CN108801740B - Iron staining reagent - Google Patents
Iron staining reagent Download PDFInfo
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- CN108801740B CN108801740B CN201810267260.3A CN201810267260A CN108801740B CN 108801740 B CN108801740 B CN 108801740B CN 201810267260 A CN201810267260 A CN 201810267260A CN 108801740 B CN108801740 B CN 108801740B
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- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 title claims abstract description 196
- 229910052742 iron Inorganic materials 0.000 title claims abstract description 98
- 239000012128 staining reagent Substances 0.000 title claims abstract description 46
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 110
- 239000000243 solution Substances 0.000 claims abstract description 91
- 239000000276 potassium ferrocyanide Substances 0.000 claims abstract description 59
- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 claims abstract description 59
- 239000012192 staining solution Substances 0.000 claims abstract description 44
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 43
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 38
- 238000004043 dyeing Methods 0.000 claims abstract description 34
- 235000015655 Crocus sativus Nutrition 0.000 claims abstract description 28
- 244000124209 Crocus sativus Species 0.000 claims abstract description 28
- 239000004248 saffron Substances 0.000 claims abstract description 28
- 235000013974 saffron Nutrition 0.000 claims abstract description 28
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims abstract description 27
- 239000004576 sand Substances 0.000 claims abstract description 23
- 239000007853 buffer solution Substances 0.000 claims abstract description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 79
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 63
- 239000008213 purified water Substances 0.000 claims description 62
- 239000007788 liquid Substances 0.000 claims description 18
- 239000003755 preservative agent Substances 0.000 claims description 17
- 230000002335 preservative effect Effects 0.000 claims description 15
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 claims description 14
- 239000008055 phosphate buffer solution Substances 0.000 claims description 10
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 6
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical compound OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 5
- 239000000834 fixative Substances 0.000 claims description 5
- ZCTXEAQXZGPWFG-UHFFFAOYSA-N imidurea Chemical compound O=C1NC(=O)N(CO)C1NC(=O)NCNC(=O)NC1C(=O)NC(=O)N1CO ZCTXEAQXZGPWFG-UHFFFAOYSA-N 0.000 claims description 5
- BUCIWTBCUUHRHZ-UHFFFAOYSA-K potassium;disodium;dihydrogen phosphate;hydrogen phosphate Chemical compound [Na+].[Na+].[K+].OP(O)([O-])=O.OP([O-])([O-])=O BUCIWTBCUUHRHZ-UHFFFAOYSA-K 0.000 claims description 5
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 4
- 239000007974 sodium acetate buffer Substances 0.000 claims description 4
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 3
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000007983 Tris buffer Substances 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 3
- QYYMDNHUJFIDDQ-UHFFFAOYSA-N 5-chloro-2-methyl-1,2-thiazol-3-one;2-methyl-1,2-thiazol-3-one Chemical group CN1SC=CC1=O.CN1SC(Cl)=CC1=O QYYMDNHUJFIDDQ-UHFFFAOYSA-N 0.000 claims description 2
- 239000007995 HEPES buffer Substances 0.000 claims description 2
- 239000008351 acetate buffer Substances 0.000 claims description 2
- CBMPTFJVXNIWHP-UHFFFAOYSA-L disodium;hydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].OP([O-])([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O CBMPTFJVXNIWHP-UHFFFAOYSA-L 0.000 claims description 2
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims description 2
- WXKPPMQZRGORPB-UHFFFAOYSA-M sodium;2-hydroxypropane-1,2,3-tricarboxylic acid;acetate Chemical compound [Na+].CC([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O WXKPPMQZRGORPB-UHFFFAOYSA-M 0.000 claims description 2
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 claims description 2
- LEAHFJQFYSDGGP-UHFFFAOYSA-K trisodium;dihydrogen phosphate;hydrogen phosphate Chemical compound [Na+].[Na+].[Na+].OP(O)([O-])=O.OP([O-])([O-])=O LEAHFJQFYSDGGP-UHFFFAOYSA-K 0.000 claims description 2
- 239000008363 phosphate buffer Substances 0.000 claims 4
- 229940061607 dibasic sodium phosphate Drugs 0.000 claims 2
- 239000007979 citrate buffer Substances 0.000 claims 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims 1
- 235000019797 dipotassium phosphate Nutrition 0.000 claims 1
- 229940111688 monobasic potassium phosphate Drugs 0.000 claims 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims 1
- 239000012064 sodium phosphate buffer Substances 0.000 claims 1
- 238000010186 staining Methods 0.000 abstract description 22
- 210000004027 cell Anatomy 0.000 abstract description 19
- 230000003834 intracellular effect Effects 0.000 abstract description 12
- 210000001185 bone marrow Anatomy 0.000 abstract description 11
- 210000003924 normoblast Anatomy 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 5
