In this article we will discuss about:- 1. Meaning and Purpose of Cross-Matching 2. Phases 3. Procedure.
Meaning and Purpose of Cross-Matching:
Before the recipient receives blood transfusion, a compatibility test must be run within the laboratory with the donor’s red cells and the recipient’s serum. This is called major cross matching.
The primary purpose of major cross match is to find out any incompatibility of donor’s cells with patient’s serum in order to avoid blood transfusion reactions. The minor cross match is rarely requested when the compatibility of the recipient’s red cells is tested against donor’s serum.
Compatibility test or cross matching is performed subsequent to the ABO grouping and Rh typing of the recipient’s and donor’s blood. It is the final criterion as to the suitability of a particular donor blood for a particular recipient.
The recipient’s blood is obtained fresh while the donor’s blood is obtained from the pilot tube. ACD anticoagulant donor’s blood should not be more than 21 days and constantly stored at 4°C.
Principle:
Serum of the recipient is tested against the red cells of the donor under different conditions in order to establish their cross matching or non-agglutination. Agglutination in any of the conditions indicates the presence of incompatible antibody in patient, natural or immune.
Phases of Cross-Matching:
The three phases of compatibility testing are listed below:
1. Saline Phase:
Where the immunologic reaction between red cells suspended in saline medium and the antibody occurs at room temperature.
2. Thermo Phase with Protein:
Where the red cells are suspended in the antibody (serum) with 22% albumin (protein) and incubated for 30 minutes at 37°C.
3. Antihuman Globulin (AHG) Phase:
Where the incubated cells are washed (to remove free globulin) and reacted with antihuman globulin serum (Comb’s reagent or ant globulin). Standard compatibility testing, however, does not include AHG phase in the laboratories of developing countries because of the high cost of the reagent and its instability.
ABO incompatibility is recognized in the saline phase while agglutination in other phases indicates the presence of immune, incomplete or irregular antibodies. If agglutination is not seen in-vitro above phases, donors and recipient’s bloods are considered to be compatible.
Specimen:
Donor’s un-clotted blood specimen is available from the pilot tube. Donor’s red cells are taken out of the clot, repeatedly washed with saline and a 5% v/v suspension is made in saline (0.1 ml packed red cells mixed with 1.9 ml of saline).
Patient’s blood is drawn fresh and collected in a sterile. Pre-labelled dry container without any anticoagulant. Separate the serum promptly. Patient’s serum is used for major cross matching and cell suspension (3 to 5% in saline) for the auto control.
Procedure:
1. Cross matching while tube 2 will be the auto control. Tube 2 receives all the treatments of tube 1, except that it does not contain donor’s cells.
2. Add 2 drops of patient’s serum in both the tubes.
3. Add 1 drop of 5% saline suspension of donor’s cells in tube I and I drop of patient’s cell suspension (5% in saline) in tube 2.
Note: The auto control is the suspension of patient’s red cells in its own serum.
4. Mix and centrifuge at 1500 rpm for 1 minute.
5. Gently dislodge the cell bottom and examine for agglutination and heterolysis. Record the results. If agglutination is noted in tube 1, ABO incompatibility is suspected.
6. To both tubes add a drop of 22% bovine albumin, mix and incubate at 37°C for 30 minutes.
Alternatively:
Incubate both tubes at 37°C for 15minutes then add a drop of 22% albumin along the side of each tube. Mix and re-incubate for another 20 to 30 minutes.
7. Centrifuge at 1500 rpm for 1 minute. Examine for agglutination and haemolysis. Record the results of agglutination with grading.
8. Wash the cells 3 to 4 times with saline, decant completely after each wash and add 2 drops of antihuman globulin serum to the sediment cells. Shake the tubes to mix the contents and then centrifuge at 1500 rpm for 1 minute.
Examine for agglutination and grade the agglutination reaction. Also look for haemolysis. Some of the antibodies bind with the complement and bring haemolysis which should be considered as an evidence of immunologic reaction (positive).
9. If there is no agglutination reaction (negative), add a drop of already sensitized cells. The sensitized or check cells must agglutinate, if the cells are adequately washed and AHG is reactive.
Procedure for Cross Matching:
The following are the procedure for cross-matching in an emergency situation:
1. Direct Emergency with No Lead Time:
Use blood group ‘O’ Rh – ve blood without cross matching. Because of its rarity in India (5% Rh-ve), most blood banks do not have O-ve blood in stock. They however, keep the list of potential donors for ready approach.
2. 15 to 30 Minutes of Lead Time:
Perform ABO and Rh blood grouping and choose group specific donor’s blood.
3. 30 to 45 Minutes of Lead Time:
Perform blood grouping of recipient and choose group specific blood instead of 30 minutes, followed by antihuman globulin test.
4. More than 45 Minutes of Lead Time:
Go through the routine procedure of complete cross matching. Where blood has been issued without cross matching or after an emergency cross matching, carry through a complete cross match in the laboratory while blood transfusion is in progress. In case an incompatibility is noted the blood transfusion should be discontinued immediately.
Anti Human Globulin (AHG) or Coombs Test:
AHG technique is very useful in recording weak immunologic reaction. It is widely used in the identification “D, compatibility testing, antibody screening and identification of sensitized cells.
AHG is produced by injecting human globulin in rabbit and purifying the anti-human globulin produced by the rabbit.
There are two types of test:
i. Direct AHG test
ii. Indirect AHG test
i. Direct AHG Test:
This recognizes sensitized RBCs when the sensitizing occurs within the body. E.g. in hemolytic disease of new born and auto immune hemolytic diseases.
ii. Indirect AHG Test/(ICT):
Sensitization of RBC is done in the Laboratory by incubating the red cells with the corresponding antibody at 37°C for 30 minutes that means test detect the presence of unsuspected antibody in serum which will react with corresponding antigen on the RBCs.
The latter get sensitized without haem-agglutination this initial phase of immune logic reaction is recognized only after treating the washed sensitized cell with AHG.
Principal:
AHG test detects the sensitized the red cells where the red cells gets coated with IgG (antibody) or globulin but do not agglutinate when the sensitized red cells come in contact with AHG reagent. They agglutinate.
Regent:
i. Anti human globulin reagent
ii. Pre-sensitize red cell (coombs control test)
iii. Saline
DCT Procedure:
i. Wash the RBC suspected of being sensitized 3-4 time with saline. Complete removal of free globulin is imported.
ii. De-cant completely at the end of task washing.
iii. Add 2 drops of AHG serum to the button cells.
iv. Mix well and centrifuge 1500 rpm for 1 minute
v. Examine for agglutination by holding against a lighted back found and taping the bottom of the tube. Hold the tube at an angle, shake gently until on cell are dislodge than tilt the tube back and forth until an even suspension of cell or agglutinate observed.
vi. If the agglutination is not seen live the tube at room temp for 10 minutes, than re-centrifuge and read. A weaker reaction antibody will delete reaction considers this is a positive.
vii. If not seen add 1 drop of pre sensitized RBC this should result in haem -agglutination of pre sensitized cell indicating that the AHG is reaction and the result is valid.
ICT:
The ICT detects cells which are sensitizing in the laboratory.
Procedure:
i. Prepare a 4% saline suspension of test cell.
ii. Add 2 drops of cell suspension in a small tube.
iii. Add 2 drops of antiserum to the cell suspension incubate in water bath at 37°C for 30 minutes.
iv. After that wash 3-4 time with large volume of saline. Decent completely after the last washing
v. Add 2 drops of AHG and mix well.
vi. Centrifuge at 1500 rpm for 1 minute
vii. Examine for haema agglutination