Secretor Status

PRINCIPLE

Approximately 80% of the population has the secretor (Se) gene. These people secrete water-soluble blood group substances in their saliva and other body fluids. Group A secretes A substance and a small amount of H, group B secretes B (and H) substance, group O secretes H substance only, and group AB secretes A, B, and a small amount of H.

To determine if a person is a secretor, the principle of AGGLUTINAITON INHIBITION is utilized, where the presence of agglutination means a negative test, and no agglutination is interpreted as a positive result.

Part I - Antibody Neutralization:

Saliva is mixed with commercial antiserum (Anti-A, Anti-B or Anti-H) and allowed to incubate briefly. If the patient is a secretor, the soluble blood group antigens in the saliva will react with and neutralize the antibodies in the commercial antiserum. It is necessary, however, to dilute the commercial antiserum so that its antibody titer more closely matches the antigen level in the saliva.

Part II - Agglutination Inhibition:

When commercial RBC of the appropriate blood group are then added to the test mixture, there should be no free antibody to agglutinate them if the patient is a secretor, because the antibodies have already reacted with the blood group antigens in the saliva. The reaction will be negative for agglutination, but is interpreted as positive for secretor status.

If the patient is a non-secretor, there will be no blood group antigens in the saliva; the antibodies in the antiserum will not be neutralized and will be free to react when the test cells are added. Therefore, agglutination is a negative test for secretor status.

 

SAMPLE

Saliva collected as described in the procedure below

REAGENTS, EQUIPMENT, AND SUPPLIES

  • Clean tube to collect saliva
  • 16 x 100 mm pyrex test tube
  • Dispo pipets
  • Boiling waterbath
  • 12 x 75 mm tubes
  • Marking pen
  • Reagent Anti-A, Anti-B or Anti-H, diluted to give a 2+ reaction
  • Reagent A1 cells, B cells or O cells (Screening cells)
  • Serofuge
  • Lighted agglutination viewer

PROCEDURE

  1. Collect 2 to 3 ml saliva in a clean 16 x 100 mm tube. Use paraffin wax or clean rubber bands to stimulate secretions - do not use gum.
     
  2. Place in a boiling waterbath for 10 minutes. This inactivates enzymes that might otherwise destroy blood group substances.
     
  3. Allow to cool briefly, then transfer to a 12 x 75 mm tube.
     
  4. Centrifuge at least 5 minutes.
     
  5. Label three 12 x 75 mm test tubes:
    A TEST;
    B TEST;
    H TEST.
     
  6. Label three more tubes:
    A CONTROL
    B CONTROL
    H CONTROL
    (These are dilution controls to ensure the anti-sera was not diluted beyond its capacity to agglutinate).
     
  7. Add one drop of the appropriate diluted antiserum to each tube:

    one drop dilute anti-A to the A test and A control

    -one drop dilute anti-B to the B test and B control

    -one drop anti-H to the H test and H control
     

  8. To each TEST tube, add one drop of clear saliva.
     
  9. To each CONTROL tube, add one drop of saline.
     
  10. Mix and incubate at room temperature 10 minutes.
     
  11. Add one drop of the appropriate reagent red cells to each tube: A1 cells for the A test and control B cells for the B test and control Screening Cell I or II for the H test and control.
     
  12. Mix and incubate at room temperature 10 minutes.
     
  13. Centrifuge the length of time for saline reactions in the serofuge.
     
  14. Using the lighted agglutination viewer, read, grade and record the reactions.
     
  15. The CONTROL tube should have agglutination for the test to be valid.

     

INTERPRETATION

  1.  Agglutination in all of the patient TEST tubes indicates a negative result for secretor status.
     
  2. If any one of the patient TEST tubes is not agglutinated, this indicates a positive test for secretor status, and the tube showing the non-agglutination should indicate the ABO type. 

REFERENCE

AABB Technical Manual, 14th Edition, 2002, Pages 674-676

Manufacturer's Directions From Special Lectin H.

Blaney, K.D. and Howard, P.R., Basic and Applied Concepts of Immunohematology,1st edition, pp. 100-101

Authored by:  Peggy Schroeder  Revised by: Peggy Jensen

 

Clinical Microbiology Syllabus