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Hdfn
1. 1
Hemolytic Disease of the FetusHemolytic Disease of the Fetus
and Newbornand Newborn
( HDFN( HDFN ))
DR. RAFIQ AHMAD
2. 2
DefinitionDefinition
» Fetal red cells become coated with IgG
alloantibody of maternal origin, directed against a
paternally inherited antigen present on the fetal
cells that is absent from maternal cells.
» The IgG coated cells may undergo accelerated
destruction both before and after birth, but the
severity of the disease can vary from serologic
abnormalities detected in an asymptomatic infant to
intrauterine death
5. 5
Mechanisms of Maternal Immunization
HDFN is often classified into three categories,
1. D hemolytic disease caused by anti-D alone or,
less often, in combination with anti-C or anti-E.
2. “Other” hemolytic disease caused by antibodies
against other antigens in the Rh system or against
antigens in other systems; anti-c and anti-K1 are
most often implicated.
3. ABO HDFN caused by anti-A,B in a group O
woman or by isolated anti-A or anti-B.
6. 6
Immunization
» Previous Transfusion
» Previous Pregnancy
- at the time of delivery –FMH
- amniocentesis.
- spontaneous or induced abortion.
- chorionic villus sampling.
- cordocentesis.
- rupture of an ectopic pregnancy.
- blunt trauma to the abdomen.
7. 7
Immunogenic Specificities
The antigen that most frequently induces
immunization is D (only 0.1ml of fetal blood can
immunize)
but any red cell antigen present on fetal cells and
absent from the mother can stimulate antibody
production
One retrospective study determined that there was a
0.24% prevalence of production of clinically
significant antibodies other than anti-D during
pregnancy (needs large volume of cells e.g.,
transfusion)
8. 8
Frequency of Immunization
Depends upon volume of cells
D sensitization- 16%
1.5% to 2% become sensitized at the time of
their first delivery, an additional 7%
become sensitized within 6 months of the
delivery, and the final 7% become
sensitized during the second affected
pregnancy
9. 9
Frequency of Immunization
In susceptible women not immunized after two D-
positive pregnancies, later pregnancies may be
affected but with diminished frequency.
Once immunization has occurred, successive
D-positive pregnancies often manifest
HDFN of increasing severity, particularly
between the first and second affected
pregnancies.
10. 10
Effect of ABO Incompatibility
ABO incompatibility between mother and
fetus has a substantial but not absolute
protective effect against maternal
immunization by virtue
The rate of immunization is decreased
from16% to between 1.5% and 2%.
11. 11
Transfusion as the Immunizing
Stimulus
It is extremely important to avoid transfusing D-
positive whole blood or red cells to D-negative
females of childbearing potential because anti-D
stimulated by transfusion characteristically causes
severe HDFN in subsequent pregnancies with a D-
positive fetus
The risk of immunization to a red cell antigen
other than D after an allogeneic red cell
transfusion has been estimated to be
1% to 2.5% in the general hospital population
12. 12
““Other” hemolytic diseaseOther” hemolytic disease
antibodiesantibodies
Experience with other alloantibodies has
not been as extensive as with anti-D; in
some series, anti-c and anti-K1 were by far
the most common causes of severe HDFN,
other than anti-D.
13. 13
ABO antibodies
The IgG antibodies that cause ABO HDFN nearly
always occur in the mother’s circulation without a
history of prior exposure to human red cells.
It can occur in any pregnancy, including the first.
It is restricted almost entirely to group A or B
infants born to group O mothers because group O
individuals make the IgG antibody, anti-A,B.
14. 14
Prenatal Evaluation
Maternal History
Information about previous pregnancies or blood
transfusions.
For a woman with a history of an infant with
hydrops fetalis due to anti-D, there is a 90% or
more chance of a subsequent fetus being similarly
affected.
In contrast, during the first sensitized pregnancy,
the risk of a hydropic fetus is 8% to 10%.
15. 15
Serologic Studies
Should be performed on all pregnant women as early
in pregnancy as possible.
Tests for:
» Blood typing for ABO and D,
» red cell antibody screening.
weak D test is not required.
partial D phenotypes, such as DVI, will also most
likely type as D negative
16. 16
Follow upFollow up
The test can be repeated at 28 weeks’ gestation
before administration of RhIG to detect
immunization that might have occurred before
28 weeks, in accordance with AABB
recommendations
17. 17
Antibody Specificity
All positive screens for red cell antibodies
require identification of the antibody
Typing the Fetus
The fetal D type can be established by using the
polymerase chain reaction (PCR) to amplify
DNA obtained from amniotic fluid, chorionic
villus samples, or by serologic typing of fetal
blood obtained by cordocentesis/amniocentesis
18. 18
Molecular MethodsMolecular Methods
Fetal DNA typing is also available for Jka/Jkb,
K1/K2, c, and E/e antigens.
