RHESUS (Rh) GROUPING SYSTEM, Rh Typing Methods

RHESUS (Rh) GROUPING SYSTEM, Rh Antigens, The D' Antigen, SALINE METHOD FOR Rh-D TYPING USING COMPLETE ANTI-D, ALBUMIN DISPLACEMENT TECHNIQUE FOR RE TYPING USING INCOMPLETE ANTI-D, ENZYME TECHNIQUES FOR RE TYPING USING INCOMPLETE ANTI-D

RHESUS (Rh) GROUPING SYSTEM

The discovery of the Rh system is based on the work by Landsteiner and Wiener in 1940 and by Levine and Stetson in 1939. A woman who delivered a still-born foetus was transfused with her husband's blood. Although both the husband and wife belonged to group O, the woman experienced a severe haemolytic reaction. 

Levine and Stetson proposed a theory that the woman's red cells were lacking in an antigen. This antigen was called new antigen, which, the child had inherited from the father. The antigen on the foetal cells stimulated the production of antibodies in the mother's blood. When she was transfused with husband's blood, these antibodies brought about the haemolytic reaction.

In 1940, Landsteiner and Wiener inoculated red cells of Rhesus monkeys into rabbits and guinea pigs. The resulting antibodies agglutinated the red cells of the monkeys and also of about 85% of the population. 

These 85% were called Rh (Rhesus) positive because they possessed the same antigen that was present on the red cells of Rhesus monkeys. The rest of the population were called Rhnegative. It was found that the antibodies to the same antigen can cause a haemolytic reaction.

		ABO GROUPING: TUBE MATHOD
	PATIENT”S RED CELLS + KNOWN ANTI SERUM		PATIENT:S SERUM +KNOWN RED CELLS
TUBE NO	ANTI –A SERUM	ANTI- B SERUM	ANTI-AB SERUM	A CELLS	B CELLS	O CELLS	BLOOD GROUP
R E A D I N	1	2	3	4	5	6
	-	-	-	+	+	-	O
	+	+	+	-	+	-	A
	-	+	+	+	-	-	B
	+	+	+	+	-	-	AB
	KEY: AGGLLUTINATION-------à+		NO AGLUTINATION ---------------à -

Rh Antigens

The Rh blood group system is much more complex than the ABO system. More than 40 antibodies have been described. Basically, there are six related blood group factors C, D, E, c, d and e; and the corresponding antibodies (except anti-d). Like ABO antigens, the Rh factors are also inherited traits. There are six Rhesus genes, C, D and E and their alleles c, d, and e. There are only eight possible combinations in which chromosomes can carry these genes. 

They are: CDe, cDE, CDE, De, Cde, cdE, CDE and cde. A shorthand system has been devised for easy identification of these combinations. The three pairs of genes are carried on the same chromosome and have three closely linked loci. Every individual has loci for six Rh genes.

The factors C, D, E, C and e (except d) are antigenic. They are capable of stimulating the production of antibodies if introduced into the body of an individual whose red cells lack them. However, the Rh antigens vary in their degree of antigenicity. The D antigen is the most immunogenic of them. 

The first transfusion of the Rh (D) antigen into an Rh(D) negative person will stimulate the production of anti-D antibodies. 

Subsequent transfusion with the Rh (D) antigen will result in a haemolytic transfusion reaction, similar to that of Levine and Stetson's patient in 1939. Therefore, it is necessary to test the blood of both the donor and the recipient for Rh (D) antigen before blood transfusion.

If the blood sample shows the presence of Rh (D) antigen, the individual is termed as Rh Positive; if it is absent, as Rh negative.

The D' Antigen

Some D antigens react weakly with the anti-D sera. This weak reactivity with anti-D sera is due to D'antigen. Some workers believe that the presence of this antigen can be the result of some genetic variation. One theory to explain D'antigen suggests that the D antigen is made up of four fractions. 

An Rh-D positive individual possesses all the four, while none is present in an Rh-D negative person. Some persons may lack one or more of these fractions and therefore their red cells are agglutinated by only some of the anti-D antisera. Such an incomplete D antigen is termed as D'. It does not usually react with complete anti-D, but it does react with incomplete anti-D antisera with a varying degree of reactivity.

While typing a blood sample for the Rh antigen, the anti-D serum will not produce agglutination with D'antigen, but will produce sensitised cells i.e. the red cells will be coated with the antibody. 

The presence of sensitised cells can be detected by the antiglobulin test. If a D person is given Rh-D positive blood, he is likely to produce anti-D antibodies, therefore, he is considered as an Rh-negative recipient. Similarly, a D' blood given to a Rh negative person will stimulate anti-D antibodies. Therefore D'blood is given only to Rh positive persons.

Rh Antibodies Unlike ABO antibodies, there are no naturally occurring Rh antibodies. All Rh-antibodies are 'immune' antibodies resulting from specific antigenic stimulation, e.g. transfusion, pregnancy or by injection of the antigen. Because there are no natural Rh-antibodies, antibody typing is not possible in the Rh system. Therefore, all Rh-typing methods depend upon antigen typing using known antiserum.

Some Rh-antibodies cannot be detected in saline suspensions of red cells. However, if a protein rich medium, such as serum or albumin is used, the antibodies can agglutinate the respective redcells. Such antibodies are called incomplete or albumin active antibodies.

The Rh-antibodies which can react even in saline solution are called complete or saline-active antibodies. There is still another class of antibodies which can be demonstrated only by means of antihuman globulin or Coomb's reagent. Such antibodies are known as incomplete univalent antibodies.

The size of the antibody molecule is largely responsible for the differences in their reactivities. The saline-active antibody molecules are of the IgM type and are the largest. The length of the IgM molecule is sufficient to cause bridging of adjacent red cells in suspension. When in suspension, red cells carry an electrical charge called the zeta potential, which causes repulsion among two adjacent red cells.

The IgM antibody molecules extend beyond the range of the zeta potential and agglutinate the red cells by binding onto their antigenic sites. The IgG molecules are smaller in size and cannot reach beyond the minimum distance between the cells, and are unable to agglutinate them. 

The zeta potential can be reduced by suspending the cells in a high protein mediume.g. patient's own serum or 22% albumin solution. 
Similarly, the antihuman globulin can detect incomplete univalent antibodies by bringing them together along with red cells to which they are attached

Rh Typing Methods

As there are no naturally occurring Rh-antibodies, Rh typing methods involve typing of red cells using known antisera against various Rh-antigens. Commercial antisera are available for C, D, E, C and e factors. Since anti-d antibody is not known to be present, it is not possible to test for Rh-d factor. Routine testing for Rh-antigens other than Rh-D is not recommended unless it is specifically indicated. All blood samples which do not give a positive reaction for Rh-D should be tested for D'antigen before reporting them as Rh-D negative. The method used for Rh-D typing depends upon the type of antibody used.

METHOD 1: SALINE METHOD FOR Rh-D TYPING USING COMPLETE ANTI-D

A. Slide Test

Specimen:- Whole blood or 50% red cell suspension prepared from clotted blood in 

patient's own serum. Reagents Anti-D antiserum (complete) Method

(1) Place a drop of antiserum on a labeled white tile.

(2) Add two drops of the specimen (whole blood or 50% red cell suspension).

(3) Mix well and place the slide on a warm viewing box, to bring the temperature of the mixture to about 37°C.

(4) Gently rock the tile back and forth for a maximum of two minutes.

(5) Examine microscopically for agglutination by transferring a small volume on a slide with a clean Pasteur pipette.

(6) Test a positive and a negative control in the same way.

Results:- Agglutination: Rh-D positive No agglutination: Rh-D negative

B. Tube Test

Specimen:-  Washed 5% red cell suspension of patient's blood. Reagent Anti-D antiserum (complete)

Method

(1) In an appropriately labelled test tube, add one drop of anti-D antiserum.

(2) Add one drop of 5% red cell suspension.

(3) Mix well and incubate at 37°C in a water bath for 30 minutes,

(4) Roll the tube gently to redisperse the cells.

(5) Check for agglutination microscopically.

If no agglutination occurs, centrifuge at 200 g for 1-2 minutes and check again.

Run known positive and negative controls simultaneously with the test.

Results :- Agglutination: Rh positive No agglutination: Rh negative.

METHOD 2: ALBUMIN DISPLACEMENT TECHNIQUE FOR RE TYPING USING INCOMPLETE ANTI-D

A 22% bovine albumin solution used in this method reduces the zeta potential (repellent electric charge), thus bringing the red cells closer, so that IgG antibody molecules can agglutinate them. Specimen 5% washed red cell suspension of patient's blood.

Reagents

1. Incomplete anti-D antiserum.

2. 22% bovine albumin. Method (1) In an appropriately labelled test tube, add one drop of incomplete anti-D and one drop of 5% red cell suspension.

(2) Mix well and incubate at 37°C water bath for 60 minutes.

(3) Without disturbing the cell button, run down one drop of 22% bovine albumin along the side of the tube onto the cells. Do not mix.

(4) Incubate further at 37°C for 30 minutes.

(5) Read microscopically for agglutination.

(6) Run positive and negative controls simultaneously with the test.

Results:- Agglutination Rh-D positive No agglutination Rh-D negative.

METHOD 3: ENZYME TECHNIQUES

Some antibodies agglutinate or lyse red cells which have been treated with proteolytic enzymes such as papain, trypsin or bromeline. These enzymes remove some negatively charged molecules from the red cell surface, allowing them to come closer for IgG molecules to agglutinate them. These proteolytic enzymes also enhance agglutination by removing a part of the hydration layer surrounding the red cell.

Note

The enzyme techniques can be used for cell typing, detection of incomplete antibodies, screening for antibodies before transfusion, and for detection of antibodies in the post-transfusion reactions.

Two-stage Papain Technique (Using Papain Treated Cells)

Specimen:- Packed red cells for cell typing.

Reagents 1. Known antiserum (e.g. incomplete anti-D for Rh-D typing).

2. 0.1% solution of papain in saline.

Technique

Step 1: Preparation of papain-treated cells 

Mix 4 drops of 0.1% papain and I drop of packed red cells in a labelled test-tube.

Incubate at 37°C in a water bath for 15 minutes. (The time may vary with the batch of papain and should be standardised.)

Wash the cells twice in saline.

Make a 5% suspension in saline (These cells can be used for up to 48 hours if stored at 4°C).

Step II: Antigen detection

(1) Mix 1 drop of papain treated cells from step 1 and 1 drop of known antiserum in a test tube.

(2) Incubate at 37°C for 20 minutes.

(3) Centrifuge at 200 g for 1 minute, or continue incubation upto 60 minutes.

(4) After centrifugation or incubation, examine macroscopically over a source of light for evidence of haemolysis in the supematant.

Then examine for agglutination by gently tapping the tube. Do not examine microscopically.

Record the presence of lysis and/or agglutination, which indicates a positive reaction.

Note

1. A similar technique can be used with ficin instead of papain.

2. The same technique can also be used for the detection of unknown antibodies in patient's serum using known control red cells.

Antiglobulin techniques are used for the detection of incomplete univalent antibodies which cannot be detected by the other methods described above. These antibodies react with the red cells, but the reaction is not observable as agglutination or haemolysis.