Rh-isoimmunization one of another cause of recurrent pregnancy loss.... dr.s.n.bera & dr. m. dash, m.k.c.g medical college, orissa

1. Rh-ISOIMMUNIZATION
DR.SURENDRA NATH BERA
DR. MITALI DASH
M K C G MEDICAL COLLEGE , ORISSA

2.  ISOIMMUNIZATION:
A process by which immune
antibodies are produced in a
person by the entry of an antigen of
another individual of same species,
the former lacking the antigen.

3. Rh- Iso imunization
Definition
known as:
Rhesus incompatibility ,Rhesus disease
RhD Hemolytic Disease of the Newborn.
-When Rh– mother gets pregnant to Rh+ fetus
—she may be sensitized to Rh antigen and
develop antibodies. These will cross the
placenta and cause hemolysis of fetal red
blood cells.

4. HISTORY
 1609-description of hydrops fetalis.
 1939-Levine and Stetson discovered
atypical aglutinin.
 1940-Landsteiner and wiener Rh-antigen.
 1941-Levine discover Rh-antidody.

5. Rhesus factor (1940):
Agglutinogen (C,D,E) - mainly D
C,D,E - dominant antigen
c,e - recessive antigen
Person lack D-antigen called Rh-ve

6. - Rh positive (85%) - homozygous (DD) (35%),
or heterozygous (Dd) (50%)
- Rh negative (15%)
- Incidence of Rh-ve in far east is about 1%
Examples of Rh factor: (CDe=R1) , (Cde=r)
(cDE=R2)
Other systems:
 Kell.
 Lweis,
 Deigo,
 luther,
 Duffy,
 MNS,

7.  Kell is most common of minor gr.
 Responsible for 10% of cases of
severe antibody-mediated anemia
 Only anti-Fy(a) antibody associated
with HDFN- may range from mild to
sever.

8. INCIDENCE OF Rh-ve
 Chinese and Japanese 1%
North American Indian 1—2%
Indo-Eurasian 2%
india 5%
African American 4 - 8%
Caucasian 15 - 16%
Basque 30 - 35%

9. Rh-isoimmunization occurs
A. Mismatched blood transfusion.
B. Rh-negative women bearing Rh-
positive fetous with feto-maternal
hemorrhage.

10. CAUSE OF FMH
Feto-maternal haemorrhage: during pregnancy
leakage of fetal cells in the maternal circulation (Rh+
fetal cells in Rh- maternal circulation)
Examples:
1.Abortion
2.APH
3.E.C.V.
4.Cordocentesis,
5.CVS,
6.Amniocentesis

11. CAUSE OF FMH
7.Severe preeclampsia
8.Ectopic pregnancy
9.Caesarean section
10.Manual removal of placenta
11.Silent feto-maternal hage

12. Pathophysiology
 Rh-isoimmunization is due to D antigen in
more than 90% of cases.
Occasionally result of other than Rh group
like anti- Kell and anti- Duffy

13. Pathophysiology
 Following feto-maternal hemorrhage.
 Initial response is forming IgM antibodies for
the short period (6wks-6month)
o Followed by production of IgG on subsequent
pregnancy which crosses placenta.
 IgG antibodies adhere to the antigen site on
the surface of erythrocytes causing hemolysis.
 The excessive removal of circulatory RBCs
leads to severe anemia and hypoxia.

14. IGM antibodies
1. Cleared by
Macrophage
2. Plasma
stem cells
Fetal Anaemia
Mother
Placental
Primary Response
•6 wks to 6 M.
•IGM.

15. Anti - D
Macroph. antigen
Presenting cell
T- helper cell
B cell
Fetal Anaemia
Mother
Placental
Secondary Response
•Small amount
•Rapid
•IgG
IgG

17.  Rh Antibodies
 Antibodies Coated Red Cells
 Destruction of Fetal Cells by Fetal RES
 Fetal Anemia
 Fetal Hypoxia and Stimulate of Erythropoitin
 Extra Medullary red Cells Synthesis
 Hepato spleenomegally
 Hepatic Cell Failure
 Hypoproteinemia, Increased Intrahepatic
Pressure, Portal hypertension
 Ascetic, Edema, hypoxia, Placental
Thickness, Polyhydramnios, Pericardial
effusion

18. Development of Rhesus antibodies:
depends on :
1- Inborn inability to respond to Rh-antigenic
stimulus.
2- Protection if ABO incompatible 110
3- Strength of Rh antigen stimumlus (CDe=R1)
4- Volume of leaking feta blood (0.1ml)
5- Immunological nonresponders found in 30%
of Rh-negative women.

19. 1- If ABO is incompatible:
 Red blood cells is easily destroyed, so
not reaching enough immunological
component to cause antibody
response and reaction.
 The risk of sensitization after ABO
incompatible pregnancy is only 2%.

