CN113945722B - Aplastic anemia diagnosis marker and application thereof in preparation of kit - Google Patents
Aplastic anemia diagnosis marker and application thereof in preparation of kit Download PDFInfo
- Publication number
- CN113945722B CN113945722B CN202111205386.6A CN202111205386A CN113945722B CN 113945722 B CN113945722 B CN 113945722B CN 202111205386 A CN202111205386 A CN 202111205386A CN 113945722 B CN113945722 B CN 113945722B
- Authority
- CN
- China
- Prior art keywords
- antibody
- ethanol
- kit
- bone marrow
- aplastic anemia
- 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.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/22—Haematology
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a diagnostic marker for aplastic anemia and application thereof in preparation of a diagnostic kit, and particularly relates to a bone marrow nerve fiber diagnostic marker, wherein the kit is used for carrying out immunohistochemical staining on nerve fibers in a bone marrow microenvironment to observe whether the nerve fibers form a fishing net-shaped characteristic or not, so that the aplastic anemia is accurately diagnosed. The method is simple and convenient, has high accuracy, and has extremely high popularization value for diagnosis of aplastic anemia.
Description
Technical Field
The invention relates to the field of medical detection, in particular to a aplastic anemia diagnostic marker and application thereof in preparation of a aplastic anemia diagnostic kit.
Background
Aplastic anemia is a bone marrow hematopoietic failure which is introduced by various etiological factors and pathogenesis. Mainly expressed as low bone marrow nucleated cell proliferation, and replaced by adipose tissue, resulting in pancytopenia. The diagnostic criteria for aplasia were as follows: decrease in whole blood cells and decrease in absolute value of reticulocytes. ② there is no splenomegaly generally. And thirdly, the marrow pathological changes of aplastic anemia are mainly hematopoietic tissue reduction, red marrow total volume reduction and fat tissue replacement. The ratio of bone marrow hematopoietic tissue to adipose tissue in normal adults is about 1: 1, and the ratio of bone marrow hematopoietic tissue to adipose tissue in aplastic patients is more than 2: 3. The hematopoietic foci are diminished in hematopoietic cells (referred to as the granulocytic, erythrocytic and megakaryocytic system) and increased in "non-hematopoietic cells" (referred to as lymphoid, plasma, tissue basophilic and reticulocyte). Bone marrow has plasma exudation, hemorrhage, lymphoproliferation, focal fibrosis and interstitial disease. Acute aplastic myelopathy develops rapidly and extensively; chronic aplasia is progressive "central atrophy" involving the ilium, then the spinous process and the sternum. Chronic aplasia still has compensatory hyperplastic foci, which are primarily erythrocytic hyperplasias with dyscrasia. Erythroid cells are not only reduced in number but also have qualitative defects. The observation of the ultrastructure shows that mature red blood cells have abnormal shapes and petal-shaped shapes; there are changes in the erythroblast plasma such as medullary changes, unbalanced development of nuclear plasma, enlargement of nuclear membrane pores, etc. The alkali-resistant hemoglobin and free protoporphyrin in the red blood cells are increased. The activity of the erythrozyme such as pyruvate kinase is reduced. The above results indicate qualitative abnormality of erythrocytes. The iron kinetics examination showed an increase in plasma iron, an increase in iron granulocytes and tissue iron, a delay in plasma iron clearance and a significant decrease in red blood cell iron uptake, suggesting a decrease in erythropoiesis. Some patients have ineffective erythropoiesis or in situ hemolysis in the bone marrow. And fourthly, diseases causing the decrease of the whole blood cells, such as paroxysmal nocturnal hemoglobinuria, refractory anemia in myelodysplastic syndrome, acute hematopoietic function stagnation, myelofibrosis, acute leukemia, malignant histiocytosis and the like can be excluded. Common antianemia drug therapy is ineffective.
To date, aplastic anemia remains an exclusive diagnosis, and myelopathology and immunohistochemical staining play a decisive role in aplastic anemia, but the characteristic pathological diagnosis markers of the disease are still lacking.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a aplastic anemia diagnostic marker and application thereof in preparing a aplastic anemia diagnostic kit.
In a first aspect of the present invention, there is provided a diagnostic marker for aplastic anemia, wherein the marker is a nerve fiber of bone marrow. The diagnostic marker exhibits a "fish net" like characteristic in bone marrow of patients with aplastic anemia.
