DETERMINING SECRETOR STATUS

I. SALIVA TEST

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 Agglutination 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.

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
  1. 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).
  1. Add one drop of the appropriate dilute 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
  1. To each TEST tube, add one drop of clear saliva.
  2. To each CONTROL tube, add one drop of saline.
  3. Mix and incubate at room temperature 10 minutes.
  4. 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.
  1. Mix and incubate at room temperature 10 minutes.
  2. Centrifuge the length of time for saline reactions in the serofuge.
  3. Using the lighted agglutination viewer, read, grade and record the reactions.
  4. 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.

II. LEWIS TYPINGS

PRINCIPLE

Lewis antigens are not intrinsic to the red cell. People who have the Lewis (Le) gene produce Lewis antigens that are carried by substances in the plasma and are adsorbed onto red blood cells.

Lewisa is initially formed, and if the patient is a non-secretor (lacks the Se gene), Lewisa substance is adsorbed onto the red cell, and the patient types as Lewisa.

If the patient is a secretor, however, the Se gene activates the H gene, which causes an additional sugar to be added to Lewisa, converting it to Lewisb. Both Lewisa and Lewisb are present in the plasma of secretors, but Lewisb adsorbs preferentially onto the red cell, and the patient types as Lewisb.

REAGENTS, EQUIPMENT, AND SUPPLIES

  • 12 x 75 mm tubes test tube rack
  • marking pen dispo pipets
  • wash bottle with physiologic saline Reagent Anti-Lewisa
  • serofuge Reagent Anti-Lewisb
  • lighted agglutination viewer

SAMPLE

Red cells collected in EDTA or heparin may be used if tested within 2 days of collection. Clotted whole blood may be tested up to 14 days from time of collection. Donor cells from a segment may be tested up to 35 days from the time of collection.

PROCEDURE

  1. Label a 12 x 75 mm tube with the patient's name, and add 2-3 drops of red cells.
  2. Wash the cells 4 times with physiologic saline.
  3. After the fourth wash, reconstitute to 3%.
  4. Label six 12 x 75 mm tubes: patient name, Lea
  • patient name, Leb
  • Lea POS Control
  • Lea NEG Control
  • Leb POS Control
  • Leb NEG Control
  1. Add one drop Anti-Lewisa to all the Lea tubes and one drop Anti-Lewisb to all the Leb tubes.
  2. Add one drop of the patient's 4x washed cells to each patient tube.
  3. Controls:
  • Add one drop of a cell known to be positive for Lewisa to the Lea POS control.
  • Add one drop of a cell known to be negative for Lewisa to the Lea NEG control.
  • Add one drop of a cell known to be positive for Lewisb to the Leb POS control.
  • Add one drop of a cell known to be negative for Lewisb to the Leb NEG control.
  1. 8. Shake all tubes gently to mix, and incubate at room temperature 5-10 minutes.
  2. 9. Centrifuge the length of time required by your serofuge for saline reactions.
  3. 10. Gently resuspend and examine MACROSCOPICALLY for agglutination, using the lighted agglutination viewer.
  4. 11. Read, grade and record results.

INTERPRETATION

If a person has the Lewis gene, he or she will type as either Lewisa positive and Lewisb negative; or Lewisa negative and Lewisb positive. The patient will not be positive for both.

Persons with the Lewis gene and the secretor gene type as Lewisa negative, Lewisb positive.

Persons with the Lewis gene but not the secretor gene type as Lewisa positive, Lewisb negative.

If a patient does not have the Lewis gene, he or she will type as Lewisa negative and Lewisb negative, regardless of secretor status.

REFERENCES

Bryant, N. An Introduction To Immunohematology, 3rd Ed. (1994), pages 115-116, 136

Ortho Diagnostics manufacturer's directions, Anti-Lewisa and Anti-Lewisb Typing Serum, revised 2/86.

AABB Technical Manual, 11th Edition, 1993, Pages 626-626

matc\secretor.lab

Author:  Peggy Schroeder, revised by Peggy Jensen

Clinical Microbiology Syllabus