Rh isoimmunisation

1. Rh isoimmunization
Dr Majaz Ahmed Khan
Fellowship in Neonatology

2. • Isoimmunization –
Production of antibodies in response to an antigen derived from another
individual of same species .
• Rh isoimmunization -
Maternal antibody production in response to fetal RBC Rh antigen , when mother
is Rh – ve , & fetus Rh + ve

3. • Rhesus factor system is the largest of all of the blood groups
• Most important antigens ----- D,C,c,E,e.
• Genes : Rh proteins are encoded by two genes on short arm of chromosome 1
Rh CE
Rh D
Rh CE encodes the C/c and E/e proteins
RHD encodes the D protein.

4. • Inheritance : RhD has dominant inheritance
C and E have co-dominance
• Zygosity : Rh positive
1. Homozygous (DD) – 40%
2. Heterozygous ( Dd) – 60%
Rh negative is -- dd

5. • Rh C, c, E, and e antigens have lower immunogenicity but they too
can cause hemolytic disease.
• Sensitization to E, c, and C antigens complicates approximately 0.3
percent of pregnancies but accounts for about 30 percent of red cell
alloimmunization cases.
• Anti-E alloimmunization is the most common, but the need for fetal
or neonatal transfusions is significantly greater with anti-c
alloimmunization than with antiE or anti-C

6. • Prevalence of Rh negativity

7. Likelihood of Isoimmunization

8. Pathogenesis of Rh-D alloimmunization

9. 1. Maternal exposure to red blood cell antigens
• D antigen is expressed as part of the RBC membrane at 38 days of gestation
• Maternal sensitization : Exposure of red cell antigens during pregnancy by feto-
maternal hemorrhage (FMH)
• FMH increases with gestational age and occurs most frequently around the
time of delivery
•Other causes of sensitization include previous blood transfusions, CVS,
amniocentesis, abortions, ECV etc.

10. Risk factors for FMH during pregnancy
• Spontaneous abortion •Ectopic pregnancy
•Elective abortion •Blunt abdominal trauma
•Amniocentesis •Fetal death in utero
•Chorionic villus sampling •Abruption &Placenta previa with
bleeding
•Antepartum hemorrhage •Cesarean delivery
•Fetal blood sampling •Manual removal of the placenta

11. • As the pregnancy progresses, the possibility of feto-maternal hemorrhage
increases:
3% in first trimester
12% in the second
46% in the third
> 50% at the time of delivery

12. 0.1 ml of antigen-positive blood is sufficient to cause sensitization
• The risk of sensitization is related to the volume of blood that passes between the
fetus and mother
For 0.1 ml of blood, the risk of sensitization is 3%
For 0.4 ml, the risk rises to 22%

13. How to look for Feto-maternal hemorrhage -Kleihauer
Betke test
• Based on acid elusion technique
• Fetal and maternal RBC’s response to citrate acid phosphate buffer solution
• Maternal cells - ghost cells
• Normal fetal cells as red refractile round cells
• Percentage of fetal cells are counted.
• Fetal blood volume = MBV × maternal Hct × % fetal cells in KB/ newborn Hct

14. Foetal RBC
Ghost
cells

15. • less accurate in two situations:
(1) cases of maternal hemoglobinopathy in which maternal red cells
carry excess fetal hemoglobin
(2) cases near or at term, at which time the fetus has already started to
produce hemoglobin A.
Fetomaternal hemorrhage can also be quantified using flow cytometry
to measure red cell size(more sensitive And accurate).
This is an automated test and is unaffected by maternal levels of fetal
hemoglobin F or by fetal levels of hemoglobin A

16. Fate of Isoimmunised pregnancies

17. Severity of disease
Mi l d d i se ase -5 0%
Little or no anaemia, minimal hyperbilirubinemia
No treatment to just Phototherapy
M o d e r a t e d i s e a s e - 2 5 %
Moderate anaemia (Hb <14g/dl), increased cord
bilirubin levels (>4mg/dl)
Intensive phototherapy or occasional DVET
S e v e r e d i s e a s e - 2 5 % Severe anaemia/ hydrops
Intrauterine transfusion

18. Sequence of events
• Firstly polyhydramnios and placentomegaly – on USG
• Hepatomegaly and pericardial effusions
• Late findings of ascites, scalp edema, pleural effusions.
• Finally, oligohydramnios.
• Fetal death if not treated.