- 210000003714 granulocyte Anatomy 0.000 abstract description 5
- 210000001616 monocyte Anatomy 0.000 abstract description 4
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- 210000002798 bone marrow cell Anatomy 0.000 description 12
- 238000000034 method Methods 0.000 description 9
- 238000005406 washing Methods 0.000 description 9
- 239000012224 working solution Substances 0.000 description 7
- 238000001035 drying Methods 0.000 description 6
- DCYOBGZUOMKFPA-UHFFFAOYSA-N iron(2+);iron(3+);octadecacyanide Chemical compound [Fe+2].[Fe+2].[Fe+2].[Fe+3].[Fe+3].[Fe+3].[Fe+3].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] DCYOBGZUOMKFPA-UHFFFAOYSA-N 0.000 description 5
- 239000002244 precipitate Substances 0.000 description 5
- 238000007447 staining method Methods 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 239000003963 antioxidant agent Substances 0.000 description 3
- 230000003078 antioxidant effect Effects 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 229960003351 prussian blue Drugs 0.000 description 3
- 239000013225 prussian blue Substances 0.000 description 3
- 208000015710 Iron-Deficiency Anemia Diseases 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
- ZINJLDJMHCUBIP-UHFFFAOYSA-N ethametsulfuron-methyl Chemical compound CCOC1=NC(NC)=NC(NC(=O)NS(=O)(=O)C=2C(=CC=CC=2)C(=O)OC)=N1 ZINJLDJMHCUBIP-UHFFFAOYSA-N 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 206010002065 Anaemia megaloblastic Diseases 0.000 description 1
- 208000032467 Aplastic anaemia Diseases 0.000 description 1
- 208000000682 Megaloblastic Anemia Diseases 0.000 description 1
- 208000009527 Refractory anemia Diseases 0.000 description 1
- 206010072684 Refractory cytopenia with unilineage dysplasia Diseases 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000000093 cytochemical effect Effects 0.000 description 1
- 210000001723 extracellular space Anatomy 0.000 description 1
- 231100001016 megaloblastic anemia Toxicity 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000011206 morphological examination Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
Images
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- 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
- G01N2001/302—Stain compositions
-
- 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 belongs to the technical field of biology, and particularly relates to an iron staining reagent. The iron dyeing reagent comprises a sand yellow dyeing solution, a fixing agent, a potassium ferrocyanide solution and a hydrochloric acid solution. The saffron staining solution contains saffron, a buffer solution and alcohol. The arenaceous yellow staining solution is prepared by adopting a buffer solution, so that the structure of a bone marrow iron staining cell is clear, and the erythroblasts, the monocytes and the granulocytes are easily distinguished, so that the observation time of internal iron is greatly shortened; in particular, the contrast of nuclear plasma can be clearer, and the intracellular iron can be more easily observed. In addition, when the fixing agent contains glutaraldehyde, the dyeing effect is better, the contrast of nuclear plasma is clearer, and intracellular iron is easier to observe.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an iron staining reagent.
Background
Iron staining is one of the cytochemical staining methods commonly used for morphological examination of bone marrow cells. It has important value for diagnosing and identifying iron-deficiency anemia, non-iron-deficiency anemia (such as nutritional megaloblastic anemia, aplastic anemia, refractory anemia and the like) and iron granulocyte erythrocytic anemia. Prussian blue staining is a commonly used staining method for displaying iron, and the detection principle is that intracellular and extracellular iron in bone marrow reacts with potassium ferrocyanide in an acid environment to form Prussian blue ferric ferrocyanide precipitates which are positioned at iron-containing parts, and the amount and the depth of blue precipitate particles are in direct proportion to the content of the intracellular and extracellular iron.
In the prior art, most of the cells are difficult to distinguish when bone marrow cell iron staining is carried out, the cell structure is unclear, and the intracellular iron observation is seriously influenced.
Chinese patent publication No. CN105136549A discloses an iron staining kit containing a nuclear red fixing compound staining solution and a staining method thereof, wherein the nuclear red fixing compound staining solution in the iron staining kit contains a detergent TritonX-100. The invention adjusts the component composition of the nuclear red fixation complex staining solution, adds the detergent TritonX-100, makes it easier to distinguish cell types when carrying out bone marrow cytoiron staining compared with the conventional nuclear red fixation complex staining solution, the cell structure is clear, the judgment of observing intracellular iron is more accurate, and simultaneously, in the staining method of the invention, the microwave mode is adopted to replace the conventional water bath mode, so that the integral staining time is greatly shortened.