In a second aspect of the present invention, an application of a bone marrow nerve fiber as a diagnostic marker in the preparation of a detection product related to aplastic anemia is provided. The bone marrow nerve fiber immunohistochemical detection kit can be used for diagnosing aplastic anemia.
In one embodiment, the aplastic anemia-associated test product is a diagnostic kit, which can be used to diagnose aplastic anemia.
In a third aspect of the present invention, a bone marrow nerve fiber immunohistochemical detection kit is provided, wherein the bone marrow nerve fiber immunohistochemical detection kit at least comprises the following components: microtubule-associated protein 2(MAP-2) antibody, Synaptophysin (SYN) antibody, Glial Fibrillary Acidic Protein (GFAP) antibody, 5-hydroxytryptamine (5-HT) antibody, gamma-aminobutyric acid (GABA) antibody, epinephrine (TH) antibody.
In one embodiment, the kit further comprises a staining agent. The kit is used for respectively staining nerve morphologies of myelinated nerve fibers and non-myelinated nerve fibers according to a rabbit anti-human/mouse microtubule-associated protein 2(MAP-2) antibody, a Synaptophysin (SYN) antibody, a Glial Fibrillary Acidic Protein (GFAP) antibody and the like; and used to determine the specific type and transmitter of the bone marrow nerve by staining with antibodies to the neurotransmitters 5-hydroxytryptamine (5-HT), gamma-aminobutyric acid (GABA), and epinephrine (TH).
In one embodiment, the kit further comprises a fixative, a deparaffinization hydrating agent, a serum blocking agent, a buffer, an antibody diluent, and a dehydration clearing agent. Preferably, the fixative is neutral buffered neutral formalin, Bouin's solution, or mDF solution; the serum blocking agent is an antibody in the serum of an animal which is from the same source as the secondary antibody, and can be combined with a site which can be combined with the secondary antibody in a tissue in advance; or calf serum, BSA, sheep serum, and not consistent with the primary antibody source; the buffer is PBS buffer; the antibody diluent can be a general antibody diluent, such as PBS, or a special antibody diluent added with sodium azide preservative and/or BSA stabilizer; the dewaxing hydration agent and the dehydration clearing agent are xylene and ethanol; the ethanol comprises absolute ethanol, 95% ethanol, 85% ethanol and 75% ethanol.
In one embodiment, the method of use of the kit comprises:
s1, taking marrow pathological tissues of a person to be tested, performing antigen retrieval after dewaxing to water, inactivating peroxidase, and sealing with 10% goat serum;
s2, respectively dropwise adding a Synaptophysin rabbit antibody, a MAP2 mouse antibody and a GFAP rabbit antibody, incubating overnight at 4 ℃, washing 3 times with PBS, and 3 minutes each time;
s3, adding a proper amount of goat anti-mouse/rabbit IgG polymer with an enhanced enzyme dropwise, incubating for 20 minutes at room temperature, washing for 3 times with PBS buffer solution, and 3 minutes each time;
s4, dyeing for 3 minutes by using a fresh DAB color developing solution, and washing with tap water to stop color development; mayer hematoxylin counterstains cell nucleuses for 3 minutes, differentiates, washes and returns blue;
s5, dehydrating and sealing the transparent piece, observing the dyeing result under an optical microscope and judging.
