CN103675298A - Immune hemolytic anemia detection kit and detection method thereof - Google Patents

本发明公开了免疫性溶血性贫血检测的试剂盒，本发明还公开了使用该试剂盒检测的方法。当直接抗人球蛋白试验阳性，抗体释放液抗体筛查试验阴性时判断是药物诱导型溶血性贫血；当直接抗人球蛋白试验阳性，血清/浆抗体筛查试验阳性，抗体释放液抗体筛查试验阳性时判断是温自身免疫性溶血性贫血（含溶血性输血反应）。根据结果准确诊断出免疫性溶血性贫血的种类，指导临床正确选择合适的药物用于对患者的治疗。该方法具有准确性好，适用于适用于试管法或微柱凝胶卡法检测。The invention discloses a kit for detecting immune hemolytic anemia, and also discloses a detection method using the kit. When the direct antiglobulin test is positive and the antibody release liquid antibody screening test is negative, it is judged to be drug-induced hemolytic anemia; when the direct antiglobulin test is positive and the serum/plasma antibody screening test is positive, the antibody release liquid antibody screening test is positive. When the test is positive, it is judged to be warm autoimmune hemolytic anemia (including hemolytic transfusion reaction). Accurately diagnose the type of immune hemolytic anemia according to the results, and guide the clinical selection of appropriate drugs for the treatment of patients. The method has good accuracy and is suitable for test tube method or microcolumn gel card method for detection.

Immune hemolytic anemia detection kit and detection method thereof

Technical field

The present invention relates to field of biological detection, especially relate to immune hemolytic anemia detection kit, the invention still further relates to the method for using this kit to detect immune 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 immune hemolytic anemia, just must carry out immune hemolytic anemia to patient and carry out detection promptly and accurately, stop using the medicine that can make patient produce immune 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, need to carry out Serological testing to drug-induced hemolytic anemia, to determine whether being drug-induced hemolytic anemia, instruct medicine that clinical selecting properly is suitable for the treatment to patient.

Detect at present immune hemolytic anemia and there is no detection kit, the content detecting is also only confined to the unexpected Antibody screening in direct antihuman globulin test, patient's blood plasma, according to the positive or negative of direct antihuman globulin test, judge whether it is immune hemolytic anemia, can not detect the reason that immune hemolytic anemia is classified and caused immune hemolytic anemia.Whether the present invention not only can detect is immune hemolytic anemia, immune hemolytic anemia can also be divided into two classes, i.e. warm autoimmune hemolytic anemia or drug-induced immune hemolytic anemia.

Summary of the invention

The object of the present invention is to provide the kit that detects immune hemolytic anemia, use this kit can accurately judge the reason of immune hemolytic anemia; 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 immune hemolytic anemia of the present invention, comprises direct antihuman globulin reagent, antibody release reagent and Antibody screening reagent.

Described direct antihuman globulin detects reagent and comprises antihuman globulin reagent (anti-IgG), antihuman globulin reagent (anti-C3d).

Described Antibody screening reagent is broad spectrum antihumanglobulin reagent (anti-IgG+C3d).

Described Antibody screening reagent checks that respectively blood plasma and antibody discharge the antibody in liquid.

Described antibody release reagent is the reagent that antibody is dissociated from red blood cell, diffuses elution reagent.

Described antibody release reagent comprises that thermolysis reagent, citric acid diffuse reagent, chloroform/triclene and diffuse that reagent, dimethylbenzene diffuse reagent, glycocoll-hydrochloric acid/disodium ethylene diamine tetraacetate diffuses reagent.

It contains direct antihuman globulin reagent two kinds, is respectively anti-IgG and anti-C ₃d, can be divided three classes immune hemolytic anemia, IgG antibody cause, C ₃d causes and IgG merging C ₃d causes.Antibody release reagent can the antibody on red blood cell be discharged into sensitization in release liquid.Utilize unexpected Antibody screening reagent, respectively antibody, erythrocyte antibody in patient's blood plasma are discharged to the antibody test in liquid, judge that immune hemolytic anemia belongs to warm autoimmune hemolytic anemia or drug induced immune hemolytic anemia, determine that immune hemolytic anemia is that warm AHD causes or the medicine that patient uses in therapeutic process causes, 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.

The detection method of kit of the present invention, comprises the steps:

Whether the first step, detect sample red blood cell to be checked in vivo by sensitization, uses direct antihuman globulin reagent;

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, utilizes Antibody screening reagent examination serum/slurry and antibody to discharge antibody in liquid;

The 4th step, according to the kind of first and third step test findings judgement immune hemolytic anemia.

The object of the invention is to detect immune hemolytic anemia by test tube method or micro-column gel block-regulations, positive when direct antihuman globulin test, when antibody discharges humoral antibody examination negative, judgement is drug induced hemolytic anemia; Positive when direct antihuman globulin test, the test of serum/slurry Antibody screening is positive, and when antibody release humoral antibody examination test is positive, judgement is warm autoimmune hemolytic anemia (containing hemolytic blood transfusion reaction).

embodiment:

Enumerate respectively test tube method kit and micro-column gel agglutination assay kit below, and illustrate that it detects using method.

Embodiment 1:

The present invention detects immune hemolytic anemia (test tube method) and comprising:

1. direct antihuman globulin reagent: comprise antihuman globulin reagent (anti-IgG), antihuman globulin reagent (anti-C ₃d).

2. antibody release reagent (glycocoll-hydrochloric acid/disodium ethylene diamine tetraacetate diffuses technology): 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. directly antihuman globulin test:

(1) sample Fresh blood sample packed red cells to be checked once obtains packed red cells with physiological sodium chloride solution washing, is mixed with the red cell suspension of 2 ~ 4% concentration with physiological sodium chloride liquid.

(2) get 3 test tubes, be labeled as respectively " IgG ", " C ₃d ", " contrast ", in the good test tube of mark, add respectively antihuman globulin reagent (anti-IgG) 100 μ l, antihuman globulin reagent (anti-C ₃d) 100 μ l, 0.9% sodium chloride solution 100 μ l(contrasts).

(3) respectively to the red cell suspension 50 μ l that add sample 2 ~ 4% to be checked in the good test tube of mark.

(4) mix, put hydro-extractor centrifugal (3000rpm * 15 second), observations record.

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. Antibody screening test:

(1) serum/slurry Antibody screening test: get 3 test tubes, be labeled as respectively " I ", " II ", " III ".In the test tube of mark, add respectively sample blood plasma 100 μ l to be checked, more respectively to 2 ~ 4% each the 50 μ l of red cell suspension that add irregular antibody examination red blood cell reagent I, II, III in the good test tube of mark.

(2) antibody discharges humoral antibody examination test: get 3 test tubes, be labeled as respectively " I ", " II ", " III ".To adding respectively in the test tube of mark antibody to discharge liquid 100 μ l, more respectively to 2 ~ 4% each the 50 μ l of red cell suspension that add irregular antibody examination red blood cell reagent I, II, III in the good test tube of mark.

(3) 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.

(4) put hydro-extractor centrifugal (3000rpm * 15 second), observations record.

Positive when direct antihuman globulin test, when antibody discharges humoral antibody examination negative, judgement is drug-induced immune hemolysis hemolytic anemia; Positive when direct antihuman globulin test, the test of serum/slurry Antibody screening is positive, and when antibody release humoral antibody examination test is positive, judgement is warm autoimmune hemolytic anemia (containing hemolytic blood transfusion reaction).

Embodiment 2:

The present invention detects immune hemolytic anemia (micro-column gel agglutination assay) and comprising:

1. direct antihuman globulin reagent: micro-column gel card comprises antihuman globulin reagent (anti-IgG) gel column, antihuman globulin reagent (anti-C ₃d) gel column, blank gel tube.

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. directly antihuman globulin test:

(1) patient's Fresh blood sample packed red cells once obtains packed red cells with physiological sodium chloride solution washing, is mixed with the red cell suspension of 0.5 ~ 1% concentration with physiological sodium chloride liquid.

(2) cut-off connects antihuman globulin test micro-column gel card, every part of clinical samples mark to be checked 3 holes.To the red cell suspension 50 μ l that add clinical samples 0.5 ~ 1% in every hole of mark.

(3) tapping mixes, and puts the supporting special centrifugal machine of micro-column gel card centrifugal, observations record.

2. antibody discharges the preparation of liquid: with embodiment 1.

3. Antibody screening test:

(1) serum/slurry Antibody screening test: get Antibody screening micro-column gel card, every part of clinical samples mark to be checked 3 holes, are labeled as respectively " I ", " II ", " III ".To 0.5 ~ 1% each the 50 μ l of red cell suspension that add irregular antibody examination red blood cell reagent I, II, III in every hole of mark, then add respectively sample blood plasma 25 μ l to be checked.

(2) antibody discharges humoral antibody examination test: get Antibody screening micro-column gel card, every part of clinical samples mark to be checked 3 holes, are labeled as respectively " I ", " II ", " III ".To 0.5 ~ 1% each the 50 μ l of red cell suspension that add irregular antibody examination red blood cell reagent I, II, III in every hole of mark, then add respectively antibody to discharge liquid 25 μ l.

(3) 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.

Positive when direct antihuman globulin test, when antibody discharges humoral antibody examination negative, judgement is drug induced hemolytic anemia; Positive when direct antihuman globulin test, the test of serum/slurry Antibody screening is positive, and when antibody release humoral antibody examination test is positive, judgement is warm autoimmune hemolytic anemia (containing hemolytic blood transfusion reaction).