19. Prevention and management of Rh
alloimmunization

21. Rh-Immunoglobulin Prophylaxis
• Efficacy :
16% of women develop sensitization after two deliveries of D positive ABO compatible
infants( vs 2% in ABO incompatible infants)
Postpartum prophylaxis decreases rate to 1-2%
Antenatal plus postnatal prophylaxis --- 0.1- 0.3 %
• Dose : 300 microgram Anti D suppress the immune response to 15 ml of D positive red cells
(or 30 ml fetal D-positive whole blood).
10 microgram dose sufficient for --- 0.5 ml of fetal RBC

22. MECHANISM OFACTION :
Rapid macrophage-mediated clearance of anti-D-coated red cells
Other mechanisms -Antigen masking
Two types- Monoclonal and polyclonal
• I.M use only (no more than five doses may be given in a 24-hour period)
• Intravenous preparation (WinRhoSDF): used for large FMH, when >5 units needed( two
ampoules may be given every 8Th hourly).

23. • First trimester : 50 microgram and protects against a FMH of 2.5 ml red cells.
• Second and third trimesters and postpartum (within 72 hours of delivery) –300
microgram(1500 I.U) (can be given till 28 days postpartum)
• Half-life of anti-D immune globulin ranges from 16 to 24 days-which is why it is
given both in the third trimester and following delivery.
• Quantifying the volume of feto-maternal bleeding, if >15 ml of red cells (30 ml whole
blood), requires more than one dose of anti-d immune globulin.
• Adequacy of response determined by positive ICT post transfusion.

24. Guidelines for anti -D Immunoglobulin.
D-negative women who
screen positive for anti-
D antibodies
Should not receive anti-
D immune
globulin
D-negative women with a
negative anti-D antibody
screen and is carrying a
fetus that is, or may be,
D-positive
Candidates for anti-D
immune globulin
D-negative women who
are carrying
D -negative fetus
Should not receive anti-D
immune
globulin
• 28 weeks of gestation
• After delivery of a D-positive neonate
• After an antepartum event associated with an increased risk of
feto-maternal bleeding

25. Management of pregnancy complicated by RhD
alloimmunization
Step wise approach :
• Determine whether fetus is at risk ??
• Follow maternal anti-D titers in at risk fetus until critical titer is reached
• Assesses for severity of anemia using MCA-PSV in fetus at risk
• Confirmation of severe anemia by cordocentesis
• Antenatal interventions : IUT, maternal IvIg therapy, Plasmapherasis

26. Management of pregnancy complicated by RhD
alloimmunization
• Determine whether fetus is at risk ??
Paternal zygosity testing
Cell free DNA testing
Amniocentesis

28. Management of pregnancy complicated by RhD
alloimmunization
Step wise approach :
• Determine whether fetus is at risk ??
• Follow maternal anti-D titers in at risk fetus until critical titer is reached
• Assesses for severity of anemia using MCA-PSV in fetus at risk
• Confirmation of severe anemia by cordocentesis
• Antenatal interventions : IUT, maternal IvIg therapy, Plasmapherasis

29. Maternal Anti-D titers
• Critical titer : Associated with a risk of development of severe anemia and
hydrops fetalis
• An anti-D titer between 16 and 32 is taken as a critical value.
• This is a screening tests, not diagnostic of severe anemia.
• A value of < 4 IU/ml is relatively safe any value > 4IU/ml needs evaluation.
• A raising level suggestive of increasing severity.
• When level is beyond > 15 IU/ml go for invasive monitoring.
•
Markham KB, Moise KJ Jr. Anti-Rh(D) Alloimmunization: Outcomes at a single institution. Am J Obstet Gynecol 2017

30. Management of pregnancy complicated by RhD
alloimmunization
Step wise approach :
• Determine whether fetus is at risk ??
• Follow maternal anti-D titers in at risk fetus until critical titer is reached
• Assesses for severity of anemia using MCA-PSV in fetus at risk
• Confirmation of severe anemia by cordocentesis
• Antenatal interventions : IUT, maternal IvIg therapy, Plasmapherasis

31. Severity of anemia using MCA-PSV in fetus
Why MCA selected-
Cerebral arteries respond rapidly to hypoxemia by increasing blood flow velocity to maintain
cerebral perfusion.
MCA is easy to identify with low inter-observer and intra-observer variability.
Anemia progresses
Stroke volume increases and blood viscosity decreases
Flow can be calculated by poiseuille’s equation

33. Some more facts about MCA PSV
• Started as early as 16-18 weeks and should be repeated at 1 to 2 week intervals.
• If the velocity is between 1.0 and 1.5 MoM and the slope is increasing—such that
the value is approaching 1.5 MoM— surveillance is generally increased to weekly
Doppler interrogation.
• MCA PSV > 1.5 MoMs for GA ----- moderate to severe fetal anemia
• Sensitivity of increased MCA-PSV > 1.5 MoMs for the prediction of moderate or severe
anemia was 100 % (95% CI 86-100) (Irrespective of hydrops)
• False-positive rate of 12 %
Mari G et al. Noninvasive diagnosis by Doppler USG of fetal anemia due to maternal red-cell alloimmunization.
Collaborative Group for Doppler assessment of the blood velocity in Anemic Fetuses. N Engl J Med 2000

34. • The false-positive rate increases significantly beyond 35 weeks, due to
the normal increase in cardiac output that develops at this gestational
age.