Disclosure of Invention
The invention aims to provide an iron staining reagent, which can make bone marrow iron staining cell structure clear and cell types easily distinguished, thereby being easier to observe iron in cells.
The invention adopts the following technical scheme:
a saffron staining solution comprises saffron and buffer solution.
Further, the buffer solution is selected from one or more of Tris buffer solution, HEPES buffer solution, phosphate buffer solution, acetate buffer solution, citric acid buffer solution and the like; preferably comprises one or more of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, potassium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, acetic acid-sodium acetate buffer solution, citric acid-sodium citrate buffer solution, citric acid-sodium acetate buffer solution, citric acid-disodium hydrogen phosphate buffer solution, etc.
Further, the pH value range of the sallow dyeing liquid is 4.5-6.5; preferably 5.2 to 6.2; further preferably 5.8.
Preferably, the buffer solution is a potassium dihydrogen phosphate-disodium hydrogen phosphate buffer solution; more preferably, the concentration of the potassium dihydrogen phosphate in the salsa yellow staining solution is 14.46g/L, and the concentration of the disodium hydrogen phosphate in the salsa yellow staining solution is 3.72 g/L.
Further, the concentration of the yellow sand in the yellow sand dyeing liquid is 0.04-5g/L, preferably 0.06-1g/L, and more preferably 0.1 g/L.
Furthermore, the sallow dyeing solution can also contain a proper amount of preservative. The preservative may be selected from one or more of ProClin series preservatives (supplied by SUPELCO corporation), sodium azide, phenol, imidazolidinyl urea (IZU), and the like; wherein the ProClin series preservative is preferably ProClin 300.
The content of the preservative in the sallow dyeing liquid is preferably 0.1-5g/L, preferably 1-4g/L, or preferably 1-2 g/L.
The yellow sand dyeing liquid can be prepared by a conventional method.
The invention surprisingly discovers that when the sallow staining solution containing the buffer solution is used for iron staining of bone marrow cells, the sallow staining solution not only can make the cell structure clear, but also can easily distinguish the erythroblasts, the monocytes and the granulocytes; but also can make the contrast of nuclear plasma clearer and make the observation of iron in cells easier.
The invention also provides an iron staining reagent (or an iron staining kit), which comprises the saffron staining solution. The iron staining reagent (or iron staining kit) also comprises one or more of a fixing agent (or fixing solution), a potassium ferrocyanide solution, a hydrochloric acid solution and the like.
Wherein, the fixing agent (or called fixing liquid), the potassium ferrocyanide solution and the hydrochloric acid solution can be prepared according to the requirements of the conventional iron dyeing method.
In order to improve the dyeing effect better, the fixing agent is preferably prepared from one or more of ethanol, formaldehyde and glutaraldehyde. Particularly, when the fixing agent contains glutaraldehyde, the dyeing effect is better, the contrast of nuclear plasma is clearer, and intracellular iron is easier to observe.
Further, the concentrations of ethanol, formaldehyde and glutaraldehyde in the fixing agent are preferably 650-900ml/L, 50-150ml/L and 10-100ml/L respectively.
The concentrations of ethanol, formaldehyde and glutaraldehyde in the fixing agent are more preferably 750-890ml/L, 60-130ml/L and 20-80ml/L respectively.
Preferably, the concentrations of ethanol, formaldehyde and glutaraldehyde in the fixing agent are 810ml/L, 90ml/L and 50ml/L respectively.
The glutaraldehyde used in the present invention is 50% glutaraldehyde, unless otherwise specified.
Further, the concentration of the potassium ferrocyanide solution is 1-200g/L, preferably 20-200g/L, more preferably 120-150g/L, and preferably 108 g/L.
In order to solve the problem that the potassium ferrocyanide solution is easy to oxidize and lose efficacy, the potassium ferrocyanide solution can also contain a proper amount of antioxidant. The antioxidant is preferably sulfurous acid, and the content of the antioxidant is preferably 5 +/-1 g/L.
Further, the concentration of the hydrochloric acid solution is 1-200g/L, preferably 20-200g/L, more preferably 120-150g/L, and preferably 108 g/L.
When the concentrations of the potassium ferrocyanide solution and the hydrochloric acid solution are the same, the potassium ferrocyanide solution and the hydrochloric acid solution can be uniformly mixed according to the volume ratio of 1:1 before use and then used, and drop dyeing or dip dyeing can be selected.
