This document discusses Rhesus isoimmunization, which occurs when a Rh-negative mother develops antibodies against Rh-positive fetal red blood cells. It covers the epidemiology, pathophysiology, causes including the "grandmother effect", investigations such as the Kleihauer-Betke and Coombs' tests, management of sensitized mothers through surveillance and potential fetal transfusions, and treatment of affected newborns including phototherapy and exchange transfusions. Prevention relies on administering Rhogam prophylaxis to unsensitized Rh-negative mothers.

1. Rhesus Isoimmunization
Presenter:
Dr. Bwire Innocent - Resident Obsgyn-KIU
Mentor:
Dr. Kato Paul - Obstetrician/Gynaecologist-HRRH

2. Outline
• Introduction to ABO And Rhesus Blood Group System
• Epidemiology
• Causes
• Grandmother effect
• Pathophysiology
• Investigations
• management
• Prevention

3. Introduction
• Isoimmunization /alloimmunization:
• Production of antibodies in response to an antigen derived from another
individual of the same species
• Rhesus Isoimmunization
• Maternal antibody production in response to fetal red blood cell Rh
antigen which occurs when mother Rh negative ,fetus Rh postive.
• Sensibilized : producing detectable antibodies only in a subsequent
pregnancy (Bowman, 1985).

4. ABO blood group system
• 4 blood types (A, B, AB & O)
• additionally classified according to the presence or absence of Rh
factor

5. The Rhesus Factor
• A group of antigens that may or may not be present on the surface of the
red blood cells. the most common Include C,c, D,d, e and E antigens
• The 2 responsible genes: RHD and RHCE are located on the short arm
of x-some 1 and are inherited together, independent of other BG genes.
• D antigen is the most immunogenic and determines Rh positivity i.e
Rh positive, those who lack the D antigen are Rh negative.
• Fetal Rh-antigen are present by 38th day after conception.

6. The Rhesus Factor
• Rh alleles: Cc, Dd and Ee. D antigen, the most potent, its presence or
absence denotes an individual to be Rh +ve or –ve.
• DD homozygous, Dd-heterozygous, dd- negative.

7. The Rhesus Factor
• Heterozygous +father: Dd, Homozygous+ father: DD , Mother dd

8. Abo Incompatibility
• ABO incompatibility describes an immune reaction that occurs in the
body if two blood samples of different, incompatible ABO types are
mixed together, when these RBC act as antigen & an immune response
is triggered.
• It affects newborns whose mothers are blood type O, and who have a
baby with type A, B or AB.
• Other blood group systems; Kidd, Diego, Kell, MNS, Duffy , Lewis,
Scianna, Dombrock, Colton, Gerbich, Lutheran, I

10. Rhesus Incompatibility

11. Epidemiology
• It is more frequent among Caucasians descent (15-17%), Africans (4-8%),
Asian descent (0.1-0.3%). (Bhutani et al 2013)
• Prevalence in Uganda is not known, found to be 2.2% in south western
Uganda (Natukunda et al 2011)
• Without anti-D immune globulin prophylaxis, a D-negative woman
delivered of a D-positive, ABO-compatible newborn has a 16% likelihood
of developing alloimmunization. 2% will become sensitized by the time of
delivery, 7 % by 6 months postpartum, and the remaining 7% will be
“sensibilized”( Boweman, 1985)

12. • Thus, affection of the baby due to Rh incompatibility is low considering
the increased number of Rh+ babies delivered to Rh-ve mothers.
• Insufficient placental transfer of fetal antigens or maternal antibodies.
• Inborn inability to respond to the Rh antigen stimulus.
• Immunological non-responder—as found in 30% of Rh-negative women.
• ABO incompatibility has a protective effect against the development of Rh
sensitization. significant if mother is type O, father is type A, B or AB
• Variable antigenic stimulus of the D antigen which depends on the Rh
genotype of the fetal blood, e.g. CDe/cde genotype.
• Volume of fetal blood entering into the maternal circulation (0.1 mL is
considered as critical sensitizing volume).