In a preferred embodiment, the method of use of the kit comprises:
s1, taking a bone marrow of a posterior superior iliac spine to be tested to prepare a slice;
s2, immunohistochemical experiment:
1) dewaxing to water: the paraffin sections are placed in two cylinders of fresh dimethylbenzene, and each cylinder is soaked for 10 min; removing excessive liquid, sequentially soaking in anhydrous ethanol, 95% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol for 5min each jar; then placing in distilled water for 5min, and finally placing in PBS buffer solution for 10 min;
2) antigen retrieval: injecting the prepared antigen retrieval liquid into a stainless steel pressure cooker, heating to boil, placing the slicing frame with the slices in the cooker, enabling the liquid to be over the slice tissues, covering the pressure cooker, timing for 90s when the pressure cooker starts to jet air, and then cooling the pressure cooker to room temperature and taking out the slices;
3) blocking endogenous peroxidase: adding appropriate amount of endogenous peroxidase blocker, incubating at room temperature for 10min, and soaking in PBS buffer solution for 3 times, each for 3 min;
4) and (3) sealing: incubating with goat serum at room temperature for 10min, and removing liquid without washing;
5) dropwise adding a proper amount of primary antibodies, wherein the primary antibodies are rabbit anti-human/mouse microtubule-associated protein 2(MAP-2) antibody, Synaptophysin (SYN) antibody, Glial Fibrillary Acidic Protein (GFAP) antibody, 5-hydroxytryptamine (5-HT) antibody, gamma-aminobutyric acid (GABA) antibody and epinephrine (TH) antibody, and standing at 4 ℃ overnight;
6) dropwise adding a reaction enhancing solution: dripping appropriate amount of reaction enhancing solution, incubating at room temperature for 10min, and soaking in PBS buffer solution for 3 times, each for 3 min;
7) dripping a proper amount of secondary antibody, namely horseradish peroxidase enzyme-labeled goat anti-rabbit IgG polymer, incubating at room temperature for 20min, and soaking for 3 times with PBS (phosphate buffer solution) for 3min each time;
8) color development: adding a proper amount of freshly prepared DAB color development solution, and incubating for 3min at room temperature;
9) counterdyeing: washing with tap water, incubating with hematoxylin staining solution for 3min, differentiating, washing, and returning to blue;
10) alcohol gradient dehydration, xylene transparent, neutral gum sealing sheet
And S3, observing by using a microscope.
In one embodiment, the diagnostic criteria for diagnosing aplastic anemia using the kit are: the bone marrow nerve fibers form a characteristic "fishnet" (or "silk net") description.
In a fourth aspect of the invention, there is provided the use of the kit as described above for the study of aplastic anemia, for non-diagnostic purposes.
Compared with the prior art, the invention has the following outstanding advantages:
1. the invention discovers that the bone marrow nerve fibers can be used for diagnosing the aplastic anemia for the first time, overcomes the technical obstacles that the aplastic anemia has no objective index for characteristic case diagnosis and seriously depends on clinical experience of a pathologist, and effectively avoids misdiagnosis and missed diagnosis caused by subjective factors of judgers;
2. the condition of the patient is effectively prevented from being delayed due to untimely diagnosis of the patient or the adoption of a mode of observing along with prevention and diagnosing while treating, and the realization of the aims of early finding, early diagnosing and early treating of the aplastic anemia is greatly promoted.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 shows the results of immunohistochemistry of paraffin-embedded aplastic anemia patients and mouse bone marrow using microtubule-associated protein 2(MAP-2), Synaptophysin (SYN), Glial Fibrillary Acidic Protein (GFAP), 5-hydroxytryptamine (5-HT), gamma-aminobutyric acid (GABA), and epinephrine (TH) antibodies. Wherein the abscissa represents different antibodies; the ordinate represents the source of the samples, up to and above, respectively normal BALB/C mice (balbc-normal), aplastic anemia BALB/C mice (balbc-AA), normal human controls (human-con), and patients with aplastic anemia (human-AA).
FIG. 2 shows immunohistochemical results of bone marrow of paraffin-embedded patients with different types of blood using microtubule-associated protein 2(MAP-2), Synaptophysin (SYN), Glial Fibrillary Acidic Protein (GFAP), 5-hydroxytryptamine (5-HT), gamma-aminobutyric acid (GABA), and epinephrine (TH) antibodies. Wherein the abscissa represents different antibodies; the ordinate represents the bone marrow of different hematological disorders, up to and above, respectively, normal human controls, leukemia, lymphoma, myelodysplastic syndrome (MDS), Multiple Myeloma (MM), chronic myeloproliferative disorder (MPD).
Detailed Description
The invention is further illustrated with reference to specific examples. It should be understood that the specific embodiments described herein are illustrative only and are not limiting upon the scope of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products which are not known to manufacturers and are available from normal sources.
The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.
Example 1 Experimental materials and principles
The reagents used in the present invention and their concentrations are shown in table 1 below:
TABLE 1
The experimental principle is as follows:
hydrogen peroxide or methanol is used to reduce the non-specific background caused by endogenous peroxidase; (ii) exposing antigen binding sites; sealing; incubating corresponding primary antibody of the protein to be detected contained in the bone marrow tissue section to be detected; incubating a fluorescein-labeled secondary antibody of the protein to be detected; sixthly, microscopic examination and photographing are carried out, and the expression condition of the protein to be detected in the tissue section is analyzed.
