CN103675297A - Medicament-induced hemolytic anemia detection kit and detection method thereof - Google Patents
Medicament-induced hemolytic anemia detection kit and detection method thereof Download PDFInfo
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- CN103675297A CN103675297A CN201310674565.3A CN201310674565A CN103675297A CN 103675297 A CN103675297 A CN 103675297A CN 201310674565 A CN201310674565 A CN 201310674565A CN 103675297 A CN103675297 A CN 103675297A
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- 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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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- G01N2800/00—Detection or diagnosis of diseases
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Abstract
The invention discloses a medicament-induced hemolytic anemia detection kit and also discloses a detection method using the kit. When plasma and antibody release liquid of a patient generate a positive reaction with corresponding medicament-sensitized erythrocytes, the plasma and the antibody release liquid of the patient generate a negative reaction with unsensitized erythrocytes; or a medicament is added into the plasma, the plasma or medicament antibodies in the antibody release liquid generates a positive reaction with normal erythrocytes, the plasma which is not added with the medicament or a dispersion solution generate a negative reaction with the normal erythrocytes. The medicament species causing the hemolytic anemia can be diagnosed accurately according to the results so as to guide to correctly select appropriate medicaments for treating the patient in clinic. The method has high accuracy, and is applicable to the detection by a test tube method or a microcolumn gel card method.
Description
Technical field
The present invention relates to field of biological detection, especially relate to drug induced hemolytic anemia detection kit, the invention still further relates to the method for using this kit detection of drugs induction type hemolytic anemia.
Background technology
Along with rolling up of types of drugs, especially antibiotics is in clinical a large amount of uses, drug induced hemolytic anemia case is rolled up, because thereby immune factor causes red blood cell considerable damage, there is clinically the haemolysis performances such as anaemia, jaundice, color of soy sauce urine, gently affect result for the treatment of, heavy delay treatment jeopardizes patient's life security.
How to avoid patient because using medicine to cause drug induced hemolytic anemia, just must carry out drug induced hemolytic anemia to patient and carry out detection promptly and accurately, stop using the medicine that can make patient produce drug induced hemolytic anemia, instruct clinically in patient selects suitable medicine, improve result for the treatment of.
At present, for drug induced hemolytic anemia, detect and all effectively carried out clinical.Its reason is that drug induced hemolytic anemia detection belongs to immunohematology sensing range, and immunohematology detection is only carried out in Blood Transfusion Dept. or the blood bank of clinical hospitals, Blood Transfusion Dept. or blood bank are only responsible for the content checking before blood transfusion, all do not carry out drug induced hemolytic anemia testing, therefore for drug induced hemolytic anemia patient, can not obtain detecting timely diagnosis, cause patient's fatal rate up to more than 30%.
Medicine causes sometimes the antibody of medicine itself is formed, the drug antibody forming can sensitization or is adsorbed on erythrocyte, causes the direct antihuman globulin test of erythrocyte positive, causes that erythrocyte destroys, produce immune hemolysis, patient's hemochrome declines.Result is: the curative effect of medication that 1, treatment patient uses is lowered or suppresses.2, patient causes anaemia because of drug-induced haemolysis.
For fear of because of drug-induced hemolytic anemia, improve the result for the treatment of of medicine, which kind of drug-induced hemolytic anemia need to carry out Serological testing to drug-induced hemolytic anemia, be to determine, instructs medicine that clinical selecting properly is suitable for the treatment to patient.
Detection of drugs induction type hemolytic anemia there is no detection kit at present, and also can not detect immune hemolytic anemia is that drug-induced detection reagent actually.The present invention is for detection of confirming that medicine causes the medicament categories of patient's hemolytic anemia, instructs medicine that clinical selecting properly is suitable for the treatment to patient.
Summary of the invention
The object of the present invention is to provide the kit of detection of drugs induction type hemolytic anemia, which kind of drug-induced hemolytic anemia uses this kit accurately to judge is; The invention also discloses the using method of using this kit, the method accuracy is good, is applicable to test tube method or micro-column gel block-regulations and detects.
