Rh sensitazation

2. Content
 History and Nomenclature
 Genetic and Biochemistry of Rh Ag
 Causes of Rh alloimmunization
 Incidence of Rh incompatible pregnancy
 FMH and Rh sensitization
 Diagnostic methods
 Clinical management
 Use of Rh immunoglobulin
 Summary
 reference

3. History
1609: First case of HDFN described
1939: Levine & Stetson described antibody
1941: Levine demonstrated causal relationship
between anti-D antibodies and HDFN
1945: Neonatal exchange transfusion began
1956: Bevis proposed amniotic fluid assessment
1961: Liley proposed amniotic fluid assessment
1963: Liley introduced intraperitoneal fetal transfusion
1968: Rh immune globulin (RhoGAM) introduced
1980’s: Real-time ultrasound
1990’s: Genetic techniques to perform fetal RBC typing

4. Nomenclature
 1940-Landsteiner produced rabbit immune sera to rhesus
monkey erythrocyte that agglutinated 85% of human RBC
-Name this the Rh factor
 Rh factor –exhibit high degree of polymorphism
-Rh –ve - women who dose not have Rh(D)
Ag
 3 systems of nomenclature
1-Fisher-Race system
2-Weiner
3-HLA like system of Rosenfield

5. Fisher Race Nom.
-commonly used in obstetrics
-3 genetic loci with two major alleles i.e -C,c,D,E,e
 Rh gene complex-described by 3 appropriate letters
-8 gene complexes could exist
-CDe/cde & CDe/cDe most prevalent
-D,C(c),E(e)-actual order of gene on
chromosome
-vast majority of incompatibility due to D
Ag

6. cont’d
2. Weiner
-Based on assumption of only one
genetic loci exist as R or r
3.Rosenfield
- Rh1-Rh48
->30 antigenic variant of Rh blood
group system
-Cw and Du are the most common

7. Genetic expression
 Only found on RBC
 Genetic locus-on short arm of chromosome 1
 Expression of D
-heterozygous
-homozygous
 Gene dosage have effect on expressing Ag

8. Biochemistry & immunology
 Rh Ag
-polypeptides
-embedded in lipid phase of RBC membrane
(transmembrane)
 appears at 38 day of embryonic life
 Function-unknown
- membrane integrity
-interact with membrane ATP

9. Cause of Rh alloimmuization
 3 things must exist for alloimmunization
1.Fetus should have Rh positive RBC &
mother should have Rh-ve RBC
2.Sufficent number of fetal RBC must
gain access to maternal circulation
3.mother-immunologic capacity to
produce Ab directed against the D Ag

10. Incidence
-Rh –ve
-15% -whites
-5-8%-American Blacks
-1-2%-Asians
-30-35%-Basques
-Zygosity
-60%-heterozygous
-40%-homozygous

11. Fetomaternal hemorrhage &Rh
sensitization
 Haemorrhage sufficient to produce sensitization
-usually occurs at delivery-15-50%
->50% of these –FMH is less than 0.1ml
 Risks for increased FMH
-C/S delivery
-multiple gestation
-APH
-Manual removal of placenta
-intrauterine manipulation
 But majority occurs with out risk

12. Cont’d
Overall
-16% of Rh -ve women alloimmunized by their 1st Rh
incompatible(ABO compatible) pregnancy if not
treated
.2% immunized by time of delivery
.7% by 6month post partum
.7% sensiblized
-50% sensitization after several incompatible
pregnancy

13. Antepartum Alloimmunization
 An overall rate of 1-2% sensitization before delivery
 FMH detected -7% FTM
-16% STM
-29% TTM
 Abortion
.spontanous-2%
.induced-4-5%
Amniocentesis,chorionic villus sampling & cord blood
sampling -11%
External cephalic version-2-6%

14. Testing for Fetomaternal haemorrhage
1.Erythrocyte rosette test
-1st screening test
-maternal RBC are mixed with anti D antibody which
coat any fetal D tve cells in the sample
Indicators-RBC bearing D Ag are then added & rosettes foam
around fetal cells as the indicator cells are attached to them
by antibodies
-presence of Rosettes-Fetal D tve cells
2.kleihauer-Betke test
-fetal RBC contain HgbF which is more resistant to acid
elution than Hgb A
-after exposure to acid-only fetal Hgb remains
-uptake of special stain-quantify on peripheral smear

16. Rh alloimmunized pregnancy
Diagnosis
-Blood / Rh(D) typing and an antibody screen should always
be performed at the first prenatal visit
-based on the presence of anti D antibody in maternal serum
Methods
1.Ab titer in saline –agglutinated by IgM
2.Ab titer in Albumin –reflects the presence of
IgM/IgG
3.Indirect coomb's test-most sensitive
-one to three dilutions higher than the results
obtained in albumin

17. Indirect coomb's test
 Any anti D antibody present will adhere to RBC
 RBCs then washed & suspended in serum containing
antihuman globulin(coombs serum)
 RBCs coated with maternal anti D will be agglutinated
by coombs test

19. Direct coombs
 Done after birth to detect the presence of maternal
antibody on the neonates RBC
 Done by placing the infants serum(RBC) in coombs
serum
 Maternal antibody is present if the cells are
agglutinated

21. First step
 Determine paternal Rh status & zygosity
Father Rh-ve - fetus Rh-ve,no further testing
Father Rh+ve -who had previously fathered Rh
negative children, heterozygous
.50% chance of Rh-ve for this fetus
Father Rh+ve with no Rh negative children assess
zygosity
.DNA analysis
.Rh antisera
if homozygous _fetus is Rh+ve no further test is
necessary

22. Second step
 Determine fetal blood type
-previously required cordocentesis
(umbilical cord blooding sampling)
-Routine at 18-20wks for Rh+ve
heterozygous father
Risk -FMH
-Fetal loss
-obsolete now days

23. currently
 DNA tests that use PCR
-possible from uncultured amniocytes
obtained from as little as
.5mg of chorionic villi
.2ml of amniotic fluid
-free fetal DNA(ffDNA)
.D typing of fetus using PCR from
maternal serum / plasma

25. Clinical management
 A maternal antibody screen should be performed
on an Rh(D) negative women with uncomplicated
pregnancy at the first visit
 A positive screen –requires further diagnostic
measure
-Amniocentesis
-ultrasound
-fetal blood sampling
-Doppler flow velocimetry

26. Antibody Titre
 Should be interpreted as screening test
 Sever EBF or perinatal death doesn't occur when
AB titre remain below a certain critical titre
-titre associated with significant risk of fetal
hydrops
-varies b/n laboratories
-usually 1:16 or 1:32
-1:8 ,considered abnormal b/c of variability in
reliability & method of titration

27. How to proceed?
1.Titre <critical value plus no hx of previously affected
delivery
-intrauterine transfusion
-hydrops fetalis
-early delivery
-neonatal hx of exchange transfusion
Follow with
-AntiD titre every 2-4wks after 20wks of gestation
-serial U/S
2.Titer >=1:16
-Amniocentesis

28. Important Consideration
-Once critical value reached, subsequent test for
titre in current or future pregnancies is
unnecessary
-titre may be stable in 80% of severely affected
pregnancies
 Some Authors –anti D antibody concentration has
better predictive value than titre
-concentration >15IU/ml,associated with sever
disease
-practiced in Europe and UK

29. Contd.
 Presence of maternal anti D AB dose not mean that
the fetus will be affected or even Rh +ve
-titre may increase b/c of amnestic response even in
Rh -ve fetus if mother is previously sensitized
 Previous obstetric Hx is very important
-fetal haemolytic disease tend to be as sever or
more sever in subsequent pregnancies
-Hx of previous IUFD/neonatal death, poor
prognosis
-previous Hx of hydropic baby, recurrence if left
untreated 80-90%

30. Amniotic Fluid Analysis
 Based on original observation of Bevis
 AF bilirubin concentration
-low compared to serum
-measured by continuous recording of
spectrophotometer
Spectrophotometer determination of AF bilirubin
correlates with severity of fetal haemolysis

31. Contd.
 Using semi logarithmic plot, the curve of optical
density of normal AF is linear b/n wave length 525-
375nm
 Bilirubin causes a shift in spectrophoto density with
peak at wave length 450nm
 The amount of shift of OD from linearity at 450nm
(Delta OD450 ) is used to estimate degree of fetal RBC
haemolysis

32. Contd.
 Sir William Liley in 1961
 On bases of Delta OD450 value in 3rd TM pregnancy(27-
41wk)
 Retrospectively correlated Delta OD450 values with
newborn outcome by dividing a semi logarithmic graph of
GA Vs Delta OD450 into 3 zones
zone I - D-ve fetus, unaffected/mild disease
zone II - mild to sever disease
lower zone II-Hgb11-13.9g/dl
upper zone II-Hgb-8-10g/dl
zone III - sever disease
Hgb < 8g/dl
death within in 7-10days

33. Contd.
 Because there is a tendency for AF bilirubin
to decrease as pregnancy advances
- boundaries of the zone slope down ward
as GA advances
 Liley’s curve has decreased PNM from 22%
to 9% over 5 years
 Single DeltaOD450 measurement is rarely
helpful unless it is very high/very low
 Establish a trend by repeating AF analysis

35. Queenan Curve
 Published in 1993, reported normal range for delta
OD450 from 14 to 40 weeks of gestation and
proposed 4 management zones.
 Also called modified Liley’s curve
 Four zones
zone 1- -ve,unaffected
zone 2-indeterminate,values don’t accurately
predict fetal Hgb
zone 3-Rh +ve affected
zone 4-IUFD risk
-prior to 27wk –Queenan curve is 10% more sensitive
than Liley’s to detect anemia

37. Fetal blood analysis
Determination of
 Hct & blood group
 direct Coombs test
 retic count
 total bilirubin
Accurate assessment of foetal anemia and IUT
Fetal Hct & delta OD450 were in
.agreement in 79% of cases
.Delta OD450 – underestimated anemia-11%
- over estimated anaemia- 10%
- Successful-95% of cases
.Fetal loss-0.5-2%

38. Role of ultrasound
 Guide to
-amniocentesis
-fetal blood sampling
-IUT
 Reliable to detect frank hydrops but not reliable in
distinguishing mild from severe haemolytic disease
 Detect -polyhydramnios-appear early
-placental thickness >4cm
-pericardial effusion
-dilatation of cardiac chambers
-chronic enlargement of spleen/liver
-visualization of both sides of fetal bowel wall
from small amount of intraabdominal
fluid, first sign of impending hydrops
-dilatation of umbilical vein

39. Non-immune Hydrops Fetalis

40. Placenta of Hydropic Pregnancy
Placenta of Normal Pregnancy

41. www.thefetus.net Fetal Ascites

42. Pericardial Effusion
Heart

43. Normal
four
Chamber
Cardiac
View

45. Body wall
edema in a
hydropic
fetus

46. Doppler flow velocimetry
 based on the fact that the anaemic fetus preserves
oxygen delivery to the brain by increasing cerebral
flow of this low viscosity blood
 Of foetal cardiac out put & RBC flow
-foetal middle cerebral artery (MCA) most
promising
-when velocity is >1.5 x of median value, moderate
to sever anaemia
 Sensitivity of MCA peak systolic velocity for
prediction of moderate-sever anaemia -100%
12%-false positivity

48. Noninvasive diagnosis of fetal anemia

49. Noninvasive diagnosis of fetal anemia
Mari et al NEJM 2000;342:9-14

50. orithm 1st Rh s

51. orithm 2nd Rh s

52. Intra uterine transfusion
 50% of Rh immunized infants don't require IUT
 Indication for transfusion
-zone III
-Rising to zone III or upper zone II
especially before 30wk
-Hgb ,2gm/dl below the average for GA
-Hct <30% -2SD below average at all GA
-Evidence of hydrops

53. Approach
1.indirect-intraperitoneal
2.direct
-umbilical vein at placental end of cord
insertion, recommended in USA
-Intrahepatic portion of umbilical vein Europe
-cardiac puncture – increased rate of fetal
loss 8%/procedure
.

54. Use of Rh immunoglobulin
 Prior to development of anti D immuneglobulin
-16% of RhD –ve women became alloimmunized
-2% with routine post partum administration of a
single dose of antiD IG
-0.1% with addition of routine antenatal
administration in the third trimester

55. Cont’d
Anti-D Ig
–is sterile solution containing IgG anti D
-manufactured from human plasma
-extracted by cold alcohol fractionation
-from plasma of increased titre D Ab
Principles
–passively administered AB will prevent active immunization
by specific Ag
-termed AB mediated immune suppression
Tested & found non reactive for
-Anti-HCV
-Anti-HIV
-Anti-HBV
-Anti-parvovirus B 19

56. cont’d
Half life-average 24 days
10mcg of Rh Ig should be given for every 1ml of fetal
blood in the circulation
If given within in 72hrs after delivery risk of
alloimmunization will decreases to1.5%
To be effective – it should be given before
primary immune response develops
-administer as soon as possible

57. Cont’d
-Antepartum
.ACOG-300mcg at 28wks
.UK 100mcg - at 28wk & 34wk
.giving only at 28wk-just as effective
-post partum-300mcg
-Abortion both spontaneous or induced
.less than 12wk-50mcg
.greater than 12wk-300mcg
-first trimester chorionic villus sampling-50mcg
-ectopic pregnancy
.prior to 12wk-50mcg
.After 12wk-300mcg

58. Guidelines for use of anti D immunoglobulin
-Amniocentesis, second trimester chorionic
villus sampling or other IU procedure-300mcg
-Hydatidiform mole-300mcg
-Fetal death in second or third trimester
pregnancy-300mcg
-Blunt trauma to the abdomen-300mcg
-Antepartum hemorrhage-300mcg
-External cephalic version-300mcg
- Fetomaternal hemorrhage-1o per ml of whole
fetal blood

59. summary
 Rh immunoglobulin should be given to Rh
negative unsensitized women at 28wk & post partum
,also during events that leads to FMH
The gene coding for the D Ag has been cloned
Measurement of AFB remains the standard for
assessment of pregnancy at risk for significant
anaemia

60. Cond.
 The timing of the 1st amniocentesis is based on
Hx,maternal anti D titer,GA,& u/s finding
 Fetal transfusion can be either intraperitoneal or
intravascular route, for hydropic fetuses
intravascular is superior
 With the reduction in Rh disease brought about
by widespread use of Rh immunoglobulin
prophylaxis, sensitization to the minor or
atypical Ags has became relatively more
common

61. REFERENCE
 Seven Gabbe obstetrics 4th ed 2002
 Williams obstetrics 22ed,2005
 Uptodate-educational CD-15.3
 ACOG Practice B’ulletin 2002
 Current obstetric & gynaecology 10th ed.2007
 American journal of obstetric & gynaecology
2006,195,1158-62
 American journal of emergency medicen(2006)
24.487-489
 International journal of GYN/OBS(2005)90,103-
106

62. Thank you