Example 2 animal modeling
1. Selection of animal species:
(1) BALB/c mouse (SPF grade)
A BALB/c mouse which is not limited by male and female, has the weight of 16-20 g and the age of 8-12 weeks and serves as a receptor mouse;
(2) DBA/2 mouse (SPF grade)
The donor mice are 8-10 weeks old DBA/2 mice.
The experiment was approved by animal ethics.
2. The modeling method comprises the following steps:
euthanasia of DBA/2 mice (n ═ 3) under USPA grade (medical) or B grade (industrial) carbon dioxide;
soaking mouse in 95% ethanol for 5 min;
aseptically taking out thymus gland and lymph nodes at axilla and inguinal region, adding RPMI-1640 culture solution, and removing surface blood stain and adherent connective tissue;
repeatedly shearing the taken tissue by a scalpel until the tissue becomes pasty, lightly grinding the tissue, and filtering the ground tissue by using a 200-mesh nylon net to form a single-cell suspension;
dripping 1 drop of trypan blue on the glass slide, and identifying that the cell activity is more than 95%; adjusting the cell concentration to 1X 10 8 /L；
BALB/c mice (n ═ 10) were irradiated systemically with gamma-rays 6.0Gy for 3 min; setting a normal control group (n is 5);
immediately injecting the cell suspension into a mouse tail vein by using an injector according to the dose of 0.2 ml/mouse within 1-4 h after the irradiation of the sublethal dose; the control group was replaced with an equal amount of PBS;
BALB/c mouse sample sampling points
Day-3 week blood sampling (blood routine blood biochemistry) X-ray living body imaging
Day 0 moulding
DAY 4 peri-frame blood sampling (blood routine blood biochemistry) X-ray living body imaging
3. The sampling method comprises the following steps:
mouse perfusion fixation method (DAY 10):
after deep anesthesia, the thoracic cavity of the mouse is cut off quickly, a 20mL syringe connected with a scalp needle is inserted through the left ventricle, the scalp needle is dull-ground, the needle is inserted from the direction forming an angle of 45 degrees with the longitudinal axis of the body, the needle point is inserted into the ascending aorta, the action is gentle, and the right auricle is cut off at the same time. 25mL of PBS containing 50ug/mL of heparin is pushed into the injector, 25mL of PBS containing 0.5% paraformaldehyde, 10% of sucrose and 50ug/mL of heparin is replaced after the pushing is finished, and the tibia, the femur, the spleen, the liver and the large intestine are taken after the filling is finished and are 5 mm; and (5) fixing paraformaldehyde.
If the animal dies within 10 days after the model building, at the death time, 5mm of tibia, femur, spleen, liver and large intestine are directly taken as aplastic tissue samples for subsequent experiments.
Example 3 selection of clinical cases
1. Selection of clinical cases
Randomly selecting 20 patients with aplastic anemia, the age of which is between 20 and 60 years and is half of male and female, belonging to auxiliary hospitals of Guangdong medical university, and signing informed consent after soliciting consent of the patients.
Selected patients all had the following clinical manifestations of aplasia:
the hemogram: the cell reduction is performed in whole blood, and the cell reduction is performed in 1 line or 2 lines in the early stage of a few cases;
② marrow elephant: the increase of oil drops is observed by naked eyes of a bone marrow smear, the vacuity of small particle microscopic examination of bone marrow, and the increase of non-hematopoietic cells and fat cells is more than 50 percent.
As a control, 10 healthy male and female volunteers of the same age group were randomly selected for sampling.
2. Bone marrow samples were taken from both patients and healthy volunteers according to the clinical standard protocol and immediately fixed in 4% paraformaldehyde fixative for subsequent experiments.
