Polybrene Test Kit

Polybrene Test Kit

Assay-kits

Product code: ACMBS-16
Specifications: 75 Tests, 150 Tests, 200 Tests, 400 Tests, 600 Tests (Box)
Intended use: It is designed to perform screening tests for complete and incomplete antibody, as well as to be applied to blood cross-matching in blood banks.
Store temperature: 5℃～25℃
Expiry date: 24 Months

More Details

PrincipleMain componentsSample requirementTest procedureMethod limitationPrecautions

Principle

The surface of red blood cells carries large amount of negative charge, so that auto-agglutination of red blood cells is avoided. When red blood cells are suspended in electrolyte, cation will be attracted by the negative charge of red blood cells. Red blood cells are surrounded by the diffused double-layer ion clouds, and form zeta potential, which determines the repellent action among red blood cells. Polybrene technique utilizes Low Ionic Medium (LIM) to lower the ionic strength of media and diminish cationic clouds around red blood cells, in order to enhance the binding of antibody between RBC and serum (or plasma). Furthermore, with addition of Polybrene reagent, which contains high valence cationic polymer and heparin neutralizer, and after dissolving, it will generate lots of positive charges to neutralize the negative charges on the surface of RBC, as well as to lower its zeta potential and shorten the distance among RBC and produce non-specific agglutination. In consequence, non-specific agglutination of normal RBC shall be dispersed by addition of Resuspending Reagent, which can neutralize polybrene, the test result is interpreted as "Negative". If RBC is sensitized by the corresponding antibody, agglutination will be produced by polybrene and shall not be dispersed, the test result is interpreted as "Positive".

Main components

Contents	Components
1. Low Ionic medium (LIM)	5% Glucose, 0.2% EDTA(2Na)
2. Polybrene reagent	0.05% Polybrene, 0.9% NaCl
3. Resuspending	3.5% Tri-sodium citrate, 1.5% Glucose

Sample requirement

1. Fresh serum, plasma with EDTA, 3%～5% RBC suspension.
2. Do not use hemolysis sample and sample contain Sodium Citrate or Heparin.

Test procedure

A. Cross-matching test

1. Mark 2 test tubes as, Major and, Minor respectively. In Major, add 2 drops of patient's serum (plasma) and 1 drop of 3～5% RBC suspension of donor. (washed or unwashed). Test procedure for the Minor is the reverse (Patient's RBC+ Donor's serum or plasma).
2. Add LIM 0.65ml into each tube and mix well. Then, add 2 drops of polybrene into both tubes.
3. Centrifuge for 10 seconds (1000g) by using BASO centrifuge (Other centrifuges: 3400rpm for 10 seconds), remove the supernatant and leave 0.1ml liquid at the tube bottom. Do not dry it.
4. Gently shake the tubes. Examine by naked eyes if agglutination of RBC occurs. If not, repeat the test.
5. Add 2 drops of resuspending solution. Gently shake and mix the tubes. Observe test result.

• If agglutination disperses within 30 seconds to 1 min, the non-specific agglutination is produced by polybrene. Hence, the patient and donor blood are compatible.

• If agglutination doesn't disperse, specific agglutination is caused by the binding of RBC antigen and antibody. In this case, the patient and donor blood are incompatible.

• If the results are equivocal, pour the specimen on a slide for microscopic examination.

B. Antibody screening test

1. Take 2 test tubes. Add 2 drops of serum (or plasma) and 1 drop of 3～5% ‚ Screened RBC into eachtube. (Screened RBC, required but not provided, contains antigens for c, E, e..etc and can be obtained from local blood transfusion centers or blood banks)
2. Add LIM 0.65ml. After mixing well, add 2 drops of polybrene. Mix thoroughly.
3. Centrifuge for 10 seconds by using BASO Centrifuge f (1000g). (Other centrifuges: 3400 rpm for 10 seconds). Remove the supernatant. Leave 0.1ml liquid at the bottom of the tube. Do not dry it.
4. Gently shake the tubes. Examine by naked eyes if agglutination of RBC occurs. If not, repeat the test.
5. Add 2 drops of resuspending solution. Gently shake and mix the tubes. Observe test result.

• If agglutination disperses, the non-specific agglutination is produced by polybrene. Hence, the antibody screening test result is Negative.

• If agglutination doesn't disperse, specific agglutination is caused by the binding of RBC antigen and antibody. In this case, the antibody screening test result is Positive. Further tests on antibody should be performed.

C. Coombs Test

1. Continue from after Cross-matching test 5th step, add 1 drop of Resuspending into test tube.
2. Wash RBC with Normal saline 1 time, after centrifuge remove supernatant, then add 3 drops of resuspending, mix well, then wash RBC 3 time with Normal saline, and then fully remove lastsupernatant.
3. Add 2 drops of anti-IgG (monospecific) serum, mix.
4. Centrifuge 3400rpm/min (1000g) for 15 seconds, observe agglutination result.
5. If agglutination, it means contain anti-IgG antibody, it must do antibody identification, and showing the donor blood are incompatible. If no agglutination, but for ensure safe of blood transfusion, must add 1 drop of Coombs (anti-IgG sensitive) control RBC, then centrifuge 3400rpm/min(1000g) for 15 seconds, the normal reaction must have over 2+ agglutination. If not, must do again.

Method limitation

This kit sensitivity is lower for irregular antibody K.

Precautions

1. Can use EDTA plasma to instead of serum.
2. If the patient serum(plasma) contain heparin, example: dialysis patient. It must add more 4～6 drops Polybrene to neutralize the heparin.
3. This kit also can use on antibody identification.
4. Please restore to room temperature before use.
5. The Coombs test is for increasing the sensitivity of Anti-K.
6. In the lower temperature, some sample contain cold agglutinin which will cause false positive result in Polybrene test. If that is the suspected, we suggest that after add Resuspending reagent, put the test tube into 37℃ water bath.
Then gently shaking the test tube, and watch the test result within 60 seconds.
7. RBC antigen test procedure is same with the antibody screen test. The testing standard serum can use the know standard serum.
8. User can use the IgG anti-D antibody (Titer≧64) testing with RhD positive RBC for positive control test, it can test effectiveness of this kit.

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