Recently interest has been generated in use of column technology for serological testing. The aims of this technology is to standardize red cell agglutination reactions and trap the agglutinates to permit simple and reliable reading.
The column consists of special microtubes containing a sephadex gel matrix. Red cells and serum or red blood cells alone are dispensed into the microtubes, incubated if necessary and then centrifuged under strictly controlled parameters.
The gel within the microtubes acts as a sieve, unagglutinated cells form a button at the bottom of the microtube and agglutinated red blood cells are trapped in the gel.
The Sephadex gel may be
Neutral
Specific reagent
AHG
Anti-A
Anti-B
Anti-D
Anti-Kell, etc.
Image : Reading and grading of haemagglutination in gel system
Advantages of gel system
- Superior in sensitivity to conventional tube test without loss of specificity.
- Easy to use.
- Requires small amount of red cells and serum for testing (1O-40m1), making it ideal for neonatal and paediatric use.
- Reactions are easily visible and can be graded
- Reactions using gel techniques are stable for at least 48 hours and the gel cards can be photocopied providing a permanent record for future reference.
Sold phase tests
These depend on the immobilization of one of the reactant so that during testing the immobilized component captures additional reactant from the liquid phase and binds them to solid phase. These techniques have been successfully developed for a range of serological testing including red cell grouping, antiglobulin testing and antibody detection.
Grouping of Cord or Newborn Infant Blood
- Wash cord red cells 3-4 times with saline to minimize errors due to Wharton’s Jelly.
- Reactions in cell grouping may be weak as ABO antigens are not fully developed at birth.
- Corresponding ABO antibodies are usually absent, therefore, only cell grouping is recommended till 6 months of age.
Testing for Al and A2 Subgroups
Anti-Al reagent is used to differentiate Al and A2 subgroups.
Sources of anti-Al reagent
Lectin - Dolichus biflorus (most commonly used)
Naturally occurring anti-Al in A2 and A2B individuals
Human anti-Al(by adsorption of group B anti-A serum by A2 cells)
Dolichus biflorus lectin reacts specifically with Al antigen and causes agglutination. It is stored at 4-8°C and may be frozen at -20°C for prolonged storage.
The group A and AR blood groups can be subtyped into Al, A2 and A2B and A2B. Approximately 8% of A2 individuals and 30% A2 B individuals develop naturally occurring anti-Al antibodies. Therefore, it may be necessary to determine the subgroups of A for both, donors and the patients. If a patient with anti-Al requires blood transfusion, it will be necessary to select blood from A2 donors.
Procedure
1. Place 1 drop of anti-Al reagent into a clean, dry test tube.
2. Add 1 drop of 2-4% saline suspension of patient’s cells.
3. Mix and leave at RT for 30-60 minutes.
4. Gently agitate and examine for agglutination.
Interpretation
Reactions showing agglutination indicate Al blood group.
Controls
Perform the test using known Al cells and A2 cells instead of patients’ cell. These will act as positive and negative controls.
Errors Encountered in ABO Grouping
Errors during ABO blood grouping usually present as discrepancies in the cell and serum grouping.
The important factors leading to such problems could be due to