Example 4 immunohistochemical analysis of bone marrow samples
1. Decalcification and dehydration of samples
The bone marrow sample wax blocks prepared in examples 2 and 3 were taken out from the 4% paraformaldehyde fixing solution, placed in a bone marrow special machine dehydration basket and then placed on a machine, and the procedures of decalcification and dehydration were as follows: 1 hour of 10% neutral formalin solution, 2 hours of 8% formaldehyde hydrochloride decalcification solution, 30 minutes of distilled water, 30 minutes of 70% alcohol I, 20 minutes of 95% alcohol II, 20 minutes of absolute ethyl alcohol I, 20 minutes of absolute ethyl alcohol II, 30 minutes of xylene II, 20 minutes of wax dipping I, 20 minutes of wax dipping II and 20 minutes of wax dipping III. The whole decalcification and dehydration time is about 8 hours.
2. Sample embedding
After dehydration, the bone marrow sample wax block was removed from the dehydrator and embedded. A small amount of paraffin is added to a small embedding mold, a part of the bone marrow sample is horizontally and flatly placed on the embedding mold, then the embedding mold is placed on a condensing table for a plurality of seconds, and then enough paraffin is supplemented. Finally, the mixture is placed in a refrigerator with the temperature of-10 ℃ for 20 minutes, and the wax block is knocked out.
3. Specimen section
After being frozen, the bone marrow wax block is sliced, the wax block is fixed on a chuck of a slicer, the slice is slowly and roughly trimmed, after the largest surface is trimmed, the largest surface is replaced with a new knife edge and sliced, each wax block is cut into at least three surfaces, and after being flattened by hot water of 50 ℃, the wax block is fished out in the center of a glass slide in a straight line; and (3) baking the cut slices in an oven at about 60-70 ℃ for more than 30 minutes, and dyeing the slices after the slices are dried.
4. Dewaxing:
EZ prep (I): dewaxing and soaking for 5 minutes in a water bath box at 65 +/-2 ℃;
EZ prep (II): dewaxing and soaking for 5 minutes in a water bath box at 65 +/-2 ℃;
EZ prep (III): dewaxing and soaking for 5 minutes in a water bath box at 65 +/-2 ℃;
flushing with running water for 2 minutes; passing distilled water for 1 minute; antigen retrieval:
preparing antigen repairing solution freshly by selecting 40ml +2L distilled water (antigen repairing working solution pH9.0) from GT100411(50X concentrated solution) of gene technology (Shanghai Co., Ltd.), and mixing uniformly in a pressure cooker;
and (3) putting the washed tissue slices into an autoclave, covering a high-pressure check safety raft, heating and boiling with high fire, adjusting to low fire, keeping boiling and spraying air for 3 minutes, turning off a switch of an electromagnetic oven in work, and moving the autoclave into cold water for cooling after 5 minutes.
After the antigen retrieval liquid in the pressure cooker is completely cooled, the pressure cooker is opened, the tissue slices are washed clean by running water and are transferred to distilled water for soaking for 2 minutes.
5. Blocking:
3％H 2 O 2 soaking for 8 minutes, and washing with running water and distilled water.
6. Dropwise addition of antibody
The sections were removed, wiped dry of water around the tissue, and a DAKO circle pen was used to draw a circle around the tissue mass to note that the circled junction was well-received, preventing false negatives due to antibody running out of the circle. Preventing tissue from being dry, and washing with PBS;
throwing off redundant PBS on the tissue slices to be detected, then dripping primary antibodies (rabbit anti-human/mouse microtubule-associated protein 2(MAP-2), Synaptophysin (SYN), Glial Fibrillary Acidic Protein (GFAP), 5-hydroxytryptamine (5-HT), gamma-aminobutyric acid (GABA) and epinephrine (TH) antibodies), and also dripping the same antibodies on positive and negative control slices, wherein the amount of the antibodies is measured by fully covering tissue blocks; PBS buffer was added dropwise to the blank control.
The section should be kept wet before adding the antibody, and drying is avoided; residual water on the tissue piece before adding the antibody cannot be excessive, so that the primary antibody is prevented from being diluted for the second time;
putting the slices into an incubation box, covering the incubation box with a cover, and putting the incubation box into a refrigerator at 4 ℃ for incubation for 18-24 hours;
taking the incubation box out of the refrigerator, and standing for 30 minutes to recover the room temperature;
the incubation box is inclined at an angle of 30-40 degrees and washed for 3 times with PBS (phosphate buffer solution), and each time lasts for 2 minutes;
and (3) throwing off redundant PBS on the section, dripping secondary antibodies (horseradish peroxidase enzyme-labeled goat anti-rabbit IgG polymers) into the section of the tissue to be detected, the positive (schwannomas) pair picture, the negative (normal person) pair picture and the blank pair picture, and putting the NovoLink polymer detection system RE7159 and the reagent which fully cover the tissue incubator into a 37 ℃ incubator to incubate for 30 minutes.
