1) Rh isoimmunization occurs when an Rh-negative mother is exposed to Rh-positive fetal blood cells, leading to production of IgG antibodies that can cross the placenta and destroy fetal red blood cells. 2) Prevention involves identifying at-risk Rh-negative mothers and administering anti-D immunoglobulin within 72 hours of potential fetal-maternal hemorrhage to suppress immunization. 3) For sensitized pregnancies, fetal anemia is monitored through Doppler ultrasound or invasive tests, and treated with intrauterine transfusions if severe anemia is detected before 35 weeks.

1. RH Iso Immunization
Mai Raafat, MD
Helwan university

2. Background
• The antigens expressed on the red blood cell determine an individual's blood group.
• There are more than 40 blood group systems (ABO, Rh, Kell, Duffy...)
• The most important of them are the ABO system (with blood types A, B, AB, and O)
and Rh system (Cc, D, Ee), because they are the most immunogenic of all the blood
group systems.
• The most immunogenic in Rh system is the D antigen. Its presence or
absence designated a person as Rh positive or negative.
• Fetal Rh antigens are present as early as 40 days after conception in contrast to A
and B antigens that are not well developed at birth.
• Most naturally occurring anti A and anti B antibodies are of IgM type which cannot
cross the placenta.
• They are not present at birth and start to be produced 2-8 months after birth.

3. • Normal plasma contains no anti-D antibodies.
• Anti-D antibodies develop only in Rh negative people only with exposure to the
D antigen.
• The primary immune response to D antigen occurs over 6 wks to 12 months. It is usually
weak and of IgM type. Upon 2nd exposure to D antigen, an amnestic immune response
occurs, which is both rapid and of IgG type.
• 85% of indeviduals are Rh +ve, and 15% are Rh -ve

5. Blood groupsystems

7. Potentially sensitizing events during pregnancy
• The immune response to D antigen varies considerably among women, and depends on several
factors such as volume, frequency, ABO incompatibility, and the immune capacity of the mother.
Early pregnancy Late pregnancy
Abortion Antepartum hemorrhage
Ectopic pregnancy External cephalic version
Vesicularmole IUFD
Chorionicvillussampling Abdominaltrauma
Amniocentesis, cordocentesis Delivery (vaginal or CS) √√√√

8. •The 1ry response takes up to 6
month to occur and consistsof the
large IgM antibodies that can’t cross
the placenta --> 1st fetus usually not
affected.
•In subsequent pregnancies if the
mother is exposed again to the D
antigen --> 2ry immune response
which is rapid & consistsof IgG that
can cross the placenta and attach to
D antigen on the surface of the
RBCs --> destruction of RBCs.

9. Some women are already sensitized in their 1st pregnancy if
1. They received Rh +ve blood or platelets before.
2. Grandmother theory: here, the woman is Rh –ve, whereas her mother was Rh
+ve. Maternal-fetal hemorrhage occurred during her birth --> passage of Rh +ve
RBCs from the grandmother to her Rh –ve daughter --> sensitization

10. Rh alloimmunization

12. Management
• The objective of antenatal management is
➢ prevention of Rh alloimmunisation in the Rh-negative unsensitized
women.
➢early detection and treatment of fetal anemia in Rh-negative sensitized
women.
• This includes history taking, clinical examination,
appropriate investigations, and treatment.

13. 1- History taking
• Some points have to be explored during history taking
-Blood group and Rh of the mother and her partner.
-All previous pregnancies, outcome, and interventions
-Previous blood transfusion.
-Previous anti-D administration.
-Vaginal bleeding.
-Maternal trauma during pregnancy.
-Previous invasive procedures.

14. 2- Clinical examination
•There is usually no specific finding on clinical examination.
3- Investigations
➢ sensitization of the mother is determined via the Indirect Coomb’s test.
➢The load of antibodies is determined by doing the antibody titer. It is done by serial
dilutions of maternal blood (1:2 then 1:4 then 1:8 then 1:16 then 1:32 etc.), the titer
is the highest dilution at which agglutination occurs. The critical titer is 1:16. this
means the presence of significant amount of anti D antibodies that can cross the
placenta and cause fetal hemolysis.