20. - If ABO is compatible:
Rh+ fetal cells  remain in circulation
(life span) until removed by (R.E.S) 
destroyed  liberating antigen (D) 
isoimmunization

21. Macroph. Antigen
Presenting Cell
T-Hellper
B-cell
Anti-D
Anti - A Anti - B
Mother
Infant
A Rh positive B Rh
Positive“O” Rh positive
Group “O” Rh Negative
Placenta

22. Fetomaternal hemorrhage as a reason
of Rh –isoimmunization has been
documented in:
6.7% in the first trimester.
 13.9% in the second trimester
 29% in the third trimester.

23.  Amount needed for sensitisation 0.1ml
FMH/ML SENSITIZATION%
0.1 1
0.5 - 1 25
>5 65

24. Complications
 Hydrops fetalis And Stillbirth.
 Icterus gravis neonatorum.
 Neonatal Jaundice.
 Compilations Of Neonatal Kernicterus
(Lethargy, Hypertonicity, Hearing Loss,
Cerebral Palsy And Learning Disability)
 Congenital Anemia of newborn.

27. ICTERUS GRAVIS NEONATORUM
 Less severe form of hemolytic
disease.
 Baby born alive without jaundice but
soon develops within 24 hrs of birth.

30. Kernicterus
Concentration of bilirubin in the
newborn blood exceeds
20mg/100ml or
in-term fetus – > 342 mmoll/L
in pre-term fetus – >205 mmoll/L,
Billirubin crosses the BBB and
damage the basal nuclei of brain.

31. Screening and diagnosis
 Maternal blood grouping, Rh-typing
and antibody screening at their 1st
pre-natal visit.
 Presence of anti-D antibodies in
serum is diagnostic of maternal Rh-
alloimmunization.

32. Antibody screening in mother
 Should be done in….
I. Rh-negative women who have
received anti-D immune globulin.
II. Rh-positive mother who have…
--blood transfusion.
--unexplained fetal loss.
--infant with unexplained jaundice.

33. Gold Standard Test
 Indirect Coombs:
-mix Rh(D)+ cells with maternal serum
-anti-Rh(D) Ab will adhere
-RBC’s then washed & suspended in
Coombs serum (antihuman globulin)
-RBC’s coated with Ab will be agglutinated
 Direct Coombs:
-mix infant’s RBC’s with Coombs serum
-maternal Ab present if cells agglutinate

34.  If indirect coombs test
is positive, the father’s
Rh should be tested.
 Serial maternal Anti D
titers should be done
every 2- 4 weeks.
 If titer is less than 1/16
the fetus is not at risk.
 If titer is more than
1/16 then severity of
condition should be
evaluated.

35.  USG : detection of hydropic change.
 CVS
 Amniocentasis
 Cordocentesis
 MCA-PSV

36. USG
 Confirmation of gestational age.
 Early detection of hydrops when finding one or
more of the following:
Ascites, pleural effusion,
pericardial effusion, skin edema.
 Increase placental size,
 Hepato-spleenomegally.

37. USG FINDINGS

38. Fetal Ascites

39. Rh- Iso imunization Body wall edema
hydropic fetus

40. MCA-PSV
 Non-invasive
 Mother not put at risk for worsening
alloimmunization
 Can be used with alloantibodies
other than RhD, including anti-Kell
antibodies

41. Biophysical surveillance
Middle cerebral artery peak velocity

42. Biophysical surveillance
Middle Cerebral Artery peak systolic velocity
B = moderate-severe
anaemia
A = mild anaemia
C = no anaemia
C

43. CVS
 To know fetal Rh genotype.
 Advantage: early detection.
 Disadvantage:
--more complicated.
--increase severity of
alloimmunization if baby Rh +

45. AMNOCENTESIS
 Methode of choice for detection of Rh factor of
fetous and amniotic fluid bilirubin.
 Critical titre/previous affected infant.
 Bilirubin correlates with fetal hemolysis.
 Spectophotometric analysis optical density of
amniotic fluid @ ▲OD 450nm.
 Data plotted on Liley curve.

47. CORDOCENTESIS
 More complicated procedure..
 Fetal Rh phenotype can be known
rapidly by blood bank serology.
 Gold standard for detection of fetal
anemia
 Greater morbidity and mortality
 2.7% total risk of fetal loss

48. Diagram of cordocentesis procedure
Cordocentesis -

49. MANAGEMENT

50.  Rh-negative women categorized in two
groups.
I. Rh-negative non-immunized women.
II. Rh-negative immunized women.
immunized against
-D-antigens
-non D-Rh antigens
-other blood group system

51. The aim of antenatal
management
 To predict which pregnancy is
at risk
 To predict whether or not the
fetus is severely affected.
 To correct anemia and reverse
hydrops by intrauterine
transfusion.
 To deliver the baby at the
appropriate time, weighing the
risks of prematurity
against these of intrauterine
transfusion.

52. OBJECTIVES
A. Non immunized women– prevention
of allo-immunization.
B. Immunized women—
a. early detection.
b. adequet treatment of
fetal anemia.

53. Rh-negative non immunized
Phenotype of
father
Rh-positive
Ante-partum sensitization
of Ab screening at
20,24,28 wks GA.
Rh-negative
Baby Rh-
negative
Manage as
normal
pregnancy

54. Cont…
Antibody screening at
20,24& 28wks ga
No anti-Ab detected
Pt should received
anti-D-immunoglobin
at 28wks of GA.
If anti-D antibodies
detected
Manage as Rh-negative
immunised women
At times of delivery to determine the
mother’s eligibility for a second dose of
anti-D immunoglobin

55. RH-NEGATIVE IMMUNIZED WOMEN

56. Management based on---
A. First affected prenancy.
B. Previous affected pregnancy.

57. FIRST AFFECTED PREGNANCY
 Should have antibody triter every 4 wks.
A. If triter≥critical level
- amniocntasis
- MCA-PSV.
B. If triter<critical level upto 36 wks of
gestation,should deliverd between 38-40wks.

58. Sudden elevation of Ab-triter
when GA>34WKS <37WKS
amniocentasis
Lung imaturity
Contineu pregnancy
if bilirubin<0.05 with
serial amniocentasis
weekly Lung maturity
Lung maturity
delivery

59. PREVIOUS AFFECTED PREGNANCY
a. Maternal anti-D triter not predict the fetal
anemia
b. MCA-PSV to determine the anemia.
c. Serial amniocentasis.

60. PREVIOUS AFFECTED PREGNANCY
a. Maternal anti-D
triter not
predict the fetal
anemia
b. MCA-PSV to
determine the
anemia.
c. Serial
amniocentasis.
TRITER LEVEL PAST
OBSTETRIC
HISTORY
% OF IUD
<64 NEGATIVE 4%
<64 POSITIVE 32%
>64 NEGATIVE 17.2%
>64 POSITIVE 68.7%

62. Suggested management after amniocentesis for ΔOD 450
Serial Amniocentesis
Lily zone I
Lower Zone II
Upper Zone II Zone III
Hydramnios & Hydrops
Repeat
Amniocentesis every
2-4 weeks
Delivery at OR near term
Repeat Amniocentesis in 7
days or FBS
Hct < 30% Hct > 30%
Intrauterine
Transfusion
Repeat Sampling
7 to 14 days
Fetal
hematocrit<30%
Fetal
hematocrit>30%
Intrauterine
Transfusion
Follow with fetal
Blood sampling
& USG
DELIVERY
WHEN LUNG
MATURE
Fetal blood sampling

63. LILEY’S CHART

64. WHITEFIELD’ ACTION LINE

65. QUEENAN CHART

66. Transfusion therapy
Intraperitoneal
 First done in 1963
 Instill blood through needle or epidural catheter
 Volume to transfuse = (G.A.-20) x 10ml.
 Generally, repeat in ~ 10 days, then every
4wks.
 Risk of death about 4% per procedure.
 Not effective in hydropic fetus.
 Some advocate combined approach (IPT and
IVT)

67. Transfusion therapy
Intravascular
 Goal is to have post-transfusion Hct 40-45%
 Can infuse about 10 ml/min
 Goal:keep Hct>25%
 Estimate requirement based on EFW and pre-
transfusion Hct
 Repeat in 1 wk., then about every 3 wkly.
 Fetal loss about 1.5% per procedure

68. Direct fetal intravascular
transfusion

69.  Other therapies:
A. Plasmapheresis : tried to remove
several liters of maternal plasma with
anti-D antibodies.
B. High dose i.v immunoglobulin :
1000mg/kg weekly.

70. MANAGEMENT DURING DELIVERY

71. Pregnant women undergo cesarean
section in isoimunization:
 Severe form of hemolytic
infant disease in the term
or 34-35 weeks after
previous antenatal
prevention of fetal hyaline
membranes syndrome;

72. Measures to be taken
 Use abdominal packs in the sides of the
uterus before opening the lower segment to
prevent spilled blood from the placenta to
inter the peritoneal cavity.
 Let the placenta to be delivered spontaneous
using control cord traction without squeezing
the uterus.
 … A void avulsions of the cord.

73.  undergo delivery in
the term of 37-38
weeks of gestation.
 Induction of labor is
performen by
prostaglandin (in the
case of “unripe”
uterine cervix) or by
intravenous oxytocin
infusion
administration (in the
case of “ripe” uterine
cervix).
Vaginal delivery in Rh-isoimmunization

74. Vaginal delivery in Rh-isoimmunization
 During labor:
 No fundal pushing in 1st or 2nd stage of labor.
 With hold inj methergin after ant. shoulder delivery.
 Early cord clamping and no milking.
 No uterine massage or squeeze in 3rd stage.
 Let the placenta to be delivered spontaneous to
avoid avulsions of the cord.
 Protect the vaginal and perineal wounds and
laceration from being exposed to the fetal blood spilled
from cord

75.  At birth
 Maternal blood
a. antibodies by indirect Comb's test ( ICT )..
b. fetal red blood cells in maternal circulation
 Cord blood sample ( Neonatal blood sample ) for
a. antibodies by Direct Comb's test ( DCT )
b. Infant blood group and rh-typing.
c. Infant bilirubin level
d. Infant Hb & Hct level

76. Rh- Iso imunization
Prevention
- Screening of all pregnant mothers to Rh D
antigen and antibody screening for Rh D
negative mother.
Word of Vincent Freda
the rule of thumb should be to administer
anti-D immunoglobin when in doubt
rather than to withhold it.

77. Prophylactic anti D immunoglobulin
To be given
 All Rh – mothers after delivery if the fetus
is Rh+
 At 28, 32 weeks of pregnancy or after
40wks if pregnancy contineued
 After abortion, amniocentesis, abruption,
ectopic pregnancy.

78. Rh- Iso imunization
Prevention The standard dose of anti D is
0.3 mg —will eradicate 15 ml of
fetal red blood cells (routine for
all Rh –ve pregnancies) within 3
days of delivery.
-If more feto-maternal bleeding is
suspected as in abruption or
ante partum hemorrhage-Do
Kleihauer –Betke test to
estimate the amount of fetal
red cells in maternal
circulation and re-calculate the
dose of the anti-D.

79. Kleihauer-Betke test
measure amount of feto-maternal haemorrhage.
PRINCIPLE: Adult hemoglobin, but not fetal hemoglobin, is soluble in a
citrate buffer with pH 3.2 and will elute out of the red cell.
 (critical volume)  isoimmunization represented by 5
fetal cells in 50 low power microscopic field of
peripheral maternal blood.
 So 1 ml is represented by 20 fetal cells

80. method
 Prepare patient blood smears
 Fix smears in 80% ethanol
 Incubate slides in citrate buffer
 Stain smears with erythrosine
 Count fetal cells on patient slid
 Red cells containing Hgb F stain bright
 pink due to erythrosin stain ,Negative
staining, adult red cells that contained Hgb
A,appear as pale, ghost cells.

81. Kleihauer Calculations
 %Fetal red cells = fetal cell counted in total slids x 100
total maternal cell
%fetal red cell x 5000ml(MBV)
volume of FMH =
100
MBV – maternal blood volume (usually 5000ml)
volume of FMH
VIAL REQUIRS =
30

83. positive cells on the
Kleihauer-Betke stain
 Post-partum mothers following a
transplacental hemorrhage
 • Newborns/infants less than 6 months
old
 • Hereditary persistence of hemoglobin F
 • Disorders that compensate with
hemoglobin F such as beta thal major or
 sickle cell disease

84.  Other test: to detect FMH
 Flow cytometry
 Rosette test
 Enzyme linked antiglobulin test
 Surrogate test

85. Immunoglobulin (RhoGAM)
prophylaxis (RhIgG)
Schedules
 Spon.abortionBefore 12wks –
RCOG,UK- no dose require
Austalian soc.- 50 μg RhIgG
Canadian soc.- 120 μg RhIgG
 2nd trimester abortion- 50 μg RhIgG
 Threatened abortion-
1st trimester - not requir
2nd trimester- 50 μg RhIgG
(should be repeated 6wkly if heavy bleeding)

86.  A minimum dose of 50 μg RhIgG is
recomendate upto 19wks+6days GA.
 After 20wks minimum dose 125 μg
RhIgG .

87.  Antepartum (RAADP)regimen.
 Two dose 500IU at 28 and 34wks GA.
 Single dose 1500IU at 28 wks GA.
 Postpartum <72 hr - 300 μg RhIgG;
IT can be given upto 28 days of delivery
0.3% require > 300 μg RhIgG

88. prophylaxis
Hydatidiform mole
complete mole-controversial.
patrial mole-anti-D to be given.

89. Following BTL-controversial.
given….
 women wants new partner desire
for IVF.
 In future if major accident occurs
and Rh- blood not available at
emergency.

90.  300 μg anti-D neutralizes 30 mL fetal
Rh-positive blood (15 mL packed fetal
RBC)

91. Management of sensitized
newborn
Mild anemia (Hb <14gm/dl,
cord bilirubin>4 mg/dl)---
Phototherapy
-Moderate to severe----
Exchange transfusion.
-Mild Hydrops improves in
88% of cases
-Severe hydrops—Mortality
is 39%