Bovine Albumin Technique
The test is similar to the 'Saline Technique' with the following difference :
To the reaction tube containing the recipient's serum add one drop of (10 to 20%) Bovine Albumin (to form a layer above the serum). Then add the suspension of donor's red cells to this tube.
The red cells are allowed to sediment through the albumin before they come in contact with the serum.
Incubate and read as for 'saline technique'.

Coombs' Technique
Carry out the 'major' matching as described under 'saline technique' with the following additions : After incubating the mixture of donor's cells and recipient's serum, wash the red cells in NS at least four times. After the final wash resuspend the cells in one drop of NS and add one drop of Coombs' Serum (Antihuman Globulin Serum). Incubate at RT for 10 minutes, centrifuge at 1000 rpm for 1 minute and read for agglutination by naked eye as well as under a microscope.

COOMBS' TEST (ANTIHUMAN GLOBULIN TEST)
INTRODUCTION
Agglutinating antibodies which are present in the serum (or plasma) are of two types :
'Complete Antibodies' which lead to agglutination of the red cells having the corresponding antigens; and 'Incomplete Antibodies' which can attach themselves to the antigen sites on the blood cells but are incapable of producing agglutination because of their 'incompleteness'.
This second type of antibodies are also known as 'Blocking Antibodies' because if a given serum contains a mixture of both types of antibodies, the incomplete ones (having a greater affinity to the antigen sites) attach themselves to the antigen sites and block the action of the complete antibodies, resulting in no obvious agglutination.
Blood bank work basically consists of finding out antigens on the red cells or finding out the presence of corresponding antibodies in the serum by using cells which are known to have certain antigens. It is apparent that the presence of blocking antibodies will lead to a great hazard as no agglutination results in spite of the presence of corresponding antigens on the cells and corresponding antibodies in the serum when these two are mixed.
The anti-sera used in blood grouping have undergone strict quality control tests and are certainly free of such blocking antibodies. The same is obviously not true of the recipient's or donor's serum which we use in grouping and cross matching tests.
In certain clinical situations (notably in the Haemolytic Disease of the Newborn) red blood cells are destroyed in the patient's body because of the presence of certain antibodies. In many such situations the antibodies present are in the 'incomplete' form. It becomes necessary to detect such 'incomplete antibodies'.
Coombs' Antihuman Globulin Test is very useful in such situations. Coombs' serum (which is an antibody prepared by an animal against human serum) is capable of leading to visible agglutination when added to cells which have their antigen sites 'coated' or 'blocked' by 'incomplete antibodies'.
Coombs' Serum will be inactivated by the presence of human serum. Red cells which are to be subjected to Coombs' test must be meticulously washed clean of human serum.
IT IS, THEREFORE, IMPERATIVE THAT RED CELLS MUST BE THOROUGHLY WASHED IN NORMAL SALINE BEFORE COOMBS' SERUM IS ADDED TO THEM.
In this Blood Bank Coombs' test is used for the 'major' cross match (that is when donor's red cells are mixed with the recipient's serum and incubated).
When red blood cells are suspected to be 'coated' by antibodies (as in the cases of the Haemolytic Disease of the Newborn) a DIRECT COOMBS' TEST is carried out.

DIRECT COOMBS' TEST
Step 1 : Set the table and label the tubes. Prepare record books.
Step 2 : Add a drop of Coombs' Serum to a small tube after appropriately labelling it.
Step 3 : Check the identity of the specimen.
Step 4 : Make suspension of the red cells to be tested in NS.
Step 5 : Wash the red cells 4 times in NS.
Step 6 : Make a 2 % suspension of the red cells.
Step 7 : Check the small tube to which Coombs' Serum has been added and add 1 drop of the red cells to be tested to it. Mix by shaking.

Step 8 : Incubate at room temperature for 30 minutes.

 
Step 9 : Spin at 1000 rpm for 1 minute.
Step 10 : Observe for agglutination with the naked eye.
Step 11 : If no agglutination is seen, read the contents under a microscope (low power).
Step 12 : Record the results.

INTERPRETATION
If agglutination is seen either in step 10 or step 11 the test is POSITIVE.
If no agglutination is seen the test is NEGATIVE.
When a person's serum is suspected to be having some 'incomplete antibodies' , such serum is mixed with cell-suspension of cells known to have antigen corresponding to the suspected antibody. After incubation at a temperature appropriate for the suspected antibody for an appropriate time the cells are washed and Coombs' Test is performed as described above. This is INDIRECT COOMBS' TEST.
During cross matching one has to be sure that the recipient's serum does not contain any antibodies which may be harmful to the donor's cells. (Because in such a situation the donor's cells may be destroyed after transfusion and may lead to haemolytic reaction.) In this Blood Bank Coomb's Test is performed on the donor's red cells after these cells have been incubated with the recipient's
(that is, the patient's) serum. The procedure will be described under 'Cross Matching'.
 

USE OF BOVINE ALBUMIN IN CROSS MATCHING
Step 1 : Set the table label the tubes and prepare record books.
Step 2 : Add one drop of appropriate serum to the tube.
Step 3 : Add one drop of 20 (or 22) % bovine albumin on top of the serum in the same tube.(Albumin should form a layer on top of serum. Do not mix).
Step 4 : Add one drop of 2% suspension of washed red blood cells so as to form a layer on top.(Now there are 3 layers in the tube).
Step 5 : Allow the cells to pass through bovine albumin and then through the serum to settle at the bottom.(Usually this takes 45 minutes. If the test is to be done urgently, centrifuge the tube without mixing).
Step 6 : Look for agglutination with the naked eye and then under a microscope.
Step 7 : Record the result.

INTERPRETATION
If agglutination is present in any of the four tubes the donor's blood is NOT COMPATIBLE with the recipient. Take another donor's sample for cross matching.
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