MLS 4240 Elutions procedure PDF

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Immunohematology Lab Manual Page 12...

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Elutions

To prepare an elution, the cells are washed free of all unbound antibody and a procedure is performed on the cells to cause the release of the antibody molecules from the cells into a solution called the “eluate”. This eluate contains the antibodies which were removed from the red blood cells and is then tested against reagent red cells with known antigens to determine the specificity. The most critical part of the procedure is though washing. Washing is performed to remove all unbound antibody from the test system. Failure to wash adequately will result in unbound antibody being present in the eluate. An aliquot of saline is saved from the last wash and serves as a negative control. If the last wash is positive, this indicates the presence of unbound antibody and the test is invalid. It is crucial to thoroughly resuspend the red cells between filling the tube with saline when washing.

Objectives: 1. 2. 3. 4. 5. 6. 7. 8.

State when an elution procedure may be performed. List 4 situations in which an elution could provide helpful information. List 3 different methods of preparing an elution. State the advantages of a Lui freeze elution over other methods. State the principle of the elution procedure. State the purpose of testing the last wash and cause of positive reactions in it. State the reasons why some eluates will react with both A and B cells. Give reactions of a cord blood work up.

A. Acid elution Sample required: EDTA red blood cells

Principle: A common and quick method of total elution of antibody to detect non-ABO antibodies is acid elution. In this method, the washed antibody-coated cells are mixed with glycine acid solution at a pH of 3. The antigen/antibody bond is disrupted, and the antibody is released into the acidic supernatant. The supernatant is collected and the

pH is neutralized so the antibody identification can be performed. Citric acid and digitonin acid are also used in similar methods. The Acid Elution is used to identify the cause of a positive Direct Antiglobulin Test or Auto Control and is especially useful when multiple antibodies may be involved. Complement alone on the RBC will give a negative result, as well as drug induced antibodies unless tested against sensitized cells (coated with the offending drug). Reagents: Based on Elu Kit II™ A. Eluting Solution B. Buffering Solution C. Concentrated Wash Solution D. Working Wash Solution: a. Using a 10 ml pipette, transfer 10 ml of Concentrated Wash Solution to a 100 ml volumetric flask and fill to the etched line with Type I water. b. Mix the solution thoroughly after preparation and prior to each use. c. Transfer the solution to an empty wash bottle and properly label and date with both preparation and expiration dates.

Procedure:

Preparation of Eluate: 1.Centrifuge the patient specimen(s) and remove all excess plasma/serum 2. Take a 1 ml aliquot of patient coated red blood cells and transfer to a clean, labeled 12 x 75 mm test tube. 3. Wash the coated red blood cells once with physiologic saline, pipetting off (instead of pouring off) the supernatant to better conserve the red blood cells. 4. Wash the coated cells 4 more times with the Working Wash Solution, saving an aliquot of the last wash supernatant in an appropriately labeled test tube, to serve as a control.

5. Transfer 1 ml (approximately 20 drops) of washed red blood cells to a clean, labeled 12 x 75 mm test tube. Add 1 ml (20 drops) of Eluting Solution and mix gently by inverting 4 times. Centrifuge immediately at 900 to 1000 rcf for 1 minute. Note: The volume of the eluting solution needs to equal the volume of washed red blood cells. If using more or less than 1 ml of washed red blood cells, adjust the volume of Eluting Solution accordingly. 6. Transfer the supernatant eluate to a clean, appropriately labeled 12 x 75 mm test tube and discard the packed red blood cells.

7. Add an equal volume of Buffering Solution to the separated acid eluate. Continue to add Buffering Solution one drop at a time until the yellow eluate turns pale blue and remains pale blue after mixing. Note: The presence of the blue indicator in the Buffering Solution facilitates the adjustment of the pH of the final eluate within the required testing range of 6.4 to 7.6. 8. Mix well and recentrifuge at 900 to 1000 rcf for 1 minute to remove any cellular debris, then transfer the eluate to a clean, appropriately labeled test tube. 9. The eluate is now ready for use in antibody detection or identification tests. Test the eluate and a sample from the last wash in parallel using the antibody screening cells. Results: 1. Negative: A. Eluate – No agglutination or hemolysis seen. B. Last Wash – No agglutination or hemolysis seen. 2. Positive A. Eluate – Agglutination or hemolysis seen. B. Last Wash – No agglutination or hemolysis seen. 3. Invalid A. Eluate – Agglutination or hemolysis seen or not seen. B. Last Wash – Agglutination or hemolysis seen.

Quality Control: In performing an antibody elution from coated red blood cells, and aliquot of the Working Wash solution if retained to be tested for antibody activity in parallel with the eluate. The purpose of this control is to assure that antibody present in the eluate has be derived from a bound state and is not unbound antibody remaining as the result of inadequate washing.

.

B. Lui Freeze-Thaw Procedure Sample required: EDTA red blood cells Reagents: 1. 2. 3. 4. 5. 6.

Test tubes 12x75 Disposable pipettes Saline A1 cells and B cells O cells Anti-IgG Check cells

Procedure: 1. You are given 20 drops of a cord cell sample from Baby X. 2. Label a tube with “eluate’’ and a tube with “LW”.(last wash). 3. Wash the 20 drops of red cells 6 times with saline. Use a pipette to remove saline between washes. 4. On the 6th wash, remove the saline and place in the tube labeled ‘LW’. 5. To the 20 drops of red cells add three drops of saline. 6. Cover tube with parafilm and rotate tube covering the sides with cells. 7. Place the tube on its side in a freezer (-20 to -70C) for 10 minutes. 8. While the red cells are freezing, label 3 tubes as “LW A1”, “LW B” , “LW O” 9. Label 3 additional tubes as “EL A1”, “EL B”, “EL O”. 10. Remove cells from freezer and thaw rapidly. 11. Centrifuge the thawed hemolysate for 60 sec.

12. Transfer the eluate (top) to the tube labeled “eluate” 13. To the tubes labeled with ‘EL”, add 2-3 drops of eluate. 14. To the tubes labeled “LW”, add 2-3 drops of the last wash . 15. Add 1 drop of the appropriate manufactured cell to each tube. 16. Mix thoroughly and centrifuge for 30 sec. 17. Read tubes macroscopically, record. Interpretation: If the last wash wash is positive with any of the test cells, the cell sample was probably under washed and the elution procedure should be repeated on a more throughly washed cell sample. Lack of agglutination of test cells by the eluate indicates absence of antibodies for the specific antigens on the test cells and the test is negative. If the last wash tubes are negative, any agglutination of reagent cells with eluate indicates recovery of antibodies from the original cells. In the case of cord blood, the

Reaction with A1 cells

Interpretation B_cells

4+

0

Immune anti-A

0

4+

Immune anti-B

4+

2+

Immune anti-A and anti-A,B

*The mother’s antibody screen must be negative so that the positive DAT is not due to an antibody other than anti-A or anti-B. If the mother has a positive antibody screen, this indicates an alloantibody directed an antigen other than the ABO system and another method of elution must be performed followed by antibody identification. If the mother is group O and the baby is group A or B, it is not unusual to obtain positive reactions with both the A and B cells. This reactivity is due to anti-A,B present in the O mothers serum that has coated the baby’s cells. This antibody will react with both A and B cells.

C.

Heat Elution:

Heat elution is a method that can be used to remove antibody from cells that demonstrate a positive direct antiglobulin test. It is the method of choice for detecting Hemolytic Disease of the Newborn caused by ABO incompatibility. Often it is possible to detect maternal anti-A and/or anti-B coated on the cord cells even though the direct antiglobulin test is negative. This procedure can also be utilized for red cells coated with other IgM antibodies. Procedure: 1. 2. 3. 4.

5.

6.

Wash at least 2 mL of cells to be eluted six times in large volumes of saline in a properly labeled tube. Save an aliquot of the final wash solution sampled from directly over the packed red cells. Do an antibody screen on the last wash, using pooled group O cells, A 1 cells, and B cells. If the antibody screen is negative, proceed with the next step; if it is positive with any one of the test cells in any phase, repeat the washing procedure in steps 1 to 3. To the washed packed cells add an equal volume of 6% bovine albumin. In cases where the direct AHG is negative, 6% albumin* equal to half the volume of packed cells may be added. Mix well by inversion. Incubate in a 56° C water bath for 10 minutes with periodic agitation.

7.

After incubation centrifuge the tube for 5 minutes in a serofuge. The supernatant should be cherry red color; if not incubate at 56° C with constant agitation for an additional 5 to 10 minutes.

8.

After centrifugation for 5 minutes the supernatant eluate should be immediately transferred to another properly labeled tube and tested with A 1, B, and group O cells using the indirect AHG technique.

*6% Albumin can be prepared using 3 mL of 22% bovine albumin and 8 mL saline.

Elutions Summary When the DAT is positive due to IgG coating of red cells, it is important to determine the specificity of the antibody. This can be achieved by performing and elution. Elutions are performed to remove antibody from red cells. The eluate is then tested against red cells with known antigen to determine the specificity of the antibody. This may help in the following investigations of:

1. 2. 3. 4.

Suspected transfusion reaction. Fetal-maternal incompatibility Drug induced pneumonia Resolving multiple antibodies in a single plasma.

There are many different types of elution methods. The methods used to remove antibody change the thermodynamics of the environment, change the structural forces between antigen and antibody, or change the structure of the red cell. Temperature dependent elutions are best at detecting IgG antibodies directed against the ABO system. The procedure selected will be determined by the situation as in the table below:

Method

Uses

Comments

Acid elution

Warm auto/ alloantibodies

Easy. Possible false positives with high titer abs.

Lui Freeze

ABO abs.

Quick, small volume required Poor recovery of other abs

Heat (56C)

ABO,IgM antibodies

Easy. Poor recovery of IgG allo/auto antibodies...