1 Blood Bank Practical Sheets

Practical NO (8): C- Rh Antibody Titration

(Antenatal Serology)

1.Rh Antibody Titration (Antenatal Serology):

1.1: Objectives:
At the end of this practical you will be familiar to:
1) The intended use and the principle of antibody titration.
2) The usual applications of titration.
3) Materials and methodology of the tests.
4) Interpretation of results.
5) Test limitation and precautions.

1.2: Intended use:

Hemolytic disease of the newborn (HDN) is a major cause of

fetal loss and death among newborn babies. A major cause of
HDN is an incompatibility of the Rh blood group between the
mother and fetus. Hemolytic disease that triggered by the D
antigen is most common. Although, other Rh antigens: c, C, E,
and e can also cause problems.

One group at increased risk of exposure and sensitization

by the D antigen are D negative mothers who give birth to D
positive infants (the child having inherited the D antigen from
the father). During delivery of the baby, Rh-positive fetal cells
enter the maternal circulation when the placenta separates
from the uterine wall. In general, only a small amount of fetal
blood (less than 30 ml whole blood) enters the mother’s
circulation, but due to the immunogenicity of the D antigen,
this is enough to cause sensitization. If a sensitized woman
becomes pregnant with another D positive baby, the baby will
be affected with severe HDN. The mother's immune response to
the fetal D antigen is to form antibodies against it (anti-D).
Initially, the maternal anti-D that is formed at the time of
sensitization is of the IgM type, which cannot cross the
placenta. In subsequent pregnancies, a repeat encounter with
the Rh D antigen stimulates the rapid production of type IgG
anti-D, which can be transported across the placenta and
enter the fetal circulation. Once in the fetal circulation, anti-D
attaches to the Rh D antigens found on the fetal RBCs, lead to
destroyed them and cause HDN.

Prepared by: Samah Awad AL-Subhi

2 Blood Bank Practical Sheets

So, we automatically perform the titer on any pregnant with

clinically significant Abs.

1.3: Principle:

Antibody titer is a laboratory test for determination the

concentration of a specific antibody in the patient's serum.

1.4: Procedure:

1.4.1: Sample:

Either plasma or serum can be titrated.

1.4.2: Reagents, Equipment, and Supplies:

 Indelible marking pen.

 Reagent cells known to have the antigen that will be
reacting with the tittered antibody (Rh D-positive RBCs
[O+] cell).
 Glass test tubes.
 Disposal pipettes.
 22% bovine albumin or LISS.
 Centrifuge.
 Lighted agglutination viewer.
 Test tube rack
 37oC incubator
 Wash bottle with physiologic saline
 Coombs serum (AHG reagent)

1.4.3: Method:

1. Verify that patient information on the sample matches

information on the worksheet.

2. Label 10 tubes according to the serial dilution: 1, 2, 4, 8,

16, 32, 64, 128, 256, 512 and the patient identification.

3. Add two drops of saline to tubes 2 through 512. No saline

in tube 1 (The first tube will be undiluted serum).

4. Add two drops of serum to both tubes 1 and 2.

5. Use a clean pipette to mix the 1/2 dilution several times

and then transfer two drops to tube 3.

6. Use a clean pipette to mix the 1/4 dilution several times

and then transfer two drops to tube 4.

Prepared by: Samah Awad AL-Subhi

3 Blood Bank Practical Sheets

7. Continue the same process for all dilutions until the last tube
(512). Remove two drops from tube 10 (512) and reserve in a clean
tube if the titration needs to be continued. If there is no need for
extra titration you can discard it.

8. Add one drops of specific red blood cells reagent (O+ cells).

9. Mix tubes thoroughly by gently shaking.

10. Centrifuge for 15 seconds at 3400 rpm

11. Gently re-suspend each cell button individually and examine

for agglutination with the aid of the lighted agglutination viewer
(Macroscopically).

12. Record the result in the appropriate place on the worksheet as

each tube is read.

13. Add two drop of LISS in each test tube and incubator for 10
minutes .If LISS not available then add two drop of 22% bovine
albumin and increase the incubation period to 30-60 minutes.

14. Centrifuge for 15 seconds at 3400 rpm and read for

agglutination as usual.

15. Record the result in the appropriate place on the worksheet as

each tube is read.

16. Wash all tubes 3 times with normal saline.

17. Immediately after the last wash, add two drops of AHG in all
test tubes.

18. Centrifuge for 15 seconds at 3400 rpm.

19. Immediately re-suspend all tubes gently and examine for

agglutination as usual.

20. Record the result in the appropriate place on the worksheet as

each tube is read.

21. Confirm negative result by adding one-drop Coombs control

cells (CCC) to tube showing no agglutination and centrifuge for 15
seconds at 3400 rpm.

22. Gently re-suspend and examine for agglutination.

Agglutination should be present in this step or the test is invalid.

Prepared by: Samah Awad AL-Subhi

4 Blood Bank Practical Sheets

23. Write a check mark in the appropriate place on the worksheet

as each tube is read.

24. Discard all materials in the appropriate trash containers.

1.4.4: Results Interpretation:

1) Observe the highest dilution that produces 1+ macroscopic

agglutination
2) If the dilution level: 1/32 (tube number 6) not 32 tube number
is considered to be significant, and it is an indication for
amniocentesis.
3) A rise in titer would need to be at least two (2) dilution increase
between the current specimen and the previous month.

1.5: Test limitation and Precautions:

1- If testing a pregnant female, each month serum should be

compared to the previous month.

2-Careful pipetting is essential.

Prepared by: Samah Awad AL-Subhi

5 Blood Bank Practical Sheets

Worksheet of practical NO (8)

Number of sample:

Results:

Rh Antibody Titration (Antenatal Serology)

Tube No. RT 37 oC AHG

phase phase phase ✔
Tube (1)

Tube (2)

Tube (3)

Tube (4)

Tube (5)

Tube (6)

Tube (7)

Tube (8)

Tube (9)

Tube (10)

Results Interpretation:

Prepared by: Samah Awad AL-Subhi

6 Blood Bank Practical Sheets

Exercise

Study Questions:

1. Define the terms “HDN”?

2. Define “Antibody titration.” as it relates to the D antigen?

3. What are the intended use and the principle of antibody titration?

Prepared by: Samah Awad AL-Subhi