Classic method: conglutination test
Before performing the analysis, the patient's blood group according to the AB0 system must be known. Two different series of anti-Rh sera compatible with the test subject's blood group are used for the study.
The procedure is performed in a Petri dish:
- Three drops of one serum series are applied to the first row, and another series to the second row.
- The test blood is added (serum to blood ratio of 10:1 or 5:1).
- Standard Rh+ erythrocytes are added as an activity control.
- Standard Rh– erythrocytes are added to control for specificity.
- The drops are gently mixed, covered with a lid, and placed in a water bath at 46–48 °C for 10 minutes.
Result evaluation
Control samples are evaluated first: agglutination must occur with positive erythrocytes and be absent with negative erythrocytes.
Interpretation of results for the patient's blood sample:
- If agglutination is present in both series of sera, the blood is considered Rh-positive (Rh+).
- If no erythrocyte clumping has occurred, the blood is Rh-negative (Rh–).
Important: unlike AB0 group determination, adding physiological saline during the reaction is strictly prohibited when assessing Rh status, as this may yield a false-negative result.
Common testing errors
Distortion of results may be a consequence of procedural violations:
- Use of anti-Rh sera with reduced activity.
- Incorrect ratio of serum to blood volumes.
- Violation of the temperature regime (insufficient or excessive heating).
- Shortened incubation time (less than 10 minutes).
- Addition of NaCl solution during the conglutination reaction.
- Ignoring activity and specificity control samples.
- Group incompatibility of the reagents used.
Express determination method
This method is used in emergency cases and does not require heating. Anti-Rh serum of group IV(AB) diluted with albumin (20–30%) or dextran (30–33%) solution is used. The colloidal medium ensures erythrocyte clumping at room temperature.
Control Rh-negative serum without antibodies is applied next to the drop of reagent. The test blood is added (its volume should be 2–3 times less than the serum), mixed for 3–4 minutes. Then a drop of saline is added, and the result is evaluated after 5 minutes.
If the reaction is doubtful (agglutination in both drops) or determining the Rh factor is impossible, only Rh-negative blood is used for emergency transfusion.
Class and exam questions
Why is it forbidden to add physiological saline in the classic method?
The addition of isotonic sodium chloride solution disrupts the conglutination reaction and can lead to a false-negative result.
Why is a dextran or albumin solution needed in the express method?
The colloidal medium from these solutions promotes erythrocyte aggregation without the need for a water bath.
What to do during an emergency transfusion if the result is doubtful?
In such a situation, only Rh-negative blood is transfused. In extreme cases, transfusion of Rh+ blood is permitted, but strictly after an individual compatibility test.
What else to study on this topic
- Mechanism of the conglutination reaction
- Causes of false agglutination
- Properties of colloidal media (dextran and albumin)
- Rh compatibility test before blood transfusion
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