15. Direct & Indirect Coomb’s test
• Fetal cord blood
• Maternal blood

16. ➢Feto-maternal hemorrhage is detected by Rosette test (qualitative) and Kleihauer-Betke test
(quantitative)
a)Rosette test
▪ Is a qualitative test that identifies whether fetal D-positive cells present in the circulation of a D-
negative woman.
▪ A sample of maternal blood is mixed with anti-D antibodies that coat any D-positive fetal cells present
in the sample.
▪ Indicator red cells bearing the D-antigen are then added, and rosettes form around the fetal cells as
the indicator cells attach to them by the second Fab arm of the D +ve antibodies.
▪ Thus, if rosette is seen under microscope, there are fetal D +ve cells in the sample

17. • Negative test means fetal RBCs are < 15 ml, then standarddose of anti D is given (300 mcg).
• Positive test means fetal RBCs are > 15 ml, then additional dose needs to be given which is calculated
by Kleihauer-Betke test.

18. b) Kleihauer-Betke test
• It is based on principle that HbF is resistant to acid
and alkali and HbA is sensitive
• Reagent used: citric acid phosphate buffer.
• A standard blood smear is prepared from
mother blood and exposed to citric acid.
• The mother RBCs elute giving an appearance
of ghost cells whereas fetal RBCs remain intact
and red in color.
• Fetal cells are counted in 25 low power field.
• The amount of fetal RBCs in ml is calculated, then
number of anti D vials, using certain formula.

19. ➢Fetal anemia is determined by:
1- Invasive techniques
a- Amniocentesisand measurementof ∆OD at 450 nm.
- The idea of this test is that the amount of bilirubin in amniotic fluid correlates
roughly with the degree of hemolysis and thus indirectly predicts the severity
of the fetal anemia.
- Spectrophotometric analysis of amniotic fluid is done between 350 nm & 75 nm.
- Normal amniotic fluid spectrophotometric analysis shows straight line.
- When bilirubin is present in amniotic fluid,
a bulge at 450 nm is seen (dotted line).
- This difference is called delta optic density & plotted
on Liley’s graph.
- the more the bilirubin, the more the bulge & ∆OD

21. b- cordocentesis
fetal blood sampling and direct measurement of fetal hematocrit (Hct).
2- noninvasive technique
➢middle cerebral artery Doppler peak systolic velocity (MCA- PSV)
- independent of etiology, fetal anemia can be detected by Doppler on the basis
of an increase in the velocity of systolic blood flow in the middle cerebral
artery.
- As the hemoglobin decreases, the anemic fetus shunts blood preferentially to
the brain to maintain adequate oxygenation. The velocity rises because of
increased cardiac output and decreased blood viscosity.
- MCA-PSV greater than 1.5 MoM is used as a screening test to identify fetuses
with severe anemia

22. MCA-PSV

24. General approach for prevention of RhD
alloimmunization
•Rh-negative women who screen positive for anti-D antibodies (by ICT ) shouldn’t
receive anti-D immune-gobulin.it is not effective once allloimmunization to D antigen
has occurred.
•All Rh-negative women who screen negative for anti-D antibodies are candidates for
anti-D immune-globulin at:
➢28 wks. of gestation
➢After delivery of a D- positive newborn.
➢After an antepartum event associated with an increased risk of fetomaternal
bleeding

25. Anti-Dimmune globulin
• It is derived from human plasma donated by individuals with high titer anti-D Ig.
• Some preparations can be given IM (RhoGam)®, and other can be given both IM & IV
(Rhophylac)®.
• Vials available in different concentrations
✓ 300 mcg = 1500 IU (1 mcg = 5 IU) àcontains sufficient Ab to suppress the immune
response to 30 ml fetal whole blood (15 ml fetal RBCs).
i.e. each ml of whole fetal blood needs 10 mcg Ig
✓50 mcg = 250 IU sufficient for 5 ml fetal whole blood (2.5 ml RBCs)
• Half life 24 days

26. •Dose
➢First trimester: because the red cell mass of the first trimester fetus is small (mean
red cell volume at 12 wks. is 1.5 ml ), a 50 mcg dose of anti-D Ig can be used for first
trimester events.
➢Second and third trimesters and postpartum:the standard 300 mcg dose is
recommended.
N.B. when anti-D IG is indicated after 20 wks, quantifying the volume of
fetomaternal bleeding (by rosette test then kleihauer Betke test)should generally be
performed to detect pregnancies with fetomaternal bleeding > 15ml of RBCs.
•Timing
➢within 72 hours of the sensitizing event

27. Intrauterine transfusion
•If MCA-PSV is > 1.5 MOM, then cordocentesis is done to determine fetal Hct.
➢ when
•If Hct is < 30% and gestationalage < 35 weeks
➢How
•Intrauterine transfusion can be done either intravascular (into the umbilical vein) or intraperitoneal.
➢What
•Fresh (<7 days), packed, o –ve, irradiated, washed blood.
➢ How much
The amount required is calculated by various formulas aiming to increase Hct to 40-50%.