35. Additional ultrasonographic assessments
1. Palcental thickness
2. Umbilical vein diameter
3. Spleen and liver size
4. Doppler assessment of velocities in descending aorta and ductus
venosus
5. Fetal ascites (Pcv< 15% and Hb< 4gm/dl) but ascites develop only in
2/3rd of fetuses with Hb< 4gm/dl.

36. Management of pregnancy complicated by RhD
alloimmunization
Step wise approach :
• Determine whether fetus is at risk ??
• Follow maternal anti-D titers in at risk fetus until critical titer is reached
• Assesses for severity of anemia using MCA-PSV in fetus at risk
• Confirmation of severe anemia by cordocentesis
• Antenatal interventions : IUT, maternal IvIg therapy, Plasmapherasis

37. Cordocentesis
• Fetal blood type, DCT, Hb, haematocrit, reticulocyte count
• Reserved for patients with elevated peak MCA Doppler velocities
• Blood should be ready for transfusion, if fetal anaemia is detected (Hct <30%)
• Complications –Fetal & perinatal death, bleeding, cord hematoma, feto-maternal
hemorrhage

38. Amnionic Fluid Spectral Analysis
• Amnionic fluid bilirubin is measured by a spectrophotometer and is
demonstrable as a change in optical density absorbance at 450 nm—
ΔOD450.
• The original Liley graph is valid from 27 to 42 weeks gestation and contains
three zones
i. Zone 1 indicates an Rh D-negative fetus or one with only mild disease.
ii. Zone 2 indicates fetal anemia, with
a. hemoglobin concentrations of 11.0 to 13.9 g/dL for values in lower
zone 2
b. those of 8.0 to 10.9 g/dL for upper zone 2.
iii. Zone 3 indicates severe anemia, with hemoglobin concentration < 8.0
g/dL.

39. • The Liley graph was subsequently modified by Queenan and
associates (1993) to include gestational ages as early as 14 weeks.
It may be considered if the MCA peak systolic velocity exceeds 1.5
MoM after 35 weeks’ gestation.
In the latter situation, if ΔOD450 assessment indicates only mild
hemolysis, delivery at 37 to 38 weeks has been recommended.

40. Management of pregnancy complicated by RhD
alloimmunization
Step wise approach :
• Determine whether fetus is at risk ??
• Follow maternal anti-D titers in at risk fetus until critical titer is reached
• Assesses for severity of anemia using MCA-PSV in fetus at risk
• Confirmation of severe anemia by cordocentesis
• Antenatal interventions : IUT, maternal IvIg therapy, Plasmapherasis

41. Antenatal Management

42. Antenatal management

43. Maternal IvIG
• Role in pregnancies less than 18-20 weeks with severe fetal anemia caused by Rh
alloimmunization.
Mechanism of action:
Feedback inhibition of maternal antibody synthesis,
Blockade of reticuloendothelial Fc receptors,
or Blockade of placental antibody transport

44. Intrauterine fetal transfusion
• Done between 18 and 35 weeks
• Two methods:
1. Intravascular transfusion
2. Intraperitoneal transfusion
• Before transfusion, a paralytic agent such as vecuronium may be given to the
fetus to minimize movements and potential needle stick trauma.

45. Type of blood required for IUT
• Red cell prerequisites :
1. Group O or ABO identical with the fetus (if known) and RhD negative
2. IAT-cross-match compatible with maternal serum and negative for the relevant
antigens determined by maternal antibody status
3. Less than 5 days old and in citrate phosphate dextrose anticoagulant
4. CMV seronegative and irradiated
5. Should have a hematocrit of up to but not more than 0.75

46. Intravascular transfusion
• Done in operating room as may require urgent delivery if complication
develops
• Most common site : near the cord insertion site into the placenta
• Indication : Hb < 2 SD below mean for Gestational age Or Hct < 30%
• Rate- 10/15 ml/min

47. Intraperitoneal transfusion
• Performed before 18 weeks (severe, early-onset hemolytic disease in the early
second trimester, a time when the umbilical vein is too narrow to readily permit
needle entry)
• Infused red blood cells are absorbed via the sub diaphragmatic lymphatic lacunae
and the right lymphatic duct

48. • In a nonhydropic fetus, the target Hematocrit is generally 40 to 50 percent.
• The volume transfused –
estimated fetal weight in grams * 0.02 for each 10-percent increase in
Hematocrit needed
• In the severely anemic fetus, less blood is transfused initially, and another
transfusion is planned for about 2 days later.
• Subsequent transfusions usually take place every 2 to 4 weeks, depending
on the Hematocrit.

49. • Another schedule is to perform a second transfusion in 10 days, the
third 2 weeks later, and any additional transfusions 3 weeks later.
• Following transfusion, the fetal Hematocrit generally decreases by
approximately 1 volume percent per day
• These include severe developmental delay in 3 percent, cerebral palsy
in 2 percent, and deafness in 1 percent.

50. Algorithm for management
of first alloimmunized
pregnancy

52. Postnatal interventions
• Delivery room resuscitation
• Exchange transfusion and Phototherapy
• Blood product transfusion
• Supportive care

53. Mode of Delivery
• Cesarean birth should be reserved for routine obstetrical indication.
• Hydrops fetalis increases the risk of birth trauma
• Alloimmunization is a relative contraindication to delayed cord clamping, even in
severe hemolytic disease of the fetus and newborn.

54. Delivery room management
1. Preparation :
If severe anemia is anticipated, unmatched type O, RhD negative packed red
blood cells should be available
Anticipation

55. Delivery room management contd…
• Prefer delivery at tertiary care center with expertise
• Cord samples- baby blood group, DCT, Hb, reticulocyte count, peripheral smear,
total serum bilirubin, direct fraction
• Cord Hb <10 mg% or TB > 5 mg/dl- indicates severe hemolysis
• Problems in DR – Difficult intubation
- May require thoracocentesis and paracentesis

56. Delivery room management contd…
• CVS stabilization may be required
• Umbilical vein provides quick central venous access
• Fluid resuscitation/ inotropic support/ partial exchange transfusion may be
required in these infants

57. NICU management
Severe anemia (HCT
<25 percent) and severe
hyperbilirubinemia
Exchange transfusion is
preferred over simple
transfusion
• If sign of volume
overload and
Hct<30 do PET first
Moderate-Severe
anemia (HCT 25-35)
and non-severe
Hyperbilirubinemia
Simple transfusion
Mild or no anemia (HCT >
35) and non-severe
Hyperbilirubinemia
No intervention
Late anemia (1-3 wks )
Continuing hemolysis,
suppression of
erythropoiesis
Symptomatic –simple
transfusion
Asymptomatic – iron
supplementation
3-6mg/kg/day

58. Post natal- Pathological jaundice
• Rh isoimmunised babies- hemolytic jaundice
• Aim:
1. Prevent TSB from rising
2. Reduce TSB levels
3. Prevent neurotoxicity
Exchan
ge
transfu
sion
IVIg
Photo
therap
y

59. Pathological jaundice- Treatment modality
• Phototherapy
• Exchange transfusion
• IvIg

60. DVET
• Umbilical catheter/ peripheral arterial line
• Leuco-reduced irradiated O neg cross matched fresh whole blood/
reconstituted PRBC
• Hematocrit - 50 to 55%
• Send pre and post DVET samples
• Repeat sample after 6 hrs of transfusion and later 12-24hrly

61. IvIG
• The use of exchange transfusion decreased significantly in the immunoglobulin
treated group (typical RR 0.35, 95% CI 0.25 to 0.49)
• The mean number of exchange transfusions per infant was also significantly
lower in the immunoglobulin treated group (MD ‐0.34, 95% CI ‐0.50 to ‐0.17

62. Prevention of affected fetus in future pregnancy
• Severity increases with subsequent pregnancy
• Avoiding conception of an RhD-positive fetus
• Following methods can be used: In vitro fertilization (IVF) with preimplantation
genetic testing, Use of a gestational carrier, Use of donor sperm from D- negative
donor

63. Long term outcome
LOTUS study:
291 children, median age of 8.2 years (Range 2-17 years)
Cerebral palsy was detected in 2.1%
Severe developmental delay in 3.1%
Bilateral deafness in 1.0%
Overall incidence of NDI was 4.8%
• In a multivariate regression analysis only severe hydrops was independently associated with
NDI (OR, 11.2; 95% CI - 1.7-92.7).
Lindenburg IT et al. LOTUS study group,. Am J Obstet Gynecol. 2012

64. Take home massage
• All pregnant mother should have blood group and antibody screen during first antenatal check up
• Early detection and treatment of isoimmunization in sensitized mother
• In alloimmunized pregnancy – regular follow up, MCA PSV monitoring , timely IUT and delivery
at tertiary hospital should be planned
• Delivery room preparation and DVET are most important postnatal strategies for increasing
outcome

65. Thank You