If not stated otherwise, the invention uses water as solvent.
Specifically, the iron staining reagent comprises a fixing agent, a potassium ferrocyanide solution, a hydrochloric acid solution and a saffron staining solution, and consists of the following components:
| fixing agent | Potassium ferrocyanide solution | Hydrochloric acid solution | Sand yellow dyeing liquid |
| 65-90% of ethanol | 0.1-20% of potassium ferrocyanide | 0.1 to 20 percent of concentrated hydrochloric acid | 0.004-0.5% of saffron yellow |
| 5 to 15 percent of formaldehyde | 0 to 0.6 percent of sulfurous acid | The balance of purified water; | 0.05 to 10 percent of buffer solution |
| 1-10% of 50% glutaraldehyde | The balance of purified water; | 0.01-0.5% of preservative; | |
| the balance of purified water; | and (4) purifying the balance of water. |
Preferably, the iron staining reagent comprises a fixing agent, a potassium ferrocyanide solution, a hydrochloric acid solution and a saffron staining solution, and consists of the following components:
| fixing agent | Potassium ferrocyanide solution | Hydrochloric acid solution | Sand yellow dyeing liquid |
| 75-89% of ethanol | 2-20% of potassium ferrocyanide | 2 to 20 percent of concentrated hydrochloric acid | 0.006-0.1% of saffron yellow |
| 6-13% of formaldehyde | 0 to 0.5 percent of sulfurous acid | The balance of purified water; | 0.2 to 6 percent of buffer solution |
| 2-8% of 50% glutaraldehyde | The balance of purified water; | 0.1-0.4% of preservative; | |
| the balance of purified water; | and (4) purifying the balance of water. |
Preferably, the iron staining reagent comprises a fixing agent, a potassium ferrocyanide solution, a hydrochloric acid solution and a saffron staining solution, and consists of the following components:
| fixing agent | Potassium ferrocyanide solution | Hydrochloric acid solution | Sand yellow dyeing liquid |
| Ethanol 81 percent | Potassium ferrocyanide 10.8% | Concentrated hydrochloric acid 10.8% | 0.01 percent of saffron yellow |
| Formaldehyde 9% | Sulphurous acid0 to 0.5 percent of acid | The balance of purified water; | 0.5 percent of buffer solution |
| 50% glutaraldehyde 5% | The balance of purified water; | 0.2 percent of preservative; | |
| the balance of purified water; | and (4) purifying the balance of water. |
The invention also comprises the application of the saffron yellow staining solution or the iron staining reagent (or the iron staining kit) in iron staining, in particular the application in iron staining of bone marrow cells and the like.
The invention also provides a method for staining bone marrow cells with iron, which comprises the following steps:
1) fixing the bone marrow smear by dripping a fixative (generally, the fixative is fully smeared on the smear for about 60 seconds), and washing with water until the bone marrow smear is dried;
2) preparing a working solution from a potassium ferrocyanide solution and a hydrochloric acid solution, and dyeing a bone marrow smear by using the working solution;
generally, the smear is applied dropwise or immersed in a working solution until the smear is covered, and then stained at about 37 ℃ for about 30 minutes; then fully washing with purified water until the water is dried or sucking the water with filter paper;
3) re-dyeing with the yellow-sand dyeing solution, washing with water, drying and performing microscopic examination.
Generally, the dyeing can be counterdyed at room temperature for about 2 minutes.
The starting materials used in the present invention are commercially available or may be prepared by methods conventional in the art.
On the basis of the common knowledge in the field, the above preferred conditions can be combined with each other to obtain the preferred embodiments of the invention.
Furthermore, the potassium ferrocyanide solution and the hydrochloric acid solution can be used for preparing a working solution; preferably, the content of the potassium ferrocyanide and the content of the hydrochloric acid in the working solution are respectively 2% -20%.
The detection principle is as follows: stored iron in normal human bone marrow is found primarily in bone marrow granules and in erythroblasts.
Iron in bone marrow reacts with potassium ferrocyanide in acidic environment to form Prussian blue ferric ferrocyanide precipitate
A starch, localized to a site containing iron.
And (4) judging a result: the nucleus of the erythroblast is bright red, the pulp is light yellow red, and the iron granules are blue-green. Cells
No blue precipitate was negative in the inner (outer) and intercellular spaces and a blue precipitate was positive.
