Antigen Typing

PRINCIPLE

Whenever a candidate for transfusion has a clinically significant antibody, whether currently detectable or detected previously, all units transfused must first be typed for the corresponding antigen.

Antigen typing of the patient is also done to confirm identity of an antibody when it is first identified.

Some prenatal workups include a request for antigen typing of the father when the mother has an antibody, to predict the likelihood of hemolytic disease of the newborn.

SAMPLE

Because a small volume of patient cells is used, a small lavender top (EDTA) or red top (clotted) tube is sufficient. The sample used for the type and crossmatch may be used for antigen typing.

REAGENTS, EQUIPMENT, AND SUPPLIES

  • Vial of reagent antiserum with manufacturer's directions
  • Heterozygous positive control cell
  • Negative control cell
  • Other materials described in the manufacturer's directions

PROCEDURE

NOTE: To ensure accuracy of results, the direction circular enclosed with each vial of antiserum must be followed closely.  The amount of antiserum, incubation time, and temperature, and type of procedure will vary from lot to lot and from manufacturer to manufacturer.
  1. Select the vial of antiserum. There is commonly special antisera available for the following antigens:

    C, c E, e
    Lewis a, Lewis b
    Kell, Cellano
    P1
    Fy a, Fy b
    M, N, S, s
    Jka, Jkb
     

  2. Select one cell known to be negative for the antigen being tested. This can usually be a screening cell but can be an identification panel cell as well.  Record which cell you are using on the worksheet.
     
  3. Select one cell known to be heterozygous positive for the antigen being tested. Use of a heterozygous cell ensures that the antiserum is reactive with weaker expressions of the antigen. This will usually have to be a panel cell. Record the cell number, lot number, expiration date, and manufacturer on the worksheet.
     
  4. Prepare a washed 3% suspension of cells to be tested. Check manufacturer's directions carefully - some methods call for multiply-washed cells, including the positive and negative controls.
     
  5. Label the following tubes:
  • NEG CONTROL
  • POS CONTROL
  • PT (or DONOR) TEST

  1. Add antiserum to each of the above tubes. (See manufacturer’s directions for number of drops)
     
  2. Add one drop of the cells selected in #2 above to the NEG control, and add one drop of the cells selected in #3 above to the POS control.
     
  3. Add one drop washed 3% patient or donor cells to be antigen-typed to the TEST tube.
     
  4. Include a patient or donor cell control:

  • label a tube CELL CONTROL
  • add one drop Rh control
  • add one drop of the cells to be antigen-typed
  • treat this tube exactly as the test - except don't add reagent antiserum - the Rh control albumin is replacing this
  1.  Follow the manufacturer's directions enclosed with the vial of antiserum. Treat test cells, the cell control, and positive and negative controls identically from this point on.
     
  2. If a Coombs procedure is used, be sure to confirm all negative results with Coombs Control Cells.
     
  3. Record all results in the proper columns on the antigen typing worksheet.

NOTES AND PRECAUTIONS

  1. For the test to be valid, the controls must be as follows:
  • cell control: negative
  • positive control: strong positive (at least 2+)
  • negative control: negative
  1. False positives may result if the cells being tested have a positive DAT. This will be detected by agglutination in the cell control tube. The antigen typings on these patients will give false positive or possibly mixed-field results. Specialized procedures may be used to remove the antibody from the patient cells so that antigen typing may be preformed. See the AABB Technical Manual.
     
  2. Be sure to perform antigen typings on a pre-transfusion sample, if possible. Antigen typings on a recently-transfused patient may produce mixed-field or false-positive results.

REFERENCE

AABB Technical Manual, 13th Edition, 1999, Pages 406-407

Manufacturer's Directions From Special Antisera.

Authored by:  Peggy Schroeder  Revised by: Peggy Jensen

 

Clinical Microbiology Syllabus