For achieving the above object, the present invention can adopt following technical proposals:
The kit of detection of drugs induction type hemolytic anemia of the present invention, comprises drug dilution liquid, antibody release reagent and Antibody screening reagent.For the drug induced hemolytic anemia patient of the direct antihuman globulin test positive, utilize serological method to be confirmed to be which kind of drug induced hemolytic anemia.Can there is positive reaction in the red blood cell that drug induced hemolytic anemia patient's blood plasma or antibody discharge its corresponding medicine sensitization of drug antibody in liquid, and with unsensitized red blood cell generation negative reaction; Or add under blood plasma or antibody release liquid conditional at medicine, drug antibody and normocyte that blood plasma or antibody discharge in liquid produce positive reaction, and do not add the blood plasma of medicine or diffuse liquid and normocyte generation negative reaction.The medicine that adopts patient to use is in the recent period prepared medicine sensitized erythrocyte and is detected corresponding antibody in patient's blood plasma; Add medicine to blood plasma or diffuse in liquid, detecting patient's blood plasma, diffuse corresponding antibody in liquid.Determine which kind of drug-induced hemolytic anemia is drug induced hemolytic anemia be, instruct medicine that clinical selecting properly is suitable for the treatment to patient, and provide foundation for the suitable medicine of selection of clinical is used for the treatment of.
Described drug dilution liquid comprises barbitol buffer solution, phosphate buffer and bovine serum albumin solution.
Described Antibody screening reagent is broad spectrum antihumanglobulin reagent, i.e. anti-IgG and anti-C3d.
Described drug induced hemolytic anemia detects uses respectively medicine sensitized erythrocyte and enzyme processing red blood cell to detect.
Described antibody release reagent is the reagent that antibody is dissociated from red blood cell, diffuses elution reagent.
Described antibody release reagent is that thermolysis reagent, citric acid diffuse reagent, chloroform/triclene and diffuse that reagent, dimethylbenzene diffuse reagent, glycocoll-hydrochloric acid/disodium ethylene diamine tetraacetate diffuses one or more in reagent.
Described enzyme is processed one or more in the red blood cell that red blood cell is bromelain, papain, cradin processing.
The detection method of kit of the present invention, comprises the steps:
The first step, selects normal person O type red blood cell, and the medicine that patient uses is in the recent period prepared medicine sensitized erythrocyte;
Second step, is used antibody release reagent to release in connection with the antibody at Surface of Erythrocytes, and Dispersal risk discharges liquid;
The 3rd step, is used medicine sensitized erythrocyte to carry out the detection of drug induced hemolytic anemia;
The 4th step, the normal person O type red blood cell that uses enzyme to process carries out the detection of drug induced hemolytic anemia.
The object of the invention is to, by test tube method or micro-column gel block-regulations detection of drugs induction type hemolytic anemia, when result meets table 1 and table 2, can determine it is this drug-induced hemolytic anemia.
Table 1 is used medicine sensitized erythrocyte to carry out the testing result of drug induced hemolytic anemia
Table 2 is used enzyme to process the testing result that red blood cell carries out drug induced hemolytic anemia
Note: "+" represents positive; " 0 " represents negative.
embodiment:
Enumerate respectively test tube method kit and micro-column gel agglutination assay kit below, and illustrate that it detects using method.
Embodiment 1:
Detection of drugs induction type hemolytic anemia of the present invention (test tube method) comprising:
1. drug dilution liquid: comprise drug dilution liquid I, drug dilution liquid II, drug dilution liquid III.
2. antibody release reagent: comprise that antibody discharges that liquid I, antibody discharge liquid II, antibody discharges liquid III.
3. Antibody screening reagent: broad spectrum antihumanglobulin reagent (anti-IgG+ C
3d).
Detection method:
1. the preparation of medicine sensitized erythrocyte:
(1), the preparation of drug solution: the medicine (as penicillin) of getting appropriate patient's use is dissolved in drug dilution liquid I(pH 9.6 barbitol buffer solutions), making drug concentration is 1mg/ml.
(2) by 0.9% sodium chloride solution washing 3 times for normal person O type packed red cells 0.5ml, obtain packed red cells.
(3) get packed red cells that step (2) obtains with being mixed with the red cell suspension of 2 ~ 4% concentration in 0.9% physiological sodium chloride solution, be non-medicine sensitized erythrocyte suspension.Separately getting the packed red cells that 0.3ml step (2) obtains mixes with 4.5ml drug solution.
(4) incubated at room is 1 hour, within every 15 minutes, mixes once.
(5) with 0.9% sodium chloride solution washing step (4) packed red cells, until the packed red cells that supernatant, without haemolysis, is obtained is with being mixed with the red cell suspension of 2 ~ 4% concentration in 0.9% sodium chloride solution, be medicine sensitized erythrocyte suspension.
2. antibody discharges the preparation of liquid:
(1) antibody discharges the preparation of liquid I: 100g disodium ethylene diamine tetraacetate (Na
2eDTA) be dissolved in distilled water or deionized water, be settled to 1000ml;
(2) antibody discharges the preparation of liquid II: 0.75g glycocoll (Gly) is dissolved in 0.9% sodium chloride solution, is settled to 100ml, with 12mol/L HCl, adjusts pH to 1.5.
The preparation of working fluid: antibody discharges liquid I to be mixed according to 1:4 volume ratio with antibody release liquid II.
(3) antibody discharges the preparation of liquid III: 12.1g trishydroxymethylaminomethane (Tris), 5.25g sodium chloride (NaCl), is dissolved in distilled water or deionized water, is settled to 100ml.
(4) get 1, test tube, add 2ml working fluid, then add 1ml with 0.9% sodium chloride solution, to wash the sample packed red cells to be checked obtaining after 5-6 time, mix.
(5) incubated at room is 2 minutes, centrifugal 1 minute of 3000rpm.
(6) supernatant is moved in another clean test tube, drip antibody and discharge liquid III, tune pH to 7.5 is reaction end.
(7) 3000rpm is centrifugal 3 minutes, separation of supernatant, and supernatant is antibody and discharges liquid.
3. use medicine sensitized erythrocyte to carry out the detection of drug induced hemolytic anemia:
(1) get 6 test tubes, be labeled as respectively " 1 ~ 6 ".In the test tube of " 1,3 " mark, add patient's blood plasma 100 μ l, in the test tube of " 2,4 " mark, add antibody to discharge liquid 100 μ l, in the test tube of " 3,6 " mark, add human normal plasma 100 μ l, respectively to each the 50 μ l of medicine sensitized erythrocyte suspension that add 2 ~ 4% in the test tube of " 1 ~ 3 " mark, in the test tube of " 4 ~ 6 " mark, add each 50 μ l of non-medicine sensitized erythrocyte suspension of 2 ~ 4% again.
(2) mix, put 37 ° of C water-baths and hatch 30 minutes.After taking out, with 0.9% sodium chloride solution washing three times, last drains, then adds respectively broad spectrum antihumanglobulin reagent (anti-IgG+ C
3d) 100 μ l.
(3) put hydro-extractor centrifugal (3000rpm * 15 second), observations record.
4. the normal person O type red blood cell that uses enzyme to process carries out the detection of drug induced hemolytic anemia:
(1) serum is processed: get 4 test tubes, be labeled as respectively " mixed liquor 1, mixed liquor 2, contrast liquid 1, contrast liquid 2 ", " mixed liquor 1 " is that patients serum mixes with 1:1 volume ratio with drug solution; " mixed liquor 2 " is that patients serum mixes with 1:1 volume ratio with drug dilution liquid; " contrast liquid 1 " is that normal human serum mixes with 1:1 volume ratio with drug solution; " contrast liquid 2 " is that normal human serum mixes with 1:1 volume ratio with drug dilution liquid II.
(2) drug induced hemolytic anemia detects specific enzyme and processes erythrocytic preparation: 1. for normal person O type red blood cell, 0.9% sodium chloride solution washs 3 times, is mixed with the red cell suspension of 2-5% concentration with 0.9% sodium chloride solution.2. red cell suspension mixes according to the volume ratio of 1:2 with 0.5% bromelain solution, mixes, and 37 ° of C water-baths are hatched 30 minutes.3. with 0.9% sodium chloride solution washing 3 times, with 0.9% sodium chloride solution, be mixed with the red cell suspension of 2 ~ 4% concentration.
(3) get 12 test tubes, be labeled as respectively " 1 ~ 12 ".In " 1,7 " test tube, add 100 μ l mixed liquors 1, in " 2,8 " test tube, add 100 μ l mixed liquors 2, in " 3,9 " test tube, add 100 μ l contrast liquid 1, in " 4,10 " test tube, add 100 μ l contrast liquid 2, in " 5,11 " test tube, add 100 μ l antibody to discharge liquid, in " 6,12 " test tube, do not add any liquid, again respectively to each the 50 μ l of normal person O type red cell suspension that add 2 ~ 4% in the test tube of " 1 ~ 6 " mark, in the test tube of " 7 ~ 12 " mark, add 2 ~ 4% enzyme to process each 50 μ l of red cell suspension.
(2) mix, put 37 ° of C water-baths and hatch 30 minutes.After taking out, with 0.9% sodium chloride solution washing three times, last drains, then adds respectively broad spectrum antihumanglobulin reagent (anti-IgG+ C
3d) 100 μ l.
(3) put hydro-extractor centrifugal (3000rpm * 15 second), observations record.
When patient's blood plasma, antibody discharge liquid and corresponding medicine sensitized erythrocyte generation positive reaction, with unsensitized red blood cell generation negative reaction; Or the blood plasma that adds medicine processes red blood cell with normocyte and enzyme and produce positive reaction, and the blood plasma that does not add medicine judges it is this drug-induced hemolytic anemia while processing red blood cell generation negative reaction (being that test findings meets table 1 and table 2) with normocyte and enzyme.
Embodiment 2:
Detection of drugs induction type hemolytic anemia of the present invention (micro-column gel agglutination assay) comprising:
1. drug dilution liquid: comprise drug dilution liquid I, drug dilution liquid II, drug dilution liquid III.
2. antibody release reagent: comprise that antibody discharges that liquid I, antibody discharge liquid II, antibody discharges liquid III.
3. Antibody screening reagent: micro-column gel card comprises broad spectrum antihumanglobulin reagent (anti-IgG+ C
3d) gel column.
Detection method:
1. the preparation of medicine sensitized erythrocyte: with embodiment 1, cell concentration is adjusted to 0.5 ~ 1%.
2. antibody discharges the preparation of liquid: with embodiment 1.
3. use medicine sensitized erythrocyte to carry out the detection of drug induced hemolytic anemia:
(1) get broad spectrum antihumanglobulin reagent (anti-IgG+C
3d) micro-column gel card, mark 6 holes, are respectively " 1 ~ 6 ".In the hole of " 1 ~ 3 " mark, add each 50 μ l of 0.5 ~ 1% medicine sensitized erythrocyte suspension, in the hole of " 4 ~ 6 " mark, add each 50 μ l of non-medicine sensitized erythrocyte suspension of 0.5 ~ 1%; In " 1,3 " hole, add patient's blood plasma 25 μ l respectively again, in " 2,4 " hole, add antibody to discharge liquid 25 μ l, in " 3,6 " hole, add human normal plasma 25 μ l.
(2) tapping mixes, and puts 37 ° of C of immune microtrabeculae couveuse and hatches 15 minutes.Take out the supporting special centrifugal machine of rearmounted micro-column gel card centrifugal, observations record.
4. the normal person O type red blood cell that uses enzyme to process carries out the detection of drug induced hemolytic anemia:
(1) serum is processed: with embodiment 1.
(2) drug induced hemolytic anemia detects specific enzyme and processes erythrocytic preparation: with embodiment 1, cell concentration is adjusted to 0.5 ~ 1%.
(3) get broad spectrum antihumanglobulin reagent (anti-IgG+C
3d) micro-column gel card, mark 12 holes, are labeled as respectively " 1 ~ 12 ".To each the 50 μ l of normal person O type red cell suspension that add 0.5 ~ 1% in the hole of " 1 ~ 6 " mark, in the hole of " 7 ~ 12 " mark, add 0.5 ~ 1% enzyme to process each 50 μ l of red cell suspension, in " 1,7 " hole, add 25 μ l mixed liquors 1 respectively again, in " 2,8 " hole, add 25 μ l mixed liquors 2, in " 3,9 " hole, add 25 μ l contrast liquid 1, in " 4,10 " hole, add 25 μ l contrast liquid 2, in " 5,11 " hole, add 25 μ l antibody to discharge liquid, in " 6,12 " hole, do not add any liquid.
(4) tapping mixes, and puts 37 ° of C of immune microtrabeculae couveuse and hatches 15 minutes.Take out the supporting special centrifugal machine of rearmounted micro-column gel card centrifugal, observations record.
When patient's blood plasma, antibody discharge liquid and corresponding medicine sensitized erythrocyte generation positive reaction, with unsensitized red blood cell generation negative reaction; Or the blood plasma that adds medicine processes red blood cell with normocyte and enzyme and produce positive reaction, and the blood plasma that does not add medicine judges it is this drug-induced hemolytic anemia while processing red blood cell generation negative reaction (being that test findings meets table 1 and table 2) with normocyte and enzyme.
Claims (8)
1. a drug induced hemolytic anemia detection kit, is characterized in that: it comprises drug dilution liquid, antibody release reagent and Antibody screening reagent.
2. drug induced hemolytic anemia detection kit according to claim 1, is characterized in that: described drug dilution liquid comprises barbitol buffer solution, phosphate buffer and bovine serum albumin solution.
3. drug induced hemolytic anemia detection kit according to claim 1 and 2, is characterized in that: described Antibody screening reagent is broad spectrum antihumanglobulin reagent, i.e. anti-IgG and anti-C
3d.
4. drug induced hemolytic anemia detection kit according to claim 1 and 2, is characterized in that: described drug induced hemolytic anemia detects uses respectively medicine sensitized erythrocyte and enzyme processing red blood cell to detect.
5. drug induced hemolytic anemia detection kit according to claim 1 and 2, is characterized in that: described antibody release reagent is the reagent that antibody is dissociated from red blood cell, diffuses elution reagent.
6. drug induced hemolytic anemia detection kit according to claim 7, is characterized in that: described antibody release reagent is that thermolysis reagent, citric acid diffuse reagent, chloroform/triclene and diffuse that reagent, dimethylbenzene diffuse reagent, glycocoll-hydrochloric acid/disodium ethylene diamine tetraacetate diffuses one or more in reagent.
7. drug induced hemolytic anemia detection kit according to claim 4, is characterized in that: described enzyme is processed one or more in the red blood cell that red blood cell is bromelain, papain, cradin processing.
8. right to use requires the detection method of the drug induced hemolytic anemia detection kit described in 1, it is characterized in that, described method comprises the steps:
The first step, selects normal person O type red blood cell, and the medicine that patient uses is in the recent period prepared medicine sensitized erythrocyte;
Second step, is used antibody release reagent to release in connection with the antibody at Surface of Erythrocytes, and Dispersal risk discharges liquid;
The 3rd step, is used medicine sensitized erythrocyte to carry out the detection of drug induced hemolytic anemia;
The 4th step, the normal person O type red blood cell that uses enzyme to process carries out the detection of drug induced hemolytic anemia.
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| CN107132323A (en) * | 2017-07-07 | 2017-09-05 | 江苏中济万泰生物医药有限公司 | Piperacillin induction hemolysis test kit and preparation method thereof |
| CN107478852A (en) * | 2017-08-31 | 2017-12-15 | 张献伟 | Antibiotics induction type hemolytic anemia micro-column gel agglutination assay detection kit |
| CN109917136A (en) * | 2019-03-13 | 2019-06-21 | 江苏力博医药生物技术股份有限公司 | The preparation method and application of cradin processing antibody screening red blood cell |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107121326A (en) * | 2017-06-06 | 2017-09-01 | 珠海贝索生物技术有限公司 | The fast acid that red blood cell is diffused diffuses method |
| CN107121326B (en) * | 2017-06-06 | 2019-10-08 | 珠海贝索生物技术有限公司 | The fast acid of red blood cell release diffuses method |
| CN107132323A (en) * | 2017-07-07 | 2017-09-05 | 江苏中济万泰生物医药有限公司 | Piperacillin induction hemolysis test kit and preparation method thereof |
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| CN109917136A (en) * | 2019-03-13 | 2019-06-21 | 江苏力博医药生物技术股份有限公司 | The preparation method and application of cradin processing antibody screening red blood cell |
| CN116297120A (en) * | 2023-03-30 | 2023-06-23 | 深圳市血液中心(深圳市输血医学研究所) | Method for detecting drug antibody in sample |
| CN116297120B (en) * | 2023-03-30 | 2023-12-01 | 深圳市血液中心(深圳市输血医学研究所) | Method for detecting drug antibody in sample |
| GB2628703A (en) * | 2023-03-30 | 2024-10-02 | Shenzhen Blood Center Shenzhen Inst Of Transfusion Medicine | Method for detecting drug antibody in specimen |
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