Compared with the prior art, the invention has the following advantages:
the sand yellow staining solution is prepared by adopting buffer solution, so that the marrow iron staining cell structure is clear and easy to realize
Greatly shortens the observation time of internal iron by distinguishing the erythroblasts, the monocytes and the granulocytes; in particular
The contrast of nuclear plasma can be clearer, and the iron in the cells can be more easily observed. In addition, when in the fixing agent
When the dye contains glutaraldehyde, the dye has better dyeing effect, is more beneficial to making the contrast of nuclear pulp clearer and is easier to observe fine
Intracellular iron.
Drawings
FIGS. 1 to 2 are graphs showing the results of experiments in examples 1 to 2, respectively.
FIGS. 3 to 6 are graphs showing the results of experiment in Experimental example 3.
FIGS. 7 to 10 are graphs showing the results of experiments in Experimental examples 4 to 7, respectively.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit 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 available from regular distributors, not indicated by the manufacturer.
The percent in the present invention means mass percent or volume percent, and the percent of solids, unless otherwise specified, means the grams of solute contained in 100mL of solution; the percentage between the liquids refers to the ratio of the volumes at 20 ℃. Unless otherwise specified, "part" in the present invention means "part by weight".
Example 1
An iron staining reagent comprises a fixing agent, a potassium ferrocyanide solution, a hydrochloric acid solution and a saffron staining solution, and consists of the following components:
| fixing agent | Potassium ferrocyanide solution | Hydrochloric acid solution | Sand yellow dyeing liquid |
| Ethanol 81 percent | Potassium ferrocyanide 10.8% | Concentrated hydrochloric acid 10.8% | 0.01 percent of saffron yellow |
| Formaldehyde 9% | The balance of purified water; | the balance of purified water; | 0.5 percent of phosphate buffer solution |
| 50% glutaraldehyde 5% | Proclin300 0.2％ | ||
| The balance of purified water; | and (4) purifying the balance of water. |
The buffer solution in the sallow staining solution is potassium dihydrogen phosphate-disodium hydrogen phosphate buffer solution; the concentration of potassium dihydrogen phosphate in the saffron staining solution is 14.46g/L, the concentration of disodium hydrogen phosphate in the saffron staining solution is 3.72g/L, and the pH value of the buffer solution is 5.8.
Example 2
An iron staining reagent comprises a fixing agent, a potassium ferrocyanide solution, a hydrochloric acid solution and a saffron staining solution, and consists of the following components:
| fixing agent | Potassium ferrocyanide solution | Hydrochloric acid solution | Sand yellow dyeing liquid |
| 90 percent of ethanol | 20 percent of potassium ferrocyanide | Concentrated hydrochloric acid 20% | 0.5 percent of saffron yellow |
| Formaldehyde 15% | The balance of purified water; | the balance of purified water; | tris buffer 10% |
| 50% glutaraldehyde 10% | Sodium azide 0.5% | ||
| The balance of purified water; | and (4) purifying the balance of water. |
Example 3
An iron staining reagent comprises a fixing agent, a potassium ferrocyanide solution, a hydrochloric acid solution and a saffron staining solution, and consists of the following components:
| fixing agent | Potassium ferrocyanide solution | Hydrochloric acid solution | Sand yellow dyeing liquid |
| 65 percent of ethanol | 0.1 percent of potassium ferrocyanide | Concentrated hydrochloric acid 0.1% | 0.004 percent of saffron yellow |
| Formaldehyde 5% | The balance of purified water; | the balance of purified water; | hepes buffer 0.05% |
| 50% glutaraldehyde 1% | 0.01 percent of phenol | ||
| The balance of purified water; | and (4) purifying the balance of water. |
Example 4
An iron staining reagent comprises a fixing agent, a potassium ferrocyanide solution, a hydrochloric acid solution and a saffron staining solution, and consists of the following components:
| fixing agent | Potassium ferrocyanide solution | Hydrochloric acid solution | Sand yellow dyeing liquid |
| 89 percent of ethanol | 20 percent of potassium ferrocyanide | Concentrated hydrochloric acid 20% | 0.1 percent of saffron yellow |
| 13 percent of formaldehyde | The balance of purified water; | the balance of purified water; | acetic acid buffer solution 6% |
| 50% glutaraldehyde 8% | Imidazolidinyl urea (IZU) 0.4% | ||
| The balance of purified water; | and (4) purifying the balance of water. |
Example 5
An iron staining reagent comprises a fixing agent, a potassium ferrocyanide solution, a hydrochloric acid solution and a saffron staining solution, and consists of the following components:
| fixing agent | Potassium ferrocyanide solution | Hydrochloric acid solution | Sand yellow dyeing liquid |
| 75 percent of ethanol | 2 percent of potassium ferrocyanide | Concentrated hydrochloric acid 2% | 0.006% of saffron yellow |
| 6 percent of formaldehyde | The balance of purified water; | the balance of purified water; | 0.2 percent of buffer solution |
| 50% glutaraldehyde 2% | 0.1 percent of preservative | ||
| The balance of purified water; | and (4) purifying the balance of water. |
Experimental example 1
Bone marrow cells were iron stained using the iron staining reagent of example 1 as follows:
1) naturally drying the bone marrow smear in the air, dripping a fixing agent, fully spreading the smear for fixing for about 60 seconds, washing with purified water, and drying or sucking the smear with filter paper;
2) dropping or immersing the smear with working solution, dyeing for 30 minutes at 37 ℃, fully washing with purified water, and drying or sucking with filter paper; wherein the working solution is prepared from a potassium ferrocyanide solution and a hydrochloric acid solution according to the volume ratio of 1: 1;
3) and re-dyeing the sallow dyeing solution at room temperature for 2 minutes, washing with purified water, drying and performing microscopic examination.