The incubation box is inclined at an angle of 30-40 degrees and washed for 3 times with PBS (phosphate buffer solution), and each time lasts for 2 minutes;
and (3) throwing off redundant PBS on the section, dripping secondary antibody (horseradish peroxidase enzyme-labeled goat anti-rabbit IgG polymer) into the tissue section and positive, negative and blank pair pictures of the patient to be detected, and putting the NovoLink polymer detection system RE7161 and the reagent which fully cover the tissue incubation box into a 37 ℃ incubator for incubation for 30 minutes.
The incubation box is inclined at an angle of 30-40 degrees and washed for 3 times with PBS (phosphate buffer solution), and each time lasts for 2 minutes;
9. color development, lining dyeing and sealing piece
Preparing DAB color development liquid: taking one plastic test tube of 5ml, respectively adding a reagent RE71632ml in the NovoLink polymer detection system kit and a reagent RE716220 mu L in the NovoLink polymer detection system kit, and uniformly mixing for later use;
taking out the section, wiping off redundant PBS around the tissue, adding the section into prepared DAB color development liquid for color development for 3-5 minutes, and observing the section under a microscope;
placing the slices in tap water to stop color development, and then flushing for 5-10 minutes by running water;
counterstaining with hematoxylin staining solution for 1 minute, and differentiating with 0.5% hydrochloric acid alcohol for 3 seconds;
flushing with running water for 5-10 min, and returning blue;
dehydrating, transparentizing, sealing and microscopic examination.
7. Observation of samples
Immunohistochemistry results using microtubule-associated protein 2(MAP-2), Synaptophysin (SYN), Glial Fibrillary Acidic Protein (GFAP), 5-hydroxytryptamine (5-HT), gamma-aminobutyric acid (GABA), and epinephrine (TH) antibodies on bone marrow of paraffin-embedded aplastic anemia patients and mice showed (see FIG. 1): structurally, the nerves that are visible in aplastic anemia appear morphologicallyWire mesh distribution(GFAP) positive expression (+) with a strong positive (+++) expression of synaptophysin. In terms of neurotransmitters, patients with aplastic anemia express 5-hydroxytryptamine negatively (-) and epinephrine positively (++). Therefore, both the nerve structure and the composition of transmitter indicate that the marrow nerve staining is remarkably different between the disease group and the normal control group.
Example 5 correlation analysis of the bone marrow nerve "fishing net signs" with other myeloid disorders
Bone marrow samples were observed in other patients with myeloid disorders in a manner similar to that described in examples 3-4, and immunohistochemical experiments were performed on bone marrow of paraffin-embedded patients with different types of blood patients using microtubule-associated protein 2(MAP-2), Synaptophysin (SYN), Glial Fibrillary Acidic Protein (GFAP), 5-hydroxytryptamine (5-HT), gamma-aminobutyric acid (GABA), and epinephrine (TH) antibodies, and the results are shown in FIG. 2: wherein the abscissa represents different antibodies; the ordinate represents the bone marrow of different hematological disorders, up to and above, respectively, normal human controls, leukemia, lymphoma, myelodysplastic syndrome (MDS), Multiple Myeloma (MM), chronic myeloproliferative disorder (MPD). In terms of structure, it is seen thatThe marrow nerves of hematopathy are all presented in shapeWire mesh shape Distribution (GFAP)Positive expression (+), different diseases are characterized in the aspects of neurotransmitter composition and the like, and the potential value of marrow nerve staining for diagnosis and treatment of blood diseases is suggested.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (7)
1. The bone marrow nerve fiber immunohistochemical detection kit is characterized by at least comprising the following components:
microtubule-associated protein 2(MAP-2) antibody, Synaptophysin (SYN) antibody, Glial Fibrillary Acidic Protein (GFAP) antibody, 5-hydroxytryptamine (5-HT) antibody, gamma-aminobutyric acid (GABA) antibody, epinephrine (TH) antibody, and horseradish peroxidase enzyme labeled goat anti-rabbit IgG polymer.
