1 Blood Bank Practical Sheets

Practical NO (10): E- Ag Phenotype Test

1.Objectives:
At the end of this practical you will be familiar to:
1. The intended use and the principle of the Ag phenotype.
2. Diagnostic significant of the Ag typing.
3. Materials and methodology of the test.
4. Interpretation of results.
5. Test limitation and precautions.

2. Ag phenotype:
2.1:Intended use:

Screening the presence antigen in donor whenever a candidate for

transfusion has a clinically significant antibody to ensure blood
transfusion free from any transfusion reaction.

2.2: Principle:

Ag phenotype done by added donor cells directed against special

antisera to determine the type of antigen, which is present on donor cells.

2.3: Diagnostic significance:

1) Determine the type of antigen that is present on donor cells.

2) Confirm identity of an antibody when it is first identified.
3) 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.

2.4: Procedure:

2.4.1: Sample:

Sample in EDTA or plain tube may be used.

2.4.2: Reagents, Equipment, and Supplies:

 Indelible marking pen.

 Vial of reagent antiserum with manufacturer's directions.
 Heterozygous positive control cell.
 Negative control cell.

Prepared by: Samah Awad AL-Subhi

2 Blood Bank Practical Sheets

 Glass test tubes.

 Disposal pipettes.
 Wash bottle with physiologic saline.
 AHG reagent (Coomb's serum).
 Test tube rack.
 37oC incubator.
 22% Bovine albumin or LISS.
 Coombs Control Cells.
 Centrifuge.
 Lighted agglutination viewer.
 Glass slide.
 Microscope.

2.4.3: Method:

1. Verify that information on the sample matches information on the

worksheet.

2. Select the vial of antiserum. There are commonly special

antisera available for the following antigens:

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

3. 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 the cell number, lot number, expiration date, and
manufacturer on the worksheet.

4. Select one cell known to be heterozygous positive for the antigen being
tested. Use of a heterozygous cell ensures that 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.

5. Prepare a washed 3% suspension of cells to be tested. Check

manufacturer's directions carefully.

Prepared by: Samah Awad AL-Subhi

3 Blood Bank Practical Sheets

6. Label the following tubes:

 NEG control.
 POS control.
 Donor test.

7. Add antiserum to each of the above tubes. (See manufacturer’s

directions for number of drops).

8. Add one drop of the cells selected in #3 above to the NEG control,
and add one drop of the cells selected in #4 above to the POS control.

9. Add one drop washed 3% donor cells to be antigen-typed to the

test tube.

10. Centrifuge for 15 seconds at 3400 rpm.

11. Examine for agglutination using the lighted agglutination

viewer (Macroscopically) and microscopically.

12. Follow the manufacturer's directions enclosed with the vial of

antiserum. Treat test cells, and positive and negative controls identically
from this point on.

13.If a Coombs procedure is used, be sure to confirm all negative

results with Coombs Control Cells.

14. Record the result in the appropriate place on the worksheet and
interpretation of it.

15. Discard all materials in the appropriate trash containers.

2.4.4:Results Interpretation:

 Agglutination indicates the presence of antigens.

 Lack of agglutination indicates the absence of antigens, and the
test is reported negative.

2.5: Test limitation and Precautions:

1. For the test to be valid, the controls must be as follows:

 Positive control: Strong positive (at least 2+).

 Negative control: Negative.

2. Antigen typing performs mainly to donors.

Prepared by: Samah Awad AL-Subhi

4 Blood Bank Practical Sheets

3.Be sure to perform antigen typing in a pre-transfusion sample (patient

not receiving blood since 3 month). Antigen typing on a recently
transfused patient may produce mixed-field or false-positive results.

4.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.

Prepared by: Samah Awad AL-Subhi

5 Blood Bank Practical Sheets

Worksheet of practical NO (9)

Number of sample:

Name of Ag:

Positive control (Heterozygous cell):

Negative control:

Results:

Ag Phenotype Test
Immediate spin Incubation phase (at Anti-human
phase (at RT) 37 oC) globulin phase

POS Control

NEG Control

Donor Test Tube

Results Interpretation:

Prepared by: Samah Awad AL-Subhi

6 Blood Bank Practical Sheets

Exercise

Study Questions:

1. Why we do the Ag typing test?

2. List clinical conditions for which the Ag typing is useful?

Prepared by: Samah Awad AL-Subhi