The experimental result is shown in figure 1, the arrows indicate intracellular iron and extracellular iron, the cell structure is clear, and the intracellular iron is easier to identify.
Experimental example 2
The bone marrow cell iron staining method is different from the method in the embodiment 1 only in the step 3) that the nuclear red fixing compound staining solution described in the embodiment 2 of Chinese patent publication No. CN105136549A is adopted; the method comprises the following specific steps: 1) and (4) dropwise adding 5-8 drops of fixing liquid into the sample, and covering the sample. After 10 minutes, washing with water to be dried for later use;
2) slowly adding a hydrochloric acid solution into a potassium ferrocyanide solution, mixing, putting a sample into the mixed solution, dyeing the sample in a water bath tank at 37 ℃ for 60 minutes, and fully washing with purified water until the sample is dried;
3) and (5) re-dyeing the nuclei for 1-2 minutes, washing with purified water, drying and performing microscopic examination.
The experimental results are shown in FIG. 2, and the arrows indicate intracellular iron and extracellular iron, so that the cells are relatively fuzzy, the cell types are not easy to be distinguished, and the observation of the intracellular iron is seriously influenced.
The experimental results show that the method in the experimental example 1 has clear cell structure, is easy to distinguish the erythroblasts, the monocytes and the granulocytes, is easy to observe iron in the cells, and greatly shortens the observation time of the iron; and the contrast of the nuclear pulp is clearer compared with that of the experimental example 2.
Experimental example 3
Bone marrow cells were iron-stained using the iron staining reagents of examples 2 to 5, respectively, in the same manner as in example 1.
The results are shown in fig. 3-6, with the darkest coloration in fig. 3 and the lightest coloration in fig. 4, and the iron particles are not well identified; fig. 5 is darker and fig. 6 is lighter. The results show that the iron staining reagent of examples 2-5 has slightly poorer staining effect than that of example 1, but the contrast of the nuclear pulp is clearer and better than that of CN 105136549A.
Experimental example 4
The method of bone marrow cell iron staining differs from example 1 only in that the saffron staining solution used in step 3) counterstaining does not contain buffer. The results are shown in FIG. 7, where needle crystals appear and the background is unclear.
Experimental example 5
The method of iron staining of bone marrow cells, which differs from example 1 only in that the fixative used in step 1) does not contain glutaraldehyde. The results are shown in FIG. 8, the cell types are blurred and indistinguishable.
Experimental example 6
Bone marrow cells were iron-stained in the same manner as in Experimental example 1 using the following iron staining reagent, and the results are shown in FIG. 9, and the staining was too light to distinguish iron particles.
| Fixing agent | Potassium ferrocyanide solution | Hydrochloric acid solution | Shahuang solution |
| Ethanol is less than 65 percent | Potassium ferrocyanide < (R) >, and its preparation method0.1％ | Concentrated hydrochloric acid is less than 0.1 percent | The sand yellow is less than 0.004 percent |
| The formaldehyde is less than 5 percent | The balance of purified water; | the balance of purified water; | purified water balance |
| (50%) glutaraldehyde < 1% | / | / | The preservative is less than 0.01 percent |
| The balance of purified water; | / | / | the buffer solution is less than 0.05 percent |
Experimental example 7
Bone marrow cells were iron-stained in the same manner as in Experimental example 1 using the following iron-staining reagent, and the results are shown in FIG. 10, and the staining was too dark to be seen.