2. The kit of claim 1, further comprising a staining agent for separately staining the nerve fibers with and without marrow according to the different types of nerve fibers, and determining the specific type of the nerve of the bone marrow and the transmitter by staining with a neurotransmitter.
3. The kit of claim 1, wherein the myeloneurofibrillary immunohistochemical detection kit further comprises a fixative, a deparaffinization hydration agent, a serum blocking agent, a buffer, an antibody diluent, and a dehydration clearing agent.
4. The kit of claim 3, wherein the fixative is neutral formalin; the serum blocking agent is goat serum; the buffer is PBS buffer; the antibody diluent is a universal antibody diluent; the dewaxing hydration agent and the dehydration clearing agent are xylene and ethanol; the ethanol comprises absolute ethanol, 95% ethanol, 90% ethanol, 80% ethanol and 70% ethanol.
5. The bone marrow nerve fiber immunohistochemical detection kit of any one of claims 1-4, wherein the method of using the kit comprises:
s1, taking a bone marrow of a posterior superior iliac spine to be tested to prepare a slice;
s2 immunohistochemical experiments:
1) dewaxing to water: the paraffin section is placed in two jars of fresh xylene, and each jar is soaked for 10 min; removing excessive liquid, sequentially soaking in anhydrous ethanol, 95% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol for 5min per jar; then placing in distilled water for 5min, and finally placing in PBS buffer solution for 10 min;
2) antigen retrieval: injecting the prepared antigen retrieval liquid into a stainless steel pressure cooker, heating to boil, putting the slicing frame with the slices in the cooker, enabling the liquid to submerge the slice tissues, covering the pressure cooker, timing for 90s when the pressure cooker starts to jet air, cooling the pressure cooker to room temperature, and taking out the slices;
3) blocking endogenous peroxidase: adding appropriate amount of endogenous peroxidase blocker, incubating at room temperature for 10min, and soaking in PBS buffer solution for 3 times, each for 3 min;
4) and (3) sealing: incubating with goat serum at room temperature for 10min, and removing liquid without washing;
5) dropwise adding a proper amount of primary antibodies, wherein the primary antibodies are rabbit anti-human/mouse microtubule-associated protein 2(MAP-2) antibody, Synaptophysin (SYN) antibody, Glial Fibrillary Acidic Protein (GFAP) antibody, 5-hydroxytryptamine (5-HT) antibody, gamma-aminobutyric acid (GABA) antibody and epinephrine (TH) antibody, and standing at 4 ℃ overnight;
6) dropwise adding a reaction enhancing solution: dripping appropriate amount of reaction enhancing solution, incubating at room temperature for 10min, and soaking in PBS buffer solution for 3 times, each for 3 min;
7) dripping a proper amount of secondary antibody, namely horseradish peroxidase enzyme-labeled goat anti-rabbit IgG polymer, incubating at room temperature for 20min, and soaking for 3 times with PBS (phosphate buffer solution) for 3min each time;
8) color development: adding a proper amount of freshly prepared DAB color development solution, and incubating for 3min at room temperature;
9) counterdyeing: washing with tap water, incubating with hematoxylin staining solution for 3min, differentiating, washing, and returning to blue;
10) alcohol gradient dehydration, xylene transparency, neutral gum sealing;
and S3, observing by using a microscope.
6. The kit of claim 5, wherein the diagnostic criteria for diagnosing aplastic anemia using said kit are: the bone marrow nerve fibers form a characteristic "fishnet" description.
7. Use of a kit according to any one of claims 1 to 6 for the study of aplastic anemia diseases, wherein said use is for non-diagnostic purposes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111205386.6A CN113945722B (en) | 2021-10-15 | 2021-10-15 | Aplastic anemia diagnosis marker and application thereof in preparation of kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111205386.6A CN113945722B (en) | 2021-10-15 | 2021-10-15 | Aplastic anemia diagnosis marker and application thereof in preparation of kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113945722A CN113945722A (en) | 2022-01-18 |
| CN113945722B true CN113945722B (en) | 2022-08-30 |
Family
ID=79330862
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111205386.6A Active CN113945722B (en) | 2021-10-15 | 2021-10-15 | Aplastic anemia diagnosis marker and application thereof in preparation of kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113945722B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006145299A (en) * | 2004-11-17 | 2006-06-08 | Kanazawa Univ | Detection method for autoantibody existing in aplastic anemia patient blood serum |
| CN203535057U (en) * | 2013-11-04 | 2014-04-09 | 北京海思特临床检验所有限公司 | Kit for detecting myelodysplastic syndromes and aplastic anemia |
| CN109187941A (en) * | 2018-08-31 | 2019-01-11 | 暨南大学 | Application of the CD4+CD70+T cell subsets in preparation auxiliary diagnosis pole aplastic anaemia kit |
-
2021
- 2021-10-15 CN CN202111205386.6A patent/CN113945722B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006145299A (en) * | 2004-11-17 | 2006-06-08 | Kanazawa Univ | Detection method for autoantibody existing in aplastic anemia patient blood serum |
| CN203535057U (en) * | 2013-11-04 | 2014-04-09 | 北京海思特临床检验所有限公司 | Kit for detecting myelodysplastic syndromes and aplastic anemia |
| CN109187941A (en) * | 2018-08-31 | 2019-01-11 | 暨南大学 | Application of the CD4+CD70+T cell subsets in preparation auxiliary diagnosis pole aplastic anaemia kit |
Non-Patent Citations (1)
| Title |
|---|
| 小鼠骨髓细胞肾上腺素β2 受体的表达及其意义;童金生等;《吉林大学学报（医学版）》;20090331;第35卷(第2期);第291页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113945722A (en) | 2022-01-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jordan | A Text-book of Histology | |
| Jirkovská et al. | Topological properties and spatial organization of villous capillaries in normal and diabetic placentas | |
| Huang et al. | Lgr6 marks epidermal stem cells with a nerve-dependent role in wound re-epithelialization | |
| CN111149767B (en) | Construction method and application of humanized skin type lupus erythematosus mouse model | |
| CN112639076A (en) | Methods and compositions for promoting cell growth and tissue repair | |
| CN103740639A (en) | Method for constructing humanized Ph chromosome positive acute lymphocytic leukemia mouse model | |
| CN107621462B (en) | Tissue clearing liquid SUT and preparation and application thereof | |
| Golberg et al. | Application of automated immunohistochemistry in anatomical research: A brief review of the method | |
| CN103994913A (en) | Fixed-decalcifying fluid for bone marrow biopsy and paraffin section method of bone marrow biopsy tissue | |
| CN113945722B (en) | Aplastic anemia diagnosis marker and application thereof in preparation of kit | |
| Aamelfot et al. | Characterisation of a monoclonal antibody detecting A tlantic salmon endothelial and red blood cells, and its association with the infectious salmon anaemia virus cell receptor | |
| JP3723204B1 (en) | Impermeable tissue quick fixative | |
| Ohe et al. | Detection of minimal bone marrow involvement of blastic plasmacytoid dendritic cell neoplastic cells-CD303 immunostaining as a diagnostic tool | |
| Downs et al. | Growth in the pre-fusion murine allantois | |
| CN104865115B (en) | Use the method for Fresh Frozen tissue detection monoclonal antibodies drug entities cross reaction | |
| Basting et al. | Fast and accurate protocol for histology and immunohistochemistry reactions in temporomandibular joint of rats | |
| CN106701886B (en) | Method for detecting influence of epithelial-mesenchymal transition process of triple-negative breast cancer cells on secretory function of endothelial cells | |
| CN105079785A (en) | Function and application of TRIM32 (Tripartite motif 32) in treating myocardial hypertrophy | |
| CN105548569A (en) | Detection method for peripheral blood VEGF of renal cancer patient | |
| Hota et al. | Stereological analysis of elastic fibers of the corpus cavernosum of rats during the aging process | |
| RU2429462C2 (en) | Optical clarification of biological tissue specimens | |
| KR102125084B1 (en) | Novel Patient-derived Xenograft Model of Glioblastoma and Use thereof | |
| Khalaniia et al. | Pathomorphology of peripheral organs of immunogenesis in cats with spontaneous feline infectious peritonitis | |
| CN112656805A (en) | Application of substance for inhibiting YTHDF1 activity in preparation of product for preventing or treating gastric cancer | |
| CN114287390B (en) | Method for establishing mouse autoimmune myelofibrosis model |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |