Rhesus factor (Rh) typing

Based on conglutination, Rh typing is performed with special antirhesus serum at the laboratory. ABO grouping usually precedes this.

The equipment and prerequisites are as follows:

1. Two different series of the standard anti-Rh serum to match the group of the blood under investigation (you may also use a compatible group of the standard washed Rh positive and Rh negative red blood cells of the same group instead).

2. Petri dish.

3. Water bath.

4. Pipette for serum.

5. Ground slide (or glass rod). Procedure

• Put three big drops of the anti-Rh serum of one serial type into the Petri dish;

• add three drops of that of the other series to arrange the drops in two parallel lines;

• place a few small drops of the test blood on the anti Rh drops in the first vertical row of both; series (in the ratio of serum to blood as 10: 1 or 5: 1);

• put the same small amount of standard Rh positive red blood cells on the serum drops in the second vertical row (to check for its strength);

• add the drops of Rh negative standard red blood cells to the serum drops in the third row (to check for its specificity);

• mix the serum and red blood cells of each row separately, with different glass rods, cover the dish and place it on the water bath at 46-48 °C;

• observe the results in 10 minutes (the room should be well lit).

Results

1. The drop with the standard Rh-positive red blood cells should give a positive reaction of agglutination.

2. The drop with Rh-negative red cells should be negative.

3. The agglutination seen with the drops in both series of the serum with red cells of the blood under examination suggests the presence of Rh factor in the blood is rhesus positive.

4. Otherwise it is Rh-negative.

It will be noted that the addition of normal saline to the serum, as is the case with ABO grouping, must be avoided, since it may counteract agglutination.

The factors that may be responsible for the mistakes in Rh typing are as follows:

• reduced activity of the standard Rh serum;

• wrong serum-blood proportions;

• inappropriate room temperature;

• reduced exposition period (less than 10 minutes);

• addition of normal saline into the serum;

• absence of testing for controls for serum strength and specificity;

• group incompatibility of the standard serum with the blood under investigation and the standard red blood cells.

In emergency, Rh typing can be performed at bedside. An «express» method of Rh typing requires special reagents - anti-Rh serum of group IV (AB) diluted in 20-30% albumin used as the conglutin, i.e. that substance that allows aggregation of red blood cells under room temperature.

Procedure

• Put a drop of the anti-Rh serum of group IV (AB) and nearly a drop of Rh-negative serum of group IV (AB) free of antibodies on a ground slide or a Petri dish;

• add to each drop of serum 2-3 times less than the patient's blood in the amounts half even less as much as those of the serum;

• mix these with a glass rod or by shaking for 3-4 minutes;

• add one drop of normal saline to each mixture;

• Observe the results in 5 minutes.

Results

1. Agglutination of the red blood cells present with the anti Rh serum and absent with the control serum implies Rh-positive blood.

2. The absence of agglutination with the serum is indicative of the patient's blood being Rh-negative.

3. In case of agglutination with both sera the reaction has to be regarded as unclear.

In emergency, transfusion of only Rh-negative blood is possible, and if it is not available and the patient's condition requires blood transfusion, Rh-positive blood may be given following cross-matching the blood for Rh compatibility.

The importance of blood grouping during blood transfusions

The antigens of blood, mainly those of ABO system and Rh, can be responsible for its immunological incompatibility. If the recipient's (i.e. patient's) blood contains antigens against to those of the red blood cells and antibodies in donor's plasma, agglutination of the red blood cells is likely to occur. This type of agglutination can be seen when similar antigens and antibodies A and 6, B and b, as well as Rh antigens and anti Rh antibodies react. For this to take place there should be sufficient amounts of antibodies, or the titre, present in the blood serum. Ottenberg's rule is based on this principle, which says that the donor's red blood cells transfused agglutinates and since the agglutinin of the transfused blood is diluted by the recipient's blood, the concentrations are not as high as to cause agglutination of the recipient's blood. Without cross-matching all recipients may therefore receive only group I (0) blood, as this blood is devoid of any agglutinogens (the holders of group I blood are referred to as universal donors). On the other hand, patients of group IV (AB) blood can receive that from donors of all the other blood groups, since the patient's blood is free of any agglutinins (the holders of group IV blood are called universal recipients). However, if large amounts of blood are needed, as is the case in uncontrolled bleeding, agglutinins of the blood transfused can cause agglutination of the patient's red blood cells. Ottenberg's rule is applicable only when the amount of blood to be transfused does not exceed 500 ml.

If Rh-positive blood is transfused for the first time to a Rh-negative patient who has not been sensitised earlier, overt incompatibility reactions are not observed, antibodies, however, being formed. Giving blood to an Rh-negative woman who has been sensitised through pregnancies with a Rh-positive foetus may result in Rh incompatibility. Transfusing Rh-negative blood to a Rh-positive recipient, one should bear in mind that production of antibodies to the weak antigen system of Rh present in the transfused blood cannot be ruled out.

According to the current principles of transfusiology, the blood transfused may be of only the same group (the ABO system and Rh group). In emergency, Ottenberg's rule can be applied.

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