| Fixing agent | Potassium ferrocyanide solution | Hydrochloric acid solution | Shahuang solution |
| Ethanol is more than 90 percent | Potassium ferrocyanide is more than 20% | Concentrated hydrochloric acid is more than 20 percent | More than 0.5 percent of saffron yellow |
| The formaldehyde is more than 15 percent | Purified water balance | Purified water balance | Purified water balance |
| (50%) glutaraldehyde > 10% | / | / | The preservative is more than 0.5 percent |
| The balance of purified water; | / | / | the buffer solution is more than 10 percent |
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (21)
1. The iron staining reagent is characterized by comprising a fixing agent, a potassium ferrocyanide solution, a hydrochloric acid solution and a yellow sand staining solution, and the iron staining reagent consists of the following components:
fixing agent:
65-90% of ethanol
5 to 15 percent of formaldehyde
1-10% of 50% glutaraldehyde
The balance of purified water;
potassium ferrocyanide solution:
0.1-20% of potassium ferrocyanide
0 to 0.6 percent of sulfurous acid
The balance of purified water;
hydrochloric acid solution:
0.1 to 20 percent of concentrated hydrochloric acid
The balance of purified water;
sand yellow staining solution:
0.004-0.5% of saffron yellow
0.05 to 10 percent of buffer solution
0.01 to 0.5 percent of preservative
The balance of purified water; wherein the pH value range of the sallow dyeing liquid is 4.5-6.5.
2. The iron staining reagent of claim 1, wherein the buffer is selected from one or more of Tris buffer, HEPES buffer, phosphate buffer, acetate buffer, and citrate buffer.
3. The iron staining reagent of claim 1, wherein the buffer is selected from one or more of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, potassium dihydrogen phosphate-disodium hydrogen phosphate buffer, acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer, citric acid-sodium acetate buffer, and citric acid-disodium hydrogen phosphate buffer.
4. The iron staining reagent of claim 1, wherein the buffer is a monobasic potassium phosphate-dibasic sodium phosphate buffer, the concentration of monobasic potassium phosphate in the saffron staining solution is 14.46g/L, and the concentration of dibasic sodium phosphate in the saffron staining solution is 3.72 g/L.
5. The iron staining reagent of any one of claims 1 to 4, wherein the pH of the saffron staining solution is in the range of 5.2 to 6.2.
6. The iron staining reagent of any one of claims 1 to 4 wherein the pH of the saffron staining solution is 5.8.
7. The iron staining reagent of any one of claims 1 to 4, wherein the concentration of saffron in the saffron staining solution is from 0.04 to 5 g/L.
8. The iron staining reagent of any one of claims 1 to 4, wherein the concentration of saffron in the saffron staining solution is from 0.06 to 1 g/L.
9. The iron staining reagent of any one of claims 1 to 4, wherein the concentration of saffron in the saffron staining solution is 0.1 g/L.
10. The iron staining reagent of claim 1 wherein the preservative is selected from one or more of ProClin series preservatives, sodium azide, phenol, imidazolidinyl urea.
11. The iron staining reagent of claim 10 wherein the ProClin series preservative is ProClin 300.
12. The iron staining reagent as claimed in claim 1 wherein the concentrations of ethanol and formaldehyde in the fixative are 650-900ml/L and 50-150ml/L respectively, and the addition amount of 50% glutaraldehyde is 10-100 ml/L.
13. The iron staining reagent as claimed in claim 1, wherein the concentrations of ethanol and formaldehyde in the fixing agent are respectively 750-890ml/L and 60-130ml/L, and the addition amount of 50% glutaraldehyde is 20-80 ml/L.
14. The iron staining reagent of claim 1 wherein the concentrations of ethanol and formaldehyde in the fixative are 810ml/L and 90ml/L, respectively, and the amount of 50% glutaraldehyde added is 50 ml/L.
15. The iron staining reagent of claim 1 wherein the concentration of the potassium ferrocyanide solution is 1-200 g/L; and/or the addition amount of the concentrated hydrochloric acid is 1-200 g/L.
16. The iron staining reagent of claim 1 wherein the concentration of the potassium ferrocyanide solution is 20-200 g/L; and/or the addition amount of the concentrated hydrochloric acid is 20-200 g/L.
17. The iron staining reagent as claimed in claim 1 wherein the concentration of the potassium ferrocyanide solution is 150 g/L; and/or the addition amount of the concentrated hydrochloric acid is 120-150 g/L.
18. The iron staining reagent of claim 1 wherein the concentration of the potassium ferrocyanide solution is 108 g/L; and/or the addition amount of the concentrated hydrochloric acid is 108 g/L.
19. The iron staining reagent is characterized by comprising a fixing agent, a potassium ferrocyanide solution, a hydrochloric acid solution and a yellow sand staining solution, and the iron staining reagent consists of the following components:
fixing agent:
75-89% of ethanol
6-13% of formaldehyde
2-8% of 50% glutaraldehyde
The balance of the purified water is the purified water,
potassium ferrocyanide solution:
2-20% of potassium ferrocyanide
0 to 0.5 percent of sulfurous acid
The balance of the purified water is the purified water,
hydrochloric acid solution:
2 to 20 percent of concentrated hydrochloric acid
The balance of the purified water is the purified water,
sand yellow staining solution:
0.006-0.1% of saffron yellow
0.2 to 6 percent of buffer solution
0.1 to 0.4 percent of preservative
The balance of purified water; wherein the pH value range of the sallow dyeing liquid is 5.2-6.2.
20. The iron staining reagent is characterized by comprising a fixing agent, a potassium ferrocyanide solution, a hydrochloric acid solution and a yellow sand staining solution, and the iron staining reagent consists of the following components:
fixing agent:
ethanol 81 percent
Formaldehyde 9%
50% glutaraldehyde 5%
The balance of the purified water is the purified water,
potassium ferrocyanide solution:
potassium ferrocyanide 10.8%
0 to 0.5 percent of sulfurous acid
The balance of the purified water is the purified water,
hydrochloric acid solution:
concentrated hydrochloric acid 10.8%
The balance of the purified water is the purified water,
sand yellow staining solution:
0.01 percent of saffron yellow
0.5 percent of buffer solution
0.2 percent of preservative
The balance of purified water; wherein the pH value of the sallow dyeing liquid is 5.8.
21. The iron staining reagent is characterized by comprising a fixing agent, a potassium ferrocyanide solution, a hydrochloric acid solution and a yellow sand staining solution, and the iron staining reagent consists of the following components:
fixing agent:
ethanol 81 percent
Formaldehyde 9%
50% glutaraldehyde 5%
The balance of the purified water is the purified water,
potassium ferrocyanide solution:
potassium ferrocyanide 10.8%
The balance of the purified water is the purified water,
hydrochloric acid solution
Concentrated hydrochloric acid 10.8%
The balance of the purified water is the purified water,
sand yellow staining solution:
0.01 percent of saffron yellow
0.5% of phosphate buffer solution, wherein the phosphate buffer solution is potassium dihydrogen phosphate-disodium hydrogen phosphate buffer solution; the concentration of potassium dihydrogen phosphate in the saffron staining solution is 14.46g/L, the concentration of disodium hydrogen phosphate in the saffron staining solution is 3.72g/L, and the pH value of the buffer solution is 5.8;
Proclin300 0.2%
and (4) purifying the balance of water.
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| CN201810267260.3A CN108801740B (en) | 2018-03-28 | 2018-03-28 | Iron staining reagent |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6082847A (en) * | 1983-10-13 | 1985-05-11 | Dai Ichi Pure Chem Co Ltd | Electrophoretic method and coloring matter reagent for detection used therein |
| CN105074422A (en) * | 2013-03-15 | 2015-11-18 | 艾瑞思国际股份有限公司 | Method and composition for staining and sample processing |
| CN105556276A (en) * | 2013-07-01 | 2016-05-04 | 思迪赛特诊断有限公司 | A method, kit and system for imaging a blood sample |
| CN106053167A (en) * | 2016-05-19 | 2016-10-26 | 四川金域医学检验中心有限公司 | Preparation method of marrow fluid smear |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1576957A1 (en) * | 2004-03-18 | 2005-09-21 | Universiteit Twente | Tissue repair using pluripotent cells |
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2018
- 2018-03-28 CN CN201810267260.3A patent/CN108801740B/en active Active
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6082847A (en) * | 1983-10-13 | 1985-05-11 | Dai Ichi Pure Chem Co Ltd | Electrophoretic method and coloring matter reagent for detection used therein |
| CN105074422A (en) * | 2013-03-15 | 2015-11-18 | 艾瑞思国际股份有限公司 | Method and composition for staining and sample processing |
| CN105556276A (en) * | 2013-07-01 | 2016-05-04 | 思迪赛特诊断有限公司 | A method, kit and system for imaging a blood sample |
| CN106053167A (en) * | 2016-05-19 | 2016-10-26 | 四川金域医学检验中心有限公司 | Preparation method of marrow fluid smear |
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