tween-20 and the ProClin950 participate in the fast red buffer solution, so that the dyeing environment is more stable, the compatibility with the antibody is good, and the antibody and other reagents can be more effectively matched to complete detection, thereby obtaining more accurate and more sensitive technical effects.
Further preferably, the fast red color developing agent comprises 20.0-40.0 g/L of primary arylamine compound and 0.5-1M of inorganic strong acid, wherein the primary arylamine compound is any one of basic fuchsin, new fuchsin and 3-amino-4-methoxybenzamide, and the inorganic strong acid is any one of hydrochloric acid, hydrobromic acid, fluoboric acid, sulfuric acid and nitric acid;
the concentration of the sodium nitrite is 8.28-16.56 g/L.
The fast red buffer solution comprises 2-3M naphthol phosphate, 12.1g/L Tris, 5.85g/L sodium chloride and 2.03g/L MgCl 2 ·6H 2 O, 0.24g/L levamisole hydrochloride, 0.3vol% Tween-20 and 0.5wt% ProClin950, wherein the naphthol phosphate is any one of naphthol AS-TR phosphate, naphthol AS-TR disodium phosphate and naphthol AS-BI phosphate disodium hydrate.
Experiments show that the fast red staining reagent has the best staining effect at the concentration.
In the invention, the hematoxylin counterstain reagent comprises hematoxylin, aluminum potassium sulfate, an oxidant II and a stabilizer V. Compared with other reagents, the hematoxylin counterstain reagent provided by the invention is used together with the primary antibody reagent, the secondary antibody reagent and other reagents, and the finally obtained cell nucleus counterstain effect is good, so that the technical effects of more accuracy, more sensitivity and small background staining problem are obtained.
In some embodiments, the oxidizing agent ii is sodium iodate and the stabilizing agent V is glycerol. Compared with other reagents, the sodium iodate and the glycerol which are used as the oxidizing agent and the stabilizing agent are applied to the hematoxylin counterstain reagent, the staining sensitivity is higher, and the staining effect is clearer and more distinct.
Further preferably, the hematoxylin counterstain reagent comprises 5g/L hematoxylin, 50g/L aluminum potassium sulfate, 0.5g/L sodium iodate and 5vol% of glycerol. Experiments show that the hematoxylin counterstain reagent has the best nuclear staining effect at the concentration.
The kit for staining the bone marrow smear megakaryocytes also comprises a fixing agent and a cleaning solution, wherein:
the fixing solution comprises methanol and acetone;
the cleaning solution comprises water, tris, sodium chloride, a surfactant and a preservative.
Compared with other reagents, the fixing solution provided by the invention has a good fixing effect on bone marrow cells, and the bone marrow cells fixed by the fixing solution are clearer in structure and more bright in dyeing when being matched with the fast red dyeing reagent; the cleaning solution provided by the invention has a good cleaning effect on the primary antibody reagent and the secondary antibody reagent, can effectively reduce the unnecessary background staining problem, and enhances the signal to noise ratio.
In some specific embodiments, in the cleaning solution, the surfactant is Tween-20, and the preservative is Proclin950.
Further preferably, the fixing solution comprises methanol and acetone in a volume ratio of 1; in the cleaning solution, the concentration of each component is 60g/L Tris,87g/L sodium chloride, 0.25vol% Tween-20 and 0.4wt% Proclin950. Experiments show that the fixing effect of the fixing liquid is best under the concentration, and the cleaning capability of the cleaning liquid is best when the cleaning liquid is diluted by 10 times in specific experiments.
In the present invention, the volume ratio of the primary antibody reagent to the secondary antibody reagent is 1, and at this volume ratio, the bone marrow cells, the primary antibody reagent and the secondary antibody reagent have the best binding capacity, so as to obtain a more accurate and sensitive technical effect.
In the invention, in the fast red staining reagent, the volume ratio of the fast red color developing agent, the fast red activating agent and the fast red buffer solution is 1.
The invention also provides a staining method of the bone marrow smear megakaryocytes, which stains the cells by adopting the staining kit of the bone marrow smear megakaryocytes.
Preferably, the dyeing method comprises the following steps:
step 1, preparing a bone marrow blood smear and fixing;
step 2, incubating by using the primary antibody reagent;
step 3, incubating with the secondary antibody reagent;
step 4, dyeing by using the fast red dyeing reagent;
and 5, counterstaining by adopting the hematoxylin counterstaining reagent.
More preferably, the bone marrow blood smear is fixed with the fixative.
More preferably, the primary antibody reagent is 80-150 μ L, and the incubation time is 30-60 min.
More preferably, the secondary antibody reagent is 80 to 150 μ L, and the incubation time is 20 to 30min.
More preferably, in the fast red staining reagent, the volume ratio of the fast red color developing agent to the fast red activating agent to the fast red buffer solution is 1.
More preferably, the hematoxylin counterstaining reagent is 80-150 mu L, and the counterstaining time is 3-5 min.
More preferably, the loading volume of the primary antibody reagent and the secondary antibody reagent is 1.
Further preferably, the dyeing method comprises the following steps:
step 1, fixing a bone marrow blood smear by using the fixing agent, wherein the fixing solution comprises methanol and acetone in a volume ratio of 1;
step 2, incubating 80 mu L of the primary antibody reagent at 35 ℃ for 60min, and cleaning by using the cleaning solution;
3, incubating the second antibody reagent by 80 mu L at 35 ℃ for 30min, and cleaning by using the cleaning solution;
step 4, adopting 80 mu L of the fast red staining reagent, incubating for 8min at room temperature, and washing with water;
and 5, adopting 80 mu L of hematoxylin counterstaining reagent, counterstaining for 3min at room temperature, and washing with water.
The present invention also provides a method for diagnosing myelodysplastic syndrome, comprising: the kit for staining the megakaryocytes of the bone marrow smear or the method for staining the megakaryocytes of the bone marrow smear are adopted to detect the sample.
The invention adopts a CD41 monoclonal antibody, a CD42b monoclonal antibody and a CD61 monoclonal antibody to be combined with a secondary antibody polymer marked by Alkaline Phosphatase (AP), and adopts a fast red staining reagent and a hematoxylin counterstaining reagent to stain the marrow cells. Compared with the prior art, the staining kit provided by the invention has the advantages of strong specificity of staining results, accurate and clear signal expression, no background non-specific staining problem, and convenient and fast staining method. The kit provided by the invention carries a full-automatic immunohistochemical staining instrument, does not need manual intervention, is simple and convenient to operate, has good staining stability, and greatly improves the staining efficiency and the laboratory standardization level
Drawings
FIG. 1 is a graph showing the results of staining a megakaryocyte cell line in example 1;
FIG. 2 is a graph showing the result of staining a sample of a bone marrow blood smear in example 2;
FIG. 3 is a graph showing the result of staining a sample of a bone marrow blood smear in example 3;
FIG. 4 is a graph showing the result of staining a sample of a bone marrow blood smear in example 4;
FIG. 5 is a graph showing the result of staining a sample of a bone marrow blood smear in example 5;
FIG. 6 is a graph showing the result of staining a sample of a bone marrow blood smear in example 6;
FIG. 7 is a graph showing the result of staining a sample of a bone marrow blood smear in comparative example 1;
FIG. 8 is a graph showing the result of staining a sample of a bone marrow blood smear in comparative example 2;
FIG. 9 is a graph showing the staining results of a bone marrow blood smear sample in comparative example 3, wherein a is the staining result observed under a low power mirror (10X) and b is the staining result observed under a high power mirror (20X);
FIG. 10 is a graph showing the result of staining a sample of a bone marrow blood smear in comparative example 4;
FIG. 11 is a graph showing the result of staining a sample of a bone marrow blood smear in comparative example 5;
FIG. 12 is a graph showing the result of staining a bone marrow blood smear sample in comparative example 6, which was derived from the same source as in comparative example 3;
FIG. 13 is a graph showing the staining results of CD41HRP-AEC of bone marrow blood smears in Experimental example 1, which is similar to the staining results of CD41HRP-AEC of bone marrow blood smears with different concentrations of hydrogen peroxide blocking agent and blocking times, wherein a is a schematic view of megakaryocyte staining and b is a schematic view of non-specific granulocyte staining;
FIG. 14 is a graph showing the staining results of CD41HRP-AEC of bone marrow smear without adding horseradish peroxidase-labeled secondary antibody in Experimental example 2, which was similar to the staining results of CD41HRP-AEC of bone marrow smear with different concentrations of hydrogen peroxide blocking agent and blocking time.
Detailed Description
The invention provides a kit for staining a bone marrow smear megakaryocyte, a staining method and application thereof, and a person skilled in the art can appropriately improve process parameters by referring to the content in the text to realize the staining. It is specifically noted that all such substitutions and modifications will be apparent to those skilled in the art and are intended to be included herein. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market. Wherein:
AP-anti-rabbit polymer (Henan Sainut Biotechnology Co., ltd., concentration 8 to 12. Mu.g/mL).
CD41 rabbit anti-human monoclonal antibody (Abcam, clone number: EPR4330, antibody titer 1
CD42b Rabbit anti-human monoclonal antibody (Henan Sainuo Biotechnology Co., ltd., clone No. EP409, antibody titer 1
CD61 rabbit anti-human monoclonal antibody (Abcam, clone number: ERP17507, antibody titer 1
The kit for staining the bone marrow smear megakaryocytes comprises the following specific components:
Figure BDA0003893772720000061
wherein the antibody diluent comprises water, tris-HCl, bovine serum albumin, sodium chloride, a nonionic surfactant, a fruit green pigment and a bacteriostatic agent, the nonionic surfactant is Tween-20, and the bacteriostatic agent is ProClin 950;
in the secondary antibody reagent, the alkaline phosphatase-labeled secondary antibody polymer is specifically an AP-rabbit resistant polymer, the protein protective agent is bovine serum albumin, the enzyme protective agent is one or a mixture of metal ions, zinc ions, magnesium ions and calcium ions, the stabilizer IV is glycerol, and the bacteriostatic agent is ProClin 950;
in the fast red color developing agent, the primary aromatic amine compound is any one of basic fuchsin, new fuchsin and 3-amino-4-methoxybenzamide, and the strong inorganic acid is any one of hydrochloric acid, hydrobromic acid, fluoroboric acid, sulfuric acid and nitric acid;
in the fast red activating agent, the nitro reagent is sodium nitrite;
in the fast red buffer solution, the naphthol phosphate is naphthol AAny one of S-TR phosphate, naphthol AS-TR disodium phosphate and naphthol AS-BI phosphate disodium hydrate, and the magnesium ion activator is MgCl 2 ·6H 2 O, taking levamisole hydrochloride as an endogenous alkaline phosphatase inhibitor, tween-20 as a scale remover and ProClin950 as a bacteriostatic agent;
in the hematoxylin counterstain reagent, an oxidant II is sodium iodate, and a stabilizing agent V is glycerol.
In the cleaning solution, the surfactant is Tween-20, and the preservative is Proclin950.
The invention also provides a dyeing method carried with the full-automatic immunohistochemical staining instrument, which comprises the following steps:
1) Sample preparation
Bone marrow cells or peripheral blood smears are prepared.
2) Fixing
Fixing for 90 seconds by adopting a fixing agent, and naturally drying to obtain a smear to be dyed.
3) Primary antibody incubation
Adding a primary antibody reagent, wherein the primary antibody reagent and the secondary antibody reagent are incubated at the temperature of 1 and 35 ℃ for 30-60 min in the volume ratio to be combined with antigen protein in the tissue section.
Note: the first anti-reagent comprises a CD41 rabbit anti-human monoclonal antibody, a CD42b rabbit anti-human monoclonal antibody and a CD61 rabbit anti-human monoclonal antibody.
4) Incubation with secondary antibody
Adding a secondary antibody reagent, and incubating for 20-30 min at 35 ℃ to ensure that the primary antibody is specifically combined with the secondary antibody.
Note: the Alkaline Phosphatase (AP) -labeled secondary antibody polymer is: the polymer AP-GAR (goat anti-rabbit IgG), the second antibody is an antibody capable of binding with the antibody, namely the antibody, and the second antibody is labeled with Alkaline Phosphatase (AP), and the main functions of the second antibody are to detect the existence of the antibody, amplify the signal of the first antibody and introduce a color development system.
5) Fast red color development
Adding fast red staining reagent of 100 μ L, incubating at room temperature for 8-15 min, and presenting in the form of red precipitate.
Note: in the fast red staining reagent, the volume ratio of the fast red developer, the fast red activator and the fast red buffer is 1. In addition, the room temperature referred to in the present application may be 18 to 30 ℃, and 25 ℃ is preferable in the specific experiment.
6) Hematoxylin counterstain
Adding hematoxylin counterstain reagent to counterstain for 3-5 min at room temperature and lining cell nucleus.
Megakaryocytes can be further marked by immunocytochemical staining of bone marrow blood smears. Qualitative abnormalities include nonlobular/lowlobular and normal-sized megakaryocytes, nonlobular or nonlobular small megakaryocytes, micromegakaryocytes, multinucleate megakaryocytes, highly lobular large megakaryocytes, cytoplasmic abnormal megakaryocytes, and damaged megakaryocytes. The immunostaining for CD41, CD42b and CD61 has important clinical value for identifying different forms of megakaryocyte abnormalities. Other abnormal megakaryocyte patterns were also detected in this study, but the megakaryocytes had a clear association with MDS. Therefore, CD41, CD42b, CD61 immunostaining is a good aid for megakaryocyte analysis.
Cell morphology positive for CD41, CD42b, CD61 marker staining: the cell membranes and cytoplasm of the megakaryocytes and the platelets show red, the positive positioning is accurate, the signal expression is clear, the dyeing result is bright and bright, and no background dyeing exists.
The technical scheme is provided with a Sonot self-research CNT330 full-automatic immunohistochemical staining instrument, supports continuous operation, three independent slide racks and 30 slides, can be continuously replaced, can immediately execute the next batch of experiments after one batch is finished, can randomly edit various immunohistochemical staining programs, and has 3 working platforms for use. The reagent dosage is as low as 80 mu L, and the reagent consumption is reduced. The full-automatic system including the immunohistochemical steps of primary antibody incubation, secondary antibody incubation, fast red color development, hematoxylin counterstaining and the like can be realized, manual intervention is not needed, the operation is simple and convenient, and the staining efficiency and the laboratory standardization level are greatly improved. The harmful waste liquid and the harmless waste liquid are separately treated, the principle of separately treating the toxic waste is met, and the laboratory evaluation and certification are facilitated.
The invention is further illustrated by the following examples:
example 1:
1. reagent preparation
1.1, primary antibody reagent: consists of pure water and the following components: CD41 rabbit anti-human monoclonal antibody (clone number: EPR4330, antibody titer 1;
1.2, secondary antibody reagent: consists of pure water and the following components: 8 μ g/mL AP-anti-rabbit polymer, 6g/L Tris, 10g/L bovine serum albumin, 8.5g/L sodium chloride, 3g/L Tween-20, 0.1wt% ProClin950, 1mM magnesium chloride, 0.1mM zinc chloride (enzyme protectant) and 40vol% glycerol, pH = (7.20. + -. 0.05);
1.3, fast red color developing agent: consists of pure water and the following components: 20.0 g/L3-amino-4-methoxybenzamide, 0.5M hydrochloric acid;
1.4, fast red activator: consists of pure water and the following components: 8.28g/L sodium nitrite;
1.5, fast red buffer: consists of pure water and the following components: 12.1g/L Tris, 5.85g/L sodium chloride, 2.03g/L MgCl 2 ·6H 2 O, 0.24g/L levamisole hydrochloride, 0.3vol% Tween-20, 0.5wt% ProClin950 and 2M naphthol AS-TR phosphate;
1.6, hematoxylin counterstain reagent: consists of pure water and the following components: 5g/L hematoxylin, 50g/L aluminum potassium sulfate, 0.5g/L sodium iodate and 5% glycerol;
1.7, cleaning solution: consists of pure water and the following components: 60g/L Tris,87g/L sodium chloride, 0.25vol% Tween-20 and 0.4wt% Proclin950, and is diluted 10 times with pure water for specific experiments.
2. The embodiment provides a dyeing method carried with a full-automatic immunohistochemical dyeing instrument, which comprises the following steps of:
1) Sample preparation
Smear samples were prepared using the purchased megakaryocyte line.
The megakaryocyte cell line is from BNCC Beinanbiota human megakaryocyte leukemia cell with the number BNCC359444.
2) Fixing
The fixing agent is a methanol-acetone solution which is prepared at present and has a volume ratio of 1. Fixing for 90 seconds by adopting a fixing agent, and naturally drying to obtain a smear to be dyed.
3) Primary antibody incubation
Adding CD41 rabbit anti-human monoclonal antibody reagent, and incubating at 35 deg.C for 60min in 80 μ L. Washing with the cleaning solution for 5 times and 2 min/time.
4) Incubation with secondary antibody
Adding 80 μ L of secondary antibody polymer labeled with Alkaline Phosphatase (AP), and incubating at 35 deg.C for 30min to allow the primary antibody to specifically bind to the secondary antibody; washing with the cleaning solution for 5 times and 2 min/time.
5) Fast red color development
Add fast red staining reagent 80 μ L (where fast red developer: fast red activator: fast red buffer =1:20 dilution ratio), incubate for 8min at room temperature, present as a red precipitate; and 5 times of deionized water washing, 1 min/time.
6) Hematoxylin counterstain
Adding 80 μ L of hematoxylin, counterstaining at room temperature for 3min, and lining-staining cell nucleus; purified water was washed 5 times for 2 s/time. Air-drying and observing under a microscope.
Example 2:
the reagents were formulated as in example 1, wherein:
in the secondary antibody reagent, the concentration of the AP-anti-rabbit polymer is 10 mu g/mL, and the enzyme protective agent is 0.1mM zinc chloride;
fast red color developing agent: consists of pure water and the following components: 40.0 g/L3-amino-4-methoxybenzamide, 1M hydrochloric acid;
fast red activator: consists of pure water and the following components: 16.56g/L sodium nitrite;
fast red buffer: consists of pure water and the following components: 12.1g/L Tris, 5.85g/L sodium chloride, 2.03g/L MgCl 2 ·6H 2 O, 0.24g/L levamisole hydrochloride, 0.3vol% Tween-20, 0.5wt% ProClin950 and 3M Naphthol AS-TR disodium salt.
The embodiment provides a dyeing method for carrying a full-automatic immunohistochemical dyeing instrument, which comprises the following steps of:
1) Sample preparation
The bone marrow blood smears are then dried thoroughly and the prepared smears are tested within a month.
2) Fixing the device
The fixing agent is a methanol-acetone solution which is prepared at present and has a volume ratio of 1. Fixing for 90 seconds by adopting a fixing agent, and naturally drying to obtain a smear to be dyed.
3) Primary antibody incubation
Adding 100 μ L of CD41 rabbit anti-human monoclonal antibody reagent, and incubating at 35 deg.C for 60min. Washing with the cleaning solution for 5 times and 2 min/time.
4) Incubation with secondary antibody
Adding 100 μ L of Alkaline Phosphatase (AP) -labeled secondary antibody polymer, and incubating at 35 deg.C for 30min to allow specific binding of the primary antibody and the secondary antibody; the cleaning solution is washed for 5 times and 2 min/time.
5) Fast red color development
Adding 100 μ L of fast red staining reagent (fast red developer: fast red activator: fast red buffer =1:20 dilution ratio), incubating at room temperature for 8min, and presenting in the form of red precipitate; the deionized water is washed for 5 times and 1 min/time.
Hematoxylin counterstain
Adding 100 μ L hematoxylin, re-staining at room temperature for 3min, and lining-staining cell nucleus; washing with purified water 5 times (2 s/time). Air-drying and observing under a microscope.
FIG. 2 shows the result of staining of lymphoid micromegakaryocyte CD41 in the case of myelodysplastic syndrome (MDS). MDS, known as lymphoid megakaryocytes, is an important distinction between MDS and other anemia-related diseases.
Example 3:
the reagents were formulated as in example 1, wherein:
in the secondary antibody reagent, the concentration of the AP-anti-rabbit polymer is 12 mu g/mL, and the enzyme protective agent is 1mM magnesium chloride;
fast red color developing agent: consists of pure water and the following components: 40.0 g/L3-amino-4-methoxybenzamide, 1M hydrochloric acid;
fast red activator: consists of pure water and the following components: 16.56g/L sodium nitrite;
fast red buffer: consists of pure water and the following components: 12.1g/L Tris, 5.85g/L sodium chloride, 2.03g/L MgCl 2 ·6H 2 O, 0.24g/L levamisole hydrochloride, 0.3vol% Tween-20, 0.5wt% ProClin950, 3M Naphthol AS-BI phosphate disodium salt hydrate.
The embodiment provides a dyeing method for carrying a full-automatic immunohistochemical dyeing instrument, which comprises the following steps of:
1) Sample preparation
The bone marrow blood smears were then dried thoroughly and the prepared smears were tested within a month.
2) Fixing the device
The fixing agent is a methanol-acetone solution which is prepared at present and has a volume ratio of 1. Fixing for 90 seconds by adopting a fixing agent, and naturally drying to obtain a smear to be dyed.
3) Primary antibody incubation
Adding CD41 rabbit anti-human monoclonal antibody reagent 150. Mu.L, and incubating at 35 deg.C for 40min. Washing with the cleaning solution for 5 times and 2 min/time.
4) Incubation with secondary antibody
Adding 150 μ L of Alkaline Phosphatase (AP) -labeled secondary antibody polymer, and incubating at 35 deg.C for 30min to allow specific binding of the primary antibody and the secondary antibody; washing with the cleaning solution for 5 times and 2 min/time.
5) Fast red color development
Add fast red staining reagent 150 μ L (fast red developer: fast red activator: fast red buffer =1:20 dilution ratio), incubate for 10min at room temperature, present as red precipitate; and 5 times of deionized water washing, 1 min/time.
6) Hematoxylin counterstain
Adding hematoxylin 150 μ L, re-staining at room temperature for 4min, and lining-staining cell nucleus; purified water was washed 5 times for 2 s/time. Air-drying and observing under a microscope.
Example 4:
the reagents were formulated as in example 1, wherein:
in the secondary antibody reagent, the concentration of the AP-anti-rabbit polymer is 8 mu g/mL, and the enzyme protective agent is 1mM magnesium chloride and 0.1mM zinc chloride;
fast red color developing agent: consists of pure water and the following components: 40.0g/L of New fuchsin, 1M hydrochloric acid;
fast red activator: consists of pure water and the following components: 16.56g/L sodium nitrite;
fast red buffer: consists of pure water and the following components: 12.1g/L Tris, 5.85g/L sodium chloride, 2.03g/L MgCl 2 ·6H 2 O, 0.24g/L levamisole hydrochloride, 0.3vol% Tween-20, 0.5wt% ProClin950 and 3M Naphthol AS-TR disodium salt.
The embodiment provides a dyeing method carried with a full-automatic immunohistochemical dyeing instrument, which comprises the following steps of:
1) Sample preparation
The bone marrow blood smears were then dried thoroughly and the prepared smears were tested within a month.
2) Fixing the device
The fixing agent is a methanol-acetone solution which is prepared at present and has a volume ratio of 1. And (5) fixing for 90 seconds by adopting a fixing agent, and naturally drying to obtain a smear to be dyed.
3) Primary antibody incubation
Adding CD41 rabbit anti-human monoclonal antibody reagent 150. Mu.L, and incubating at 35 deg.C for 30min. Washing with the cleaning solution for 5 times and 2 min/time.
4) Incubation with secondary antibody
Adding 150 μ L of Alkaline Phosphatase (AP) -labeled secondary antibody polymer, and incubating at 35 deg.C for 20min to allow the primary antibody and the secondary antibody to bind specifically; washing with the cleaning solution for 5 times and 2 min/time.
5) Fast red color development
Add fast red staining reagent 150 μ L (fast red developer: fast red activator: fast red buffer =1:20 dilution ratio), incubate for 15min at room temperature, present as a red precipitate; the deionized water is washed for 5 times and 1 min/time.
6) Hematoxylin counterstain
Adding hematoxylin 150 μ L, counterstaining at room temperature for 5min, and lining-staining cell nucleus; purified water was washed 5 times for 2 s/time. Air-drying and observing under a microscope.
Example 5:
the reagents were formulated as in example 1, wherein:
in the secondary antibody reagent, the concentration of the AP-anti-rabbit polymer is 8 mug/mL, and the enzyme protective agent is 1mM magnesium chloride;
fast red color developing agent: consists of pure water and the following components: 20.0g/L of New fuchsin, 1M hydrochloric acid;
fast red activator: consists of pure water and the following components: 8.28g/L sodium nitrite;
fast red buffer: consists of pure water and the following components: 12.1g/L Tris, 5.85g/L sodium chloride, 2.03g/L MgCl 2 ·6H 2 O, 0.24g/L levamisole hydrochloride, 0.3vol% Tween-20, 0.5wt% ProClin950 and 2M naphthol AS-TR phosphate.
The embodiment provides a dyeing method for carrying a full-automatic immunohistochemical dyeing instrument, which comprises the following steps of:
1) Sample preparation
The bone marrow blood smears are then dried thoroughly and the prepared smears are tested within a month.
2) Fixing the device
The fixing agent is a methanol-acetone solution prepared in situ according to a volume ratio of 1. Fixing for 90 seconds by adopting a fixing agent, and naturally drying to obtain a smear to be dyed.
3) Primary antibody incubation
Adding 100. Mu.L of rabbit anti-human CD42b monoclonal antibody reagent, and incubating at 35 ℃ for 60min. The cleaning solution is washed for 5 times and 2 min/time.
4) Incubation with a second antibody
Adding 100 μ L of Alkaline Phosphatase (AP) -labeled secondary antibody polymer, and incubating at 35 deg.C for 30min to allow the primary antibody and the secondary antibody to bind specifically; the cleaning solution is washed for 5 times and 2 min/time.
5) Fast red color development
Add fast red staining reagent 100 μ L (fast red developer: fast red activator: fast red buffer =1:20 dilution ratio), incubate for 8min at room temperature, present as red precipitate; and 5 times of deionized water washing, 1 min/time.
6) Hematoxylin counterstain
Adding 100 μ L hematoxylin, re-staining at room temperature for 3min, and lining-staining cell nucleus; purified water was washed 5 times for 2 s/time. Air-drying and observing under a microscope.
Example 6:
the reagents were formulated as in example 1, wherein:
in the secondary antibody reagent, the concentration of the AP-anti-rabbit polymer is 10 mug/mL, and the enzyme protective agent is 0.1mM zinc chloride;
fast red color developing agent: consists of pure water and the following components: 40.0g/L of New fuchsin, 1M hydrochloric acid;
fast red activator: consists of pure water and the following components: 16.56g/L sodium nitrite;
fast red buffer: consists of pure water and the following components: 12.1g/L Tris, 5.85g/L sodium chloride, 2.03g/L MgCl 2 ·6H 2 O, 0.24g/L levamisole hydrochloride, 0.3vol% Tween-20, 0.5wt% ProClin950 and 3M naphthol AS-TR phosphate.
The embodiment provides a dyeing method carried with a full-automatic immunohistochemical dyeing instrument, which comprises the following steps of:
1) Sample preparation
The bone marrow blood smears are then dried thoroughly and the prepared smears are tested within a month.
2) Fixing
The fixing agent is a methanol-acetone solution prepared in situ according to a volume ratio of 1. Fixing for 90 seconds by adopting a fixing agent, and naturally drying to obtain a smear to be dyed.
3) Primary antibody incubation
Add CD61 rabbit anti-human monoclonal antibody reagent 80 u L,35 degrees C were incubated for 60min. The cleaning solution is used for washing 5 times, the first two times are 0 min/time, and the last three times are 1 min/time.
4) Incubation with secondary antibody
Adding 80 μ L of secondary antibody polymer labeled with Alkaline Phosphatase (AP), and incubating at 35 deg.C for 30min to allow the primary antibody to specifically bind to the secondary antibody; the cleaning solution is washed for 5 times, the first two times are 0 min/time, and the second three times are 1 min/time.
5) Fast red color development
Add fast red staining reagent 80 μ L (fast red developer: fast red activator: fast red buffer =1:20 dilution ratio), incubate for 8min at room temperature, present as red precipitate; the deionized water is used for rinsing for 5 times, the first two times are 0 min/time, and the last three times are 1 min/time.
6) Hematoxylin counterstain
Adding 80 μ L of hematoxylin, counterstaining at room temperature for 3min, and lining-staining cell nucleus; purified water was washed 5 times for 2 s/time. Air-drying and observing under a microscope.
The dyeing results of examples 1 to 6 are summarized in the following table.
Figure BDA0003893772720000121
Figure BDA0003893772720000131
Comparative example 1:
this comparative example provides a conventional rui-giemsa staining method comprising the steps of:
the Rui's-Jimsa staining solution used in this comparative example was purchased from Saint Biotechnology, inc. of Henan.
1) Sample preparation
After the bone marrow blood smear is made, it is quickly shaken or fanned dry in air.
2) Dripping Rui's-Jimsan A liquid
About 0.5-0.8 mL of Rayleigh-Giemsa A liquid is dripped on the smear, and the dyeing liquid is allowed to cover the whole specimen for dyeing for 1min.
3) Dripping Ruishi-Jiemsa B liquid
Dripping Raynaud-Giemsa B on the liquid A (the dripping amount is 2-3 times of the liquid A), blowing out waffle wind with a nozzle or a ear washing ball to make the liquid surface ripple, mixing the two liquids sufficiently, and dyeing for 8min.
4) Washing with water
The dye solution can not be poured out first during washing, and the dye solution is washed away by running water to prevent sediments from being deposited on the specimen, and the specimen is dried and examined under the microscope.
FIG. 7 is a graph showing the results of staining of a bone marrow blood smear by Rayleigh-Giemsa in comparative example 1, which shows blue deep staining of binuclear megakaryocytes and nuclei.
Comparative example 2:
in the comparative example, a commercially available China fir general SP kit (chain enzyme avidin-biotin method detection system, product number: SP-9000) is adopted, an HRP-DAB color development system is adopted, and the bone marrow blood smear is stained with CD41, and the manual operation steps are as follows:
1) Sample preparation
The bone marrow blood smears were then dried thoroughly and the prepared smears were tested within a month.
2) Fixing the device
The fixing agent is a methanol-acetone solution prepared in situ according to a volume ratio of 1. Fixing for 90 seconds by adopting a fixing agent, and naturally drying to obtain a smear to be dyed.
3) Blocking endogenous peroxidase
Adding 100 μ L of endogenous peroxidase blocking agent, incubating at room temperature for 10min, and washing with cleaning solution for 3 times (3 min/time).
4) Normal goat serum working solution for dropwise adding and sealing
Adding 100 μ L of blocking normal goat serum working solution, incubating at 37 deg.C for 10min, pouring off serum, and washing.
5) Primary antibody incubation
Add CD41 rabbit anti-human monoclonal antibody reagent 100 u L,37 degrees C were incubated for 60min. The cleaning solution is washed for 3 times and 3 min/time.
6) Dropwise adding biotin to mark goat anti-mouse/rabbit IgG
Adding 100 mu L of goat anti-mouse/rabbit IgG labeled with biotin, incubating at 37 ℃ for 10min, washing with a cleaning solution for 3 times and 3 min/time.
7) Dropwise adding horse radish peroxidase labeled streptokinase ovalbumin working solution
Adding 100 μ L of horseradish peroxidase labeled streptavidin working solution, and incubating at 37 deg.C for 10min. The cleaning solution is washed for 3 times and 3 min/time.
8) DAB color development
DAB substrate: DAB buffer =1 at 20 dilution ratio, incubated for 8min at room temperature, and presented as a brown precipitate; and 3 times of deionized water washing for 3 min/time.
9) Hematoxylin counterstain
Counter-staining for 3min at room temperature, lining-staining cell nucleus, washing with purified water, air-drying, and observing under microscope.
FIG. 8 is a staining result of a bone marrow blood smear sample in comparative example 2, which shows that the megakaryocyte cell membrane and the positive part of cytoplasm can be stained brown under low power microscope with a commercial SP kit (detection system of streptoenzyme-ovalbumin-biotin method), but the nuclear blue is not clear, DAB brown positive cells are not easy to be distinguished from erythrocytes, and the contrast is not as clear as when AP red staining, which is more obvious when smear is thicker, sample edge part and erythrocytes are stacked. In addition, more punctate brown stains can be seen in the visual field, which are nonspecific staining of neutrophils and are more obvious under a high power microscope.
Comparative example 3:
in the comparative example, a commercially available SP kit for Chinese fir (detection system of streptoenzyme avidin-biotin method, product number: SP-9000) was used, and an HRP-AEC color development system was used to dye a bone marrow blood smear for CD41 staining, and the manual operation steps were as follows:
1) Sample preparation
The bone marrow blood smears are then dried thoroughly and the prepared smears are tested within a month.
2) Fixing
The fixing agent is a methanol-acetone solution which is prepared at present and has a volume ratio of 1. Fixing for 90 seconds by adopting a fixing agent, and naturally drying to obtain a smear to be dyed.
3) Blocking endogenous peroxidase
Adding 100 μ L of endogenous peroxidase blocking agent, incubating at room temperature for 10min, and washing with cleaning solution for 3 times (3 min/time).
4) Normal goat serum working solution for dropwise adding and sealing
Adding 100 μ L of normal goat serum working solution for blocking, incubating at 37 deg.C for 10min, decanting, and washing.
5) Primary antibody incubation
Add CD41 rabbit anti-human monoclonal antibody reagent 100 u L,37 degrees C were incubated for 60min. The cleaning solution is washed for 3 times and 3 min/time.
6) Dropping biotin labeled goat anti-mouse/rabbit IgG
Adding 100 μ L of goat anti-mouse/rabbit IgG labeled with biotin, incubating at 37 deg.C for 10min, washing with washing solution for 3 times, and 3 min/time.
7) Dropwise adding horse radish peroxidase labeled streptokinase ovalbumin working solution
Adding 100 μ L of horseradish peroxidase labeled streptavidin working solution, and incubating at 37 deg.C for 10min. The cleaning solution is washed for 3 times and 3 min/time.
8) AEC color development
Adding 850 mu L of deionized water into a small test tube, then respectively adding 50 mu L of AEC buffer solution, 50 mu L of AEC substrate and 50 mu L of AEC chromogen, and uniformly mixing to obtain 1mL of AEC color development working solution. Incubating at room temperature for 8min, and presenting in the form of red precipitate; rinsing with deionized water for 3 times (3 min/time).
9) Hematoxylin counterstain
Counter-staining for 3min at room temperature, lining-staining cell nucleus, washing with purified water, air-drying, and observing under microscope.
FIG. 9 is a graph showing the staining results of the bone marrow smear sample in comparative example 3, wherein a is the HRP-AEC staining of megakaryocytes visible to the naked eye under a low power microscope (10X), and a more non-specific staining of granulocytes in a dotted distribution is also visible. b is nonspecific staining of granulocytes under high power lens (20X).
Comparative example 4:
this comparative example used a commercially available SP kit for Chinese fir (detection system by streptavidin-biotin method, cat # SP-9000) and an HRP-AEC color system to stain bone marrow blood smears with CD41, and this example was carried out without the addition of secondary antibodies by hand as follows:
1) Sample preparation
The bone marrow blood smears were then dried thoroughly and the prepared smears were tested within a month.
2) Fixing
The fixing agent is a methanol-acetone solution which is prepared at present and has a volume ratio of 1. Fixing for 90 seconds by adopting a fixing agent, and naturally drying to obtain a smear to be dyed.
3) Blocking endogenous peroxidase
Adding 100 μ L of endogenous peroxidase blocking agent, incubating at room temperature for 10min, and washing with cleaning solution for 3 times (3 min/time).
4) Normal goat serum working solution for dropwise adding and sealing
Adding 100 μ L of normal goat serum working solution for blocking, incubating at 37 deg.C for 10min, decanting, and washing.
5) Primary antibody incubation
Add CD41 rabbit anti-human monoclonal antibody reagent 100 u L,37 degrees C were incubated for 60min. The cleaning solution is washed for 3 times and 3 min/time.
6) AEC color development
Adding 850 mu L of deionized water into a small test tube, then respectively adding 50 mu L of AEC buffer solution, 50 mu L of AEC substrate and 50 mu L of AEC chromogen, and uniformly mixing to obtain 1mL of AEC color development working solution. Incubating at room temperature for 8min, and presenting in the form of red precipitate; and 3 times of deionized water washing for 3 min/time.
7) Hematoxylin counterstain
Counter-staining for 3min at room temperature, lining-staining cell nucleus, washing with purified water, air-drying, and observing under microscope.
Comparative examples 2, 3 and 4 CD41 immunocytochemical staining was performed using a commercially available SP kit (streptavidin-biotin detection system) HRP staining system, HRP-DAB staining, HRP-AEC staining, and HRP-AEC staining without adding a secondary antibody, respectively, and as a result, as shown in fig. 8, 9 and 10, non-specific staining of granulocytes was observed in all samples of bone marrow smears, because erythrocytes, granulocytes, monocytes and eosinophils are rich in endogenous peroxidase, and complete blocking of endogenous peroxidase of blood and myeloid leukocytes in bone marrow smears was very difficult. Therefore, the granulocyte is non-specifically stained, and the cytoplasm is abnormal and the nucleus is shrunk slightly. When the blood membrane is thick and the red blood cell amount at the edge part is large, the endogenous peroxidase is not completely sealed, the red blood cell background is close to the positive expression color of CD41, and the red blood cell background is not easy to distinguish.
Comparative example 5:
the comparative example adopts a CD41 antibody detection kit (detection system of streptokinase-avidin-biotin method) of Tianjin mineral Bo Biotechnology Limited company to carry out immunohistochemical staining on a bone marrow blood smear, and the manual operation steps are as follows:
1) Sample preparation
The bone marrow blood smears were then dried thoroughly and the prepared smears were tested within a month.
2) Fixing
The fixing agent is a methanol-acetone solution which is prepared at present and has a volume ratio of 1. Fixing for 90 seconds by adopting a fixing agent, and naturally drying to obtain a smear to be dyed.
3) Primary antibody incubation
50 μ L of biotinylated CD41 antibody reagent (coverage area about 1.5 cm. Times.1.5 cm. About.1.5 cm. Times.3.0 cm) was added and incubated at room temperature for 50min. The cleaning solution is washed for 2 times and 5 min/time.
4) Incubation with secondary antibody
Adding dropwise basic phosphatase-labeled streptavidin working solution 50 μ L, and incubating at room temperature for 40min. The cleaning solution is washed for 2 times and 5 min/time.
5) Firm red color development
1mL of fast red buffer solution is taken to dissolve 2mg of fast red (prepared at present, effective within 10 min), and 300 mu L of fast red buffer solution is dripped on the specimen to develop the color for 20 minutes. Rinsing with deionized water for 3 times (3 min/time).
6) Hematoxylin counterstain
Adding 100 μ L hematoxylin dropwise, counterstaining at room temperature for 30min, lining with nucleus, and washing with clear water.
7) Return blue
Returning blue with 150 μ L of blue returning liquid for 5-10 s, washing with clear water, drying, and observing under a microscope.
FIG. 11 is a graph showing the staining results of the bone marrow smear sample in comparative example 5, which shows that the area of the area to be defined before manual staining is about 1.5cm × 1.5cm to 1.5cm × 3.0cm, the amount of the antibody reagent added is small, and the micro-megakaryocytes are easy to miss the examination under the condition of less cells, so that false negative may occur. Biotinylated CD41 primary antibody (30-50) mu L, alkaline phosphatase labeled streptoenzyme ovalbumin secondary antibody (30-50) mu L, firm red developing solution 300 mu L, hematoxylin 100 mu L and blue returning solution 150 mu L, wherein the addition amounts are different, and the memory is not convenient after the instructions are separated. And 2mg of firm red powder is dissolved in 1mL, errors are easy to occur when the solid red powder is weighed and dissolved, the repeatability is poor, the concentration of prepared liquid can not be ensured to be completely consistent during each dyeing, and the dyeing stability is difficult to ensure. The color development time is relatively long, the hematoxylin counterstain time is longer, and after the hematoxylin counterstain, the blue color is darker, and the red blood cells are also stained into blue gray. The effect of returning blue liquid is slightly improved, but the effect is slight. Especially for some samples with relatively thick bone marrow blood smears, the positive megakaryocytes hidden under the stacked red blood cells are difficult to identify and are easy to generate false negative. The overall steps are complicated, the operation is complex, the time is long, and the consistency of the dyeing result is difficult to ensure.
Comparative example 6:
the bone marrow blood smear sample from the same source as in comparative example 3 was manually stained with the second antibody, AP, of the second antibody polymer of Sexanote Biotech Co., ltd. In the present invention using the same reagents as in example 2 by the following specific method:
1) Sample preparation
The bone marrow blood smears were then dried thoroughly and the prepared smears were tested within a month.
2) Fixing the device
The fixing agent is a methanol-acetone solution which is prepared at present and has a volume ratio of 1. Fixing for 90 seconds by adopting a fixing agent, and naturally drying to obtain a smear to be dyed.
3) Primary antibody incubation
Add 100. Mu.L of CD41 rabbit anti-human monoclonal antibody reagent and incubate for 60min at room temperature. The cleaning solution is washed for 3 times and 3 min/time.
4) Incubation with a second antibody
Adding 100 μ L of Alkaline Phosphatase (AP) -labeled secondary antibody polymer, and incubating at room temperature for 30min to allow the primary antibody to specifically bind to the secondary antibody; the cleaning solution is washed for 3 times and 3 min/time.
5) Fast red color development
Fast red color developing agent: fast red activator: fast red buffer =1, 20 dilution ratio, incubation for 8min at room temperature, presented as red precipitate; rinsing with deionized water for 3 times (3 min/time).
6) Hematoxylin counterstain
Re-staining for 3min at room temperature, and lining-staining cell nuclei; rinsing with deionized water for 3 times (3 min/time). Air-drying and observing under a microscope.
FIG. 12 is a graph showing the result of staining a bone marrow blood smear sample obtained in comparative example 6, wherein the bone marrow blood smear sample has the same source as that of comparative example 3, and the positive expression of the CD41 immunocytochemistry of the bone marrow blood smear on megakaryocytes by AP red staining is clear, and the location is accurate. The bright red positive signal generated by the alkaline phosphatase substrate is clearer and brighter than the staining of the traditional peroxidase DAB and AEC, and the nonspecific staining phenomenon of the neutrophils is avoided. The operation procedure is simple, the use is convenient, and the dyeing stability is good. The positive staining of megakaryocytes is clearly compared with the hematoxylin cytonucleus counterstaining, and the blue returning treatment is not needed;
the dyeing results of comparative examples 2 to 6 are summarized in the following table.
Figure BDA0003893772720000171
Experimental examples 1 and 2 are intended to further illustrate the drawbacks of conventional horseradish peroxidase staining system selection for immunohistochemical staining of bone marrow blood smears, and the advantages of the non-biotin alkaline phosphatase staining system selection of the present application.
Test example 1:
in the test example, a Xenopus self-developed horse radish peroxidase-labeled goat anti-rabbit polymer AEC color development system is adopted, and the HRP-AEC staining effect of a bone marrow blood smear is contrastively analyzed by using 80% methanol aqueous solution to prepare hydrogen peroxide blocking agents with different concentrations and blocking time. The manual operation steps for CD41 staining were as follows:
1) Sample preparation
The bone marrow blood smears were then dried thoroughly and the prepared smears were tested within a month.
2) Fixing
The fixing agent is a methanol-acetone solution which is prepared at present and has a volume ratio of 1. Fixing for 90 seconds by adopting a fixing agent, and naturally drying to obtain a smear to be dyed.
3) Blocking endogenous peroxidase
Adding hydrogen peroxide blocking agent (prepared from 80% methanol solution, shown in the table below) of 100 μ L, incubating at room temperature for 10min/20min/30min, and washing with washing solution for 3 times and 3 min/time.
4) Primary antibody incubation
Add CD41 rabbit anti-human monoclonal antibody reagent 100 u L, room temperature incubation for 60min. The cleaning solution is washed for 3 times and 3 min/time.
5) Incubation with a second antibody
Adding 100 μ L of goat anti-rabbit polymer labeled with horseradish peroxidase, and incubating at room temperature for 30min. The cleaning solution is washed for 3 times and 3 min/time.
6) AEC color development
Adding 850 mu L of deionized water into a small test tube, then respectively adding 50 mu L of AEC buffer solution, 50 mu L of AEC substrate and 50 mu L of AEC chromogen, and uniformly mixing to obtain 1mL of AEC color development working solution. Incubating at room temperature for 8min, and presenting in the form of red precipitate; rinsing with deionized water for 3 times (3 min/time).
7) Hematoxylin counterstain
Counter-staining for 3min at room temperature, lining-staining cell nucleus, washing with purified water, air-drying, and observing under microscope.
Test example 2:
the experimental example was performed without the addition of horseradish peroxidase-labeled goat anti-rabbit polymer secondary antibody, and the manual procedure for CD41 staining was as follows.
1) Sample preparation
The bone marrow blood smears were then dried thoroughly and the prepared smears were tested within a month.
2) Fixing
The fixing agent is a methanol-acetone solution which is prepared at present and has a volume ratio of 1. Fixing for 90 seconds by adopting a fixing agent, and naturally drying to obtain a smear to be dyed.
3) Blocking endogenous peroxidase
Adding hydrogen peroxide blocking agent 100 μ L (prepared from 80% methanol solution, shown in the table below) with different concentrations, incubating at room temperature for 10min/20min/30min, and washing with cleaning solution for 3 times and 3 min/time.
4) Primary antibody incubation
Add CD41 rabbit anti-human monoclonal antibody reagent 100 u L, room temperature incubation for 60min. The cleaning solution is used for washing for 3 times and 3 min/time.
5) Blank control (TBS Wash instead of HRP-labeled Secondary antibody Polymer)
100. Mu.L of TBS wash was added dropwise and incubated at room temperature for 30min. The cleaning solution is used for washing for 3 times and 3 min/time.
6) AEC color development
Adding 850 mu L of deionized water into a small test tube, then respectively adding 50 mu L of AEC buffer solution, 50 mu L of AEC substrate and 50 mu L of AEC chromogen, and uniformly mixing to obtain 1mL of AEC color development working solution. Incubating at room temperature for 8min, and presenting in the form of red precipitate; and 3 times of deionized water washing for 3 min/time.
7) Hematoxylin counterstain
Counter-staining for 3min at room temperature, lining-staining cell nucleus, washing with purified water, air-drying, and observing under microscope.
FIG. 13 shows the results of staining in test example 1 in a and b, FIG. 14 shows the results of staining in test example 2, and the results of staining bone marrow blood smears with different concentrations of hydrogen peroxide blocking agent and different blocking times in test examples 1 and 2 with CD41HRP-AEC are similar to those in FIGS. 13 and 14. FIG. 13, a, shows that the staining with HRP-AEC can mark the megakaryocytes positive for CD41, the megakaryocytes are stained brick red, but there is still non-specific staining of neutrophils, as shown in FIG. 13, b. FIG. 14 shows that when TBS wash was used in place of horseradish peroxidase labeled secondary antibody (blank control), there should be no CD41 positive staining of any cells, but there was a non-specific staining of neutrophils as shown in FIG. 14, indicating that although different concentrations of hydrogen peroxide were used to block different times, the endogenous peroxidase in the bone marrow smear could not be completely blocked.
The following table shows the effect of peroxidase blocking agent concentration and blocking time on CD41 staining results. As shown in the table below, the time for sealing endogenous peroxidase by using the 30% hydrogen peroxide sealing agent is shorter, the sealing can be basically completed within 3min at room temperature, but the practicability is not strong due to overhigh concentration, and the final dyeing effect is not obviously different from that of the 3% hydrogen peroxide sealing agent.
a) When the concentration of the hydrogen peroxide sealing agent is 0.3-2%, sealing treatment is carried out for 10-30 min, and when no secondary antibody is added, a plurality of granulocytes can be stained in a non-specific way under a low power microscope. When the concentration of the hydrogen peroxide sealant is 3%, sealing treatment is carried out for 10-20 min, and when no secondary antibody is added, the non-specific staining of the granulocytes under the high power lens is still obvious, and the non-specific staining of the granulocytes under the low power lens is not obvious. Indicating that the endogenous peroxidase in the bone marrow blood smear is still difficult to completely block.
b) Because the individual difference of the bone marrow blood smear sample is large, some blood films are thin, and some blood films are thick. For a bone marrow blood smear with a thin blood film, the reaction speed is too fast and difficult to control when a sealant with higher concentration is sealed, and the smear removal and cell aggregation deformation are easily caused. The hydrogen peroxide blocking agent concentration cannot be too high.
c) The dyeing results of comparative examples 1 to 5 and test examples 1 to 2 confirmed that: when bone marrow blood smears are stained with HRP-DAB and HRP-AEC, megakaryocytes can be normally stained, but a non-specific staining phenomenon of granulocytes cannot be avoided, so that result interpretation is influenced. The AP alkaline phosphatase staining has clear positive expression on megakaryocytes, accurate positioning and better specificity, and the preferred AP red staining method of the kit for staining the small megakaryocytes of the bone marrow blood smears is AP red staining.
Figure BDA0003893772720000191
Endogenous peroxidases are widely found in hemoglobin of erythrocytes, myeloperoxidase (MPD) of neutrophils, monocytes, eosinophils. The content of endogenous peroxidase in erythrocytes and granulocytes of bone marrow blood smears is particularly high, and complete blocking of these endogenous peroxidases is very difficult, and at the same time blocking may cause denaturation of some antigenic determinants. According to a conventionally adopted DAB (horseradish peroxidase) staining system, a bone marrow blood smear is subjected to sealing treatment of 3% or even 10% hydrogen peroxide (prepared by 80% methanol solution), cells are stacked into clusters, the smear removal rate is high, endogenous peroxidase cannot be completely eliminated, so that the final staining result shows non-specific staining of neutrophils, and the accurate interpretation of the result is influenced. And methanol-H has been considered by researchers 2 O 2 The blocking treatment is too intensive and may cause some antigens to denature. Therefore, for bone marrow blood smears, it is preferred to use non-peroxidase-labeled antibodies, such as alkaline phosphatase. The bright red positive signal generated by the alkaline phosphatase substrate is clearer and brighter than the traditional DAB staining by peroxidase.
The above is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, a plurality of modifications and embellishments can be made without departing from the principle of the present invention, and these modifications and embellishments should also be regarded as the protection scope of the present invention.

Claims (10)

1. Kit for staining megakaryocytes of bone marrow smears, characterized in that it comprises: a primary antibody reagent, a secondary antibody reagent, and a staining agent;
the primary anti-agent comprises an antibody and an antibody diluent, wherein the antibody is selected from at least one of a CD41 monoclonal antibody, a CD42b monoclonal antibody or a CD61 monoclonal antibody;
the secondary antibody reagent comprises a secondary antibody polymer marked by alkaline phosphatase;
the staining agent comprises a fast red staining agent and a hematoxylin counterstaining agent.
2. The kit of claim 1, wherein the antibody diluent comprises water, tris-HCl, bovine serum albumin, sodium chloride, tween-20, viridin, and ProClin950.
3. The kit of claim 2, wherein the antibody diluent consists of water and 6g/L Tris-HCl, 15g/L bovine serum albumin, 8.5g/L sodium chloride, 0.1vol% Tween-20, 0.01wt% physodin, and 0.15wt% proclin950, and has a pH of 7.60 ± 0.05.
4. The kit of claim 1, wherein the secondary antibody reagents comprise water, alkaline phosphatase-labeled secondary antibody polymer, tris, bovine serum albumin, sodium chloride, tween-20, proClin950, an enzyme protecting agent which is a metal ion, and glycerol, the enzyme protecting agent comprising a mixture of one or more of zinc ions, magnesium ions, and calcium ions.
5. The kit of claim 4, wherein the secondary reagents are anti-rabbit polymer labeled with water and 8-12 μ g/mL alkaline phosphatase, 6g/L Tris, 10g/L bovine serum albumin, 8.5g/L sodium chloride, 3g/L Tween-20, 0.1wt% ProClin950, 0.1-1 mM enzyme protector, and 40vol% glycerol, the enzyme protector is a metal ion, the enzyme protector comprises one or more mixtures of zinc ions, magnesium ions, and calcium ions, and the pH of the secondary reagents is 7.20 ± 0.05.
6. The kit of claim 1, wherein the fast red staining reagent comprises: fast red color developing agent, fast red activating agent and fast red buffer solution;
the fast red color developing agent comprises an aromatic primary amine compound and inorganic strong acid, wherein the aromatic primary amine compound is any one of basic fuchsin, new fuchsin and 3-amino-4-methoxybenzamide, and the inorganic strong acid is any one of hydrochloric acid, hydrobromic acid, fluoboric acid, sulfuric acid and nitric acid;
the fast red activating agent is sodium nitrite solution;
the fast red buffer solution comprises naphthol phosphate, tris, sodium chloride and MgCl 2 ·6H 2 O, levamisole hydrochloride, tween-20 and ProClin950, wherein the naphthol phosphate is any one of naphthol AS-TR phosphate, naphthol AS-TR disodium phosphate and naphthol AS-BI phosphate disodium hydrate.
7. The kit of claim 6, wherein the fast red staining reagent comprises: fast red color developing agent, fast red activating agent and fast red buffer solution;
the fast red color developing agent comprises 20.0-40.0 g/L of aromatic primary amine compound and 0.5-1M of inorganic strong acid, wherein the aromatic primary amine compound is any one of basic fuchsin, new fuchsin and 3-amino-4-methoxybenzamide, and the inorganic strong acid is any one of hydrochloric acid, hydrobromic acid, fluoboric acid, sulfuric acid and nitric acid;
the fast red activating agent comprises 8.28-16.56 g/L of sodium nitrite solution;
the fast red buffer solution comprises 2-3M naphthol phosphate, 12.1g/L Tris, 5.85g/L sodium chloride and 2.03g/L MgCl 2 ·6H 2 O, 0.24g/L levamisole hydrochloride, 0.3vol% Tween-20 and 0.5wt% ProClin950, wherein the naphthol phosphate is naphthol AS-TR phosphate, naphthol AS-TR disodium phosphate or naphthol AS-BI disodium phosphateAny one of hydrates;
in the fast red staining reagent, the volume ratio of the fast red color developing agent to the fast red activating agent to the fast red buffer is 1.
8. The kit of claim 1, wherein the hematoxylin counterstain reagent comprises 5g/L hematoxylin, 50g/L aluminum potassium sulfate, 0.5g/L sodium iodate, and 5vol% glycerol.
9. A method for staining a bone marrow smear megakaryocyte, which comprises staining a cell with the kit according to any one of claims 1 to 8.
10. Dyeing process according to claim 9, characterized in that it comprises the following steps:
step 1, preparing a bone marrow blood smear and fixing;
step 2, incubating by using the primary antibody reagent;
step 3, incubating by using the secondary antibody reagent;
step 4, dyeing by adopting the fast red dyeing reagent;
and 5, counterstaining by adopting the hematoxylin counterstaining reagent.
CN202211271162.XA 2022-10-17 2022-10-17 Kit for staining megakaryocyte of bone marrow smear, staining method and application Pending CN115615786A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202211271162.XA CN115615786A (en) 2022-10-17 2022-10-17 Kit for staining megakaryocyte of bone marrow smear, staining method and application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202211271162.XA CN115615786A (en) 2022-10-17 2022-10-17 Kit for staining megakaryocyte of bone marrow smear, staining method and application

Publications (1)

Publication Number Publication Date
CN115615786A true CN115615786A (en) 2023-01-17

Family

ID=84862220

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202211271162.XA Pending CN115615786A (en) 2022-10-17 2022-10-17 Kit for staining megakaryocyte of bone marrow smear, staining method and application

Country Status (1)

Country Link
CN (1) CN115615786A (en)

Similar Documents

Publication Publication Date Title
JP3503890B2 (en) Improved immunohistochemical staining method and reagents therefor
Swan et al. Bone marrow core biopsy specimens in AL (primary) amyloidosis: a morphologic and immunohistochemical study of 100 cases
JPH03266999A (en) Reagent for automatic flow sightmetry measurement of sub-population of at least one leukocyte from whole blood and measuring method using said reagent
CN112964877A (en) Immunohistochemical multiple staining kit and staining procedure for identifying mantle cell lymphoma
JP2005017133A (en) Antigen detecting method using immunohistochemical staining method
Mason Intracellular lysozyme and lactoferrin in myeloproliferative disorders.
CN111766385A (en) Immunohistochemical kit for rapidly identifying lung cancer and sclerosing lung cell tumor in operation
Oosterwijk et al. Development of a preparation and staining method for fetal erythroblasts in maternal blood: simultaneous immunocytochemical staining and FISH analysis
Ng et al. Is triple immunostaining with 34βE12, p63, and racemase in prostate cancer advantageous? A tissue microarray study
CN112305213A (en) Rich-fractionation double-dyeing kit and use method thereof
Shukla et al. Mast cell ultrastructure and staining in tissue
Yam et al. Immunocytochemical characterization of human blood cells
CN115615786A (en) Kit for staining megakaryocyte of bone marrow smear, staining method and application
CN110320087B (en) Pigment-containing tissue immunohistochemical staining kit and staining method
CN116381240A (en) Double-dyeing immunohistochemical kit and application
JPH0566548B2 (en)
US5945291A (en) Method for distinguishing viable, early apoptotic, late apoptotic, and necrotic cells
CN111089967B (en) Application of skin tissue immune cell in-situ detection kit for lupus erythematosus typing
Borgström et al. Clinical significance of A, B, H isoantigen deletion of urothelial cells in bladder carcinoma
CN112255407A (en) Novel serum alpha-fetoprotein heteroplasmon 3 determination kit and preparation method and application thereof
CN111458503A (en) Antibody chip for separating and counting blood cells and preparation method thereof
Gloghini et al. A combined cytochemical and immunocytochemical method for simultaneous visualization of cytoplasmic enzyme reactivity and cell surface antigens in cell suspensions
Miller et al. Improved avidin-biotin immunoperoxidase method for terminal deoxyribonucleotidyl transferase and immunophenotypic characterization of blood cells
CN113504092A (en) Helicobacter pylori staining kit and application thereof
Zarbo et al. Abc‐immunoperoxidase staining of cytologic preparations: Improvement of specificity

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination