Blood typing: standard red blood cell method

Collection and preparation of biological material

To correctly perform antibody detection, a specialist needs to obtain the liquid part of the patient's blood, devoid of its own formed elements. The preparation process includes two main stages:

Material collection. Venous blood of the patient is collected in the treatment room. To properly carry out the reaction, a volume of 3–4 ml is required.
Obtaining serum. The collected blood is subjected to mandatory centrifugation. This physical procedure is necessary to separate the cellular sediment. As a result of centrifugation, the researcher obtains serum, which will serve as the patient's main material for the analysis (the use of plasma, lysate, or sediment is not provided for in this method).

Methodology and technique of the reaction

The typing procedure itself requires attentiveness and compliance with time intervals. The workflow is as follows:

First, one large drop of the test patient's serum is applied to a specially marked white tile.
Standard red blood cells of known groups — I, II, and III — are added to the serum. It is important to remember that red blood cell mass of a known group acts as the reagent, rather than standard sera or monoclonal antibodies (anti-A and anti-B).
Strict proportion: the volume of the added drop of standard red blood cells must be exactly five times smaller than the volume of the applied drop of the test serum.
The reagents are gently mixed using the corner of a clean microscope slide.
After mixing, the tile must be continuously rocked for 3 minutes.
Then, one drop of an isotonic NaCl solution is added to the reacting mixture.
The final evaluation of the result (presence or absence of cell agglutination) is performed a total of 5 minutes after the start of the reaction.

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Interpretation of research results

Evaluation of blood group affiliation is based on recording the phenomenon of agglutination — specific clumping of standard red blood cells under the influence of antibodies contained in the patient's serum.

Group I(0): Antibodies causing agglutination with standard red blood cells II(A) and III(B) are present in the serum. There is no reaction with cells I(0).
Group II(A): The serum produces agglutination exclusively with standard red blood cells III(B). When red blood cells I(0) and II(A) are added, no clumping occurs.
Group III(B): Agglutination is clearly recorded only with red blood cells II(A). In drops with standard red blood cells I(0) and III(B), the reaction is completely absent.
Group IV(AB): Group antibodies are absent in the serum, therefore agglutination is absent with all standard red blood cells (I, II, and III).

Briefly: answers to topical questions

For what purpose is isotonic NaCl solution added to the reacting mixture?

Isotonic sodium chloride (NaCl) solution is added to the reacting mixture as a preventive measure against false agglutination, i.e., a false-positive result. This action helps prevent pseudo-agglutination, which occurs due to the drying of the drop and the formation of red blood cell «rouleaux», creating the appearance of a reaction when it is actually absent.

Which specific agglutinins (antibodies) are contained in the serum of a patient with blood group II(A)?

The blood serum of a patient with group II(A) contains exclusively the $\beta$ agglutinin (antibody). According to the incompatibility rule of the AB0 system, the simultaneous presence of homologous components in a person's blood is impossible. Therefore, since agglutinogen A is located on the membrane of this group's red blood cells, normally only the heterologous $\beta$ agglutinin is present in the blood serum.

At what indoor air temperature should the blood typing reaction be performed?

The blood typing reaction must be performed at an air temperature strictly above 15 °C. Compliance with this temperature regime is a mandatory measure to prevent cold agglutination. At temperatures below 15 °C, a false-positive result may occur, where true agglutination is absent, but the reaction appears visible.

How to memorize

Serum never causes agglutination with red blood cells of its own group and group I(0).

Class and exam questions

What specific patient material is needed for this method?

The study requires clean patient serum, which is obtained by centrifuging 3–4 ml of venous blood.

In what ratio should the reagents be mixed?

Mixing is done in a 5:1 ratio. A drop of the patient's serum must be 5 times larger than a drop of standard red blood cells.

When is the isotonic solution added and why is it needed?

A drop of isotonic NaCl solution is added after 3 minutes of rocking the tile. The final result is evaluated at the 5th minute.

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What else to study on this topic

Centrifugation mechanisms and differences between serum and plasma
Causes of false agglutination and the role of isotonic NaCl solution
Errors in non-compliance with reagent proportions
Comparison of the standard red blood cell method with the use of anti-A and anti-B reagents

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Blood typing using standard washed red blood cells