13. Early pregnancy loss
abortion- elective or spont.
molar pregnancy
Ectopic pregnancy
Fetal death (any trimester)
Procedures
Fetal blood sampling, CVS, Amniocentesis
Cesarean Section/ vaginal delivery
blood transfusion /bone marrow transplants
injections with contaminated needles
Idiopathic :
? Vanishing twin, grandmother theory
Other
Idiopathic
Maternal trauma/APH
Manual placental removal
External version
Causes of Fetomaternal Hemorrhage that May Incite Rh Isoimmunization

14. The Grandmother Effect
• In virtually all pregnancies, small amounts of mat. blood enter the fetal
circulation. PCR has been used to identify mat D+ve DNA in peripheral
blood from preterm and full-term D-ve newborns (Lazar, 2006).
• Thus, it’s possible for a RhD- female fetus exposed to mat. rhD+ red cells
to develop sensitization. When such an individual reaches adulthood, she
may produce anti-D antibodies even before or early in her 1st pregnancy.
• mechanism called the grandmother effect or theory bse the fetus in the
current pregnancy is jeopardized by mat. ab that were initially provoked
by his or her grandmother’s erythrocytes.

15. Pathophysiology
• There is normally no mixing of fetal & maternal blood during pregnancy.
• When the placenta begins to separate & the chorionic villi tear, the risk of
feto-maternal transfusion is increased.
• Rh antigens of the fetal red cells (0.1ml) stimulate the prodn of maternal
antibodies(become detectable after 6 months)
• The 1st encounter may not result in Ig M but no sensitization, on 2nd
encounter Ig G are produced and these cross the placenta

16. Pathophysiology
• maternal IgG crosses the placenta barrier & enters into fetal circulation
while IgM does not cross. Once formed the antibodies are permanent.
• If the fetus is Rh positive, the antibody becomes attached to the antigen
sites on the surface of the erythrocytes.
• The affected cells are rapidly removed from the circulation by the RES
resulting in fetal anemia.
• Hyperplasia of the placental tissue occurs in an effort to increase the
transfer of oxygen but the available fetal red cells

19. Fetal effects
• There is no risk 1st trimester abortion in RH Isoimmunization unless
a/w other congenital anomalies
• Effects of RH Isoimmunization is not evident till second trimester of
pregnancy only after 2nd trimester the reticuloendothelial system of
fetus is developed and RH antigens are formed in utero
• It has been found that the amount of FMH is very minute (around 0.03
mL) during 1st and 2nd trimester however it may be as high as 25 mL
during 3rd trimester (Puangsricharern 2007 : Bhatnagar 1980)

20. Fetal effects
• Destruction of fetal RBC’s cause fetal anaemia which is continuous during
intrauterine life. Due to anemia, the fetal growth is retarded
• Erythroblastosis fetalis. Overproduction of immature fetal and neonatal red
cells, now called Hemolytic disease of the fetus and newborn(HDFN)
• Hydrops fetalis: abnormal collection of fluid in more than one area of the
fetal body is termed hydrops fetalis (scalp edema, pleural effusion,
pericardial effusion, ascites, and skin edema). Buddha’ sign on ultrasound.
• tissue hypoxia and acidosis eventually lead to intra uterine fetal death.

21. Neonatal Effects
• Congenital hemolytic anemia: slow hemolysis , the anemia develops
slowly within 1st few weeks of life, the jaundice is not usually evident.
Hemolysis continues up to 6 weeks due to antibody presence
• Hyperbilirubinemia, if timely actions are not taken the hyperbilirubinemia
results in deposition of bilirubin in the basal cerebral ganglia leading to
kernicterus. Due to relative immaturity of function of the liver
• Icterus gravid neonatorum. The fetus is born alive without jaundice but
develops it within 24 h.
• Progressive anemia can lead to congestive cardiac failure and death

22. Maternal effects
• There is increased incidence of:
• Preeclampsia;
• Polyhydramnios;
• Big size baby with its hazards;
• Hypofibrinogenemia -prolonged retention of dead fetus in uterus;
• Postpartum hemorrhage - big placenta and blood coagulopathy;
• “maternal syndrome”- generalized edema, proteinuria and pruritus due
to cholestasis.

23. Investigations
• Blood grouping and Rhesus typing
• Indirect Combs test (ICT): maternal antibody screen
• Paternal zygosity testing (PCR)
• Cell free fetal DNA testing
• Kleihauer-Betke test: amount of fetal blood in maternal circulation.
• amnionic fluid Spectrophotometry: also known as the ΔOD450 test, is
no longer recommended (SMFM, 2015)
• Fetal blood sampling
• Cord blood examn: ABO , Rh, DCT, Hb and Bilirubin

24. If severe fetal anemia, a sinusoidal heart rate pattern may develop

25. Direct Coombs’ Test
• used to detect these antibodies or complement proteins that are bound
to the surface of RBC.
• A blood sample is taken & the RBCs are washed & then incubated
with antihuman globulin[also known as coombs’ reagent].
• If this produces agglutination [clumping together] of RBCs, The
Direct Coombs’ Test Is Positive.

27. Indirect Coombs’ Test
• It detects antibodies against RBCs that are present unbound in the
patient’s serum.
• In this case serum is extracted from the blood sample taken from the
patient.
• Then the serum is incubated with the RBC of known reference values
from other patient blood samples.
• If this produces agglutination of RBCs, The Indirect Coombs’ Test Is
Positive.

29. KB test
• Fetal red cells in the maternal circulation can be identified by use of
the acid elution principle first described by Kleihauer, Brown, and
Betke
• Fetal erythrocytes contain HbF, which is more resistant to acid elution
than HbA. After exposure to acid, only fetal hemoglobin remains.
• Fetal red cells can then be identified by uptake of a special stain and
quantified on a peripheral smear
• If there are 80 fetal RBCs in 50 low power fields in mat peripheral
blood films, it rep. a transplacental hemorrhage to the extent of 4 mL
of fetal blood.

31. Diagnosis
 The essentials of diagnosis are:
1. The mother must be Rh negative.
2. Presence of maternal Rh antibodies as shown by a positive
indirect coombs’ test on mother’s blood.
3. Maternal antibody titre posing risk to fetus.
4. History of birth of previous affected baby.
5. Fetal cord blood shows low level of Hb at birth & raised
bilirubin

32. Management
• Fetal Risk Assessment
• maternal Antibody titer
surveillance
• Ultrasound monitoring of the
fetal MCA PSV if a critical
antibody titer is reached
• Delivery timing and mode

34. Critical titer
• The critical titer is the level at which significant fetal anemia could
potentially develop.
• This may be different for each antibody, is determined individually by
each laboratory, and usually ranges between 1:8 and 1:32.
• the critical titer for anti-D antibodies is 1:16, a titer ≥1:16 indicates the
possibility of severe hemolytic disease.
• In any pregnancy in which the antibody titer has reached a critical
value, there is no benefit to repeating the titer.

35. Management of Rh-ve Non-sensitized mother
• Initial evaluation of alloimmunization risk with blood group and Rh factor
, mat antibody screening with indirect coombs test at booking visit
• Monthly ICT till delivery , if positive manage as sensitized
• At 28 weeks give prophylactic 300-μg of anti-D and within 72 hrs after
delivery if rhesus positive newborn
• If immune anti-D ab is detected, prophylaxis is no longer necessary

37. Management Of rh-ve Sensitized Mother
• Sensitization IS by the detection of Rh antibodies in mat, circulation using ICT .
• If alloimmunization is detected and the titer is below the critical value, the titer is
generally repeated every 4 weeks for the duration of the pregnancy (ACOG, 2019a).
• Anti-D antibody titres determined and if titre < 4IU/ml is unlikely to produce severe
fetal disease, if > 4 iu/ml but < 15 iu/ml correlates with a moderate risk of HDFN , if
> 15 iu/ml can cause severe HDFN.
• After the 1st affected pregnancy, the ability to predict fetal anemia from the mat. anti-
D antibody titers is lost and now these pregnancies shd be monitored by MCA-PSV

39. When to administer RHOGAM?
• It binds to fetal RBCs and prevents stimulation of maternal immune system.
• It should be given at 28 wks to all unsensitized Rh-ve mother and postpartum
within 72 hours if the baby’s Blood group is Rh positive.
• In case, where the specified time limit is over (>72hour) She may be given
up to 28 days after delivery to avoid sensitization.
• One 300-μg dose is given for each 15 mL of fetal red cells or 30 mL of fetal
whole blood to be neutralized. 10μg of anti- D should be given for every
additional 0.5 mL of fetal RBCs in maternal circulation.

40. • .

41. fetal transfusion (intra-uterine transfusion).
• Fetal blood sampling is generally performed if the MCA PSV exceeds the threshold
for severe anemia, with plan for concurrent IUT as needed.
• If the MCA PSV exceeds 1.5 MoM or if hydrops develops or fetal Hct <30% or the
Hb is at least 2 g/dl below the mean for normal fetuses of corresponding GA.
• fresh O-negative doubly packed cells is given to the fetus by intravascular or
intraperitoneal route. The volume transfused = (Gest age – 20) × 10 mL
• Fetal transfusion is typically performed prior to 34 to 35 WOG. Later in gestation,
the benefits of transfusion may be outweighed by the risks of delaying delivery..

42. Obstetric management
• Aim is to carry beyond 34 weeks. Mildly affected fetus – May wait till
term and for spontaneous onset of labour.
• Moderately affected fetus – either vaginally or by LSCS before term.
• Severely affected fetus – LSCS is preferred when fetal parameters poor
• Care at delivery: Close monitoring through out the first stage, Clamp the
cord immediately, Gentle handling of the uterus, Cord blood is collected
for neonatal group and type, Hb, haematocrit, bilirubin and direct
Coombs
• Avoid the following: Uterine massage, Manual removal of placenta ,
Ergometrine

43. Postnatal Management
• The neonates may be born with anemia, hepato-spleenomegaly &
ascites and rapidly develop hyperbilirubinemia.
• These infants are hypo-coagulable & have a tendency of developing
hypoglycemia.
• The presence of positive DCT is sufficient to confirm the diagnosis in
an Rh positive neonate born to a sensitized Rh negative women.

44. Treatment of neonatal anemia
• In those infants who exhibit only anemia, fresh packed red cells are
given to correct this defect.
• The management of hydrops fetalis which is the several form of the
disorder is usually done by the rapid correction of anemia by small
exchange transfusion 20 to 40 ml/kg body wt raises the hematocrit
sufficiently.

45. Treatment Of Neonatal Jaundice.
• Phototherapy exposure of neonates to light with wave length of 420-
470mm results in addition of bilirubin & conversion to products that
are water soluble, are not neurotoxic and which can be excreted via
stool and urine.
• It takes 12-24 hrs to be effective.

46. Exchange transfusion:
• Helps by removing unconjugated bilirubin & Rh positive cells coated with
antibodies.
• Traditionally double volume exchange transfusion done via umbilical vein
using group specific negative blood cross matched with mothers serum
effectively.
• Indications: Rh-positive with direct Coombs’ test positive babies having:
• (i) Cord blood bilirubin level > 4 mg/dl and hemoglobin level is < 11 gm/dl.
• (ii) Rising rate of bilirubin over 1 mg/dl/hour despite phototherapy.
• (iii) Total bilirubin level is 20 mg/dl or more.

47. Prevention Of Maternal Isoimmunization
• There are 3 ways of preventing a women from producing rhesus
antibodies.
 Avoiding transfusion of Rh positive blood.- avoids sensitization
 Prevention of avoidable feto-maternal transfusion- avoid
transplacental amniocentesis
Administration of ANTI D immunoglobulin

48. Thank you for listening
queries, supplements are welcome
References
• Williams Obstetrics, 26th edition, 2022
• RCOG, The Management of Women with Red Cell Antibodies during
Pregnancy. Green-top Guideline No. 65, May 2014.
• DC Dutta’s, Text book of obstetrics,8th ed, 2015
• Smart Study Series: Obstetrics and Gynecology, 3/e Bhojani
• Up To Date 2023
• Agarwal, K., Rana, A. & Ravi, A.K. Treatment and Prevention of Rh
Isoimmunization.(2014). https://doi.org/10.1007/s40556-014-0013-z
• Gabbes obs/self asssesments obstetrics, Holland/brews Manuel of obstetrics