A more recent development in fetal RhD typing
involves the isolation of free fetal DNA in the
maternal serum. Although not routinely available in
at this time, this will likely replace amniocentesis for
Fetal genotyping in the near future
19. 19
Maternal Antibody Titer
The antibody titer should be established in the
first trimester to serve as a baseline, and the
specimen should be frozen for future
comparisons
Critical titer for anti-D - 1:16-32
for anti- K1 1- 8
For antibodies other than anti-D, and ant-K1
critical titers have not been identified
20. 20
Amniotic Fluid Analysis
A good index of intrauterine hemolysis
and fetal well-being is the level of bilirubin
pigment found in amniotic fluid obtained
by amniocentesis
Amniotic fluid is obtained by inserting
a long needle through the mother’s abdominal wall and uterus
into the uterine cavity under continuous ultrasound guidance.
wavelengths of 350 to 700 nm. Peak absorbance of bilirubin
is at 450nm.
21. 21
Liley’s GraphLiley’s Graph
This method is
applicable to
pregnancies from
27 weeks through
term.
The ΔOD450 value is plotted on a graph against the estimated length of gestationThe ΔOD450 value is plotted on a graph against the estimated length of gestation
22. 22
Queenan’s graphQueenan’s graph
This method is
applicable to
pregnancies from
14 weeks through
term.
when amniocentesis or cordocentesis is performed for any reason on a
D-negative woman who does not have anti-D, Rh immunoprophylaxis
should be given
23. 23
Percutaneous Umbilical Blood
Sampling (PUBS) or Cordocentesis
» A needle into an umbilical blood
vessel, preferably the vein at its
insertion into the placenta, and
obtain a fetal blood sample.
» It allows direct measurement of
Hematologic and biochemical
variables. The fetal mortality of
intrauterine fetal blood sampling
has been reported to be 1%to 2%, and the procedure
carries a high FMH
24. 24
Doppler Flow Studies
» Because fetal anemia results in increased
cardiac output, several investigators have
measured various blood velocities in fetal
vessels using Doppler ultrasonography to
determine the clinical status of the fetus
in a noninvasive manner
» There is good correlation between middle
cerebral artery (MCA) peak velocity, fetal
hemoglobin, and ΔOD450 reading
» MCA Doppler is associated with a high
false-positive rate for the diagnosis of
fetal anemia after 35 weeks gestation
25. 25
Suppression of Maternal
Alloimmunization
» Intensive plasma exchange and the administration of
intravenous immunoglobulin (IGIV) is being used to
reduce the maternal antibody titer.
» Plasma exchange can reduce antibody levels by as
much as 75%. Unfortunately, rebound usually follows
because the IgG antibody is mostly extravascular and
antigen exposure may be ongoing.
» AABB and the American Society for Apheresis (ASFA)
categorize plasma exchange as treatment Category III
because its efficacy and safety
26. 26
Intrauterine Transfusion
IT can be performed by the intraperitoneal
route (IPT) or the direct intravascular
approach (IVT) by the umbilical vein. In
many instances, IVT is the procedure of
choice, but there may be problems of access
that make IPT preferable
27. 27
It is usually done after 20th week of gestation;
Once initiated, transfusions are usually
administered periodically until delivery.
It carries a 1%to 2% risk of perinatal loss, it
should be performed only after careful clinical
evaluation
Intrauterine Transfusion
28. 28
Techniques
IPT is performed through a needle
passed, with ultrasonographic
monitoring, through the mother’s
abdominal wall into the abdominal
cavity of the fetus
Transfused red cells enter the fetal
circulation through lymphatic channels that drain the
peritoneal
cavity.
In IVT, the umbilical vein is penetrated under ultrasound
guidance and a blood sample is taken to verify positioning
in the fetal vasculature.
29. 29
Selection of Red Cells
» The red cells used should be group O, D-negative, or
negative for the antigen corresponding to the mother’s
antibody if the specificity is not anti-D.
» Blood for intrauterine transfusion should be as fresh as
possible, Irradiated, CMV reduced, and HbS negative
» Red cells of mother, washed in saline, with final Hct
of 75%-85% or deglycerolized, and irradiated to prevent
graft vs host disease have also been used.
30. 30
Volume Administered
For IPT, a volume calculated by the formula
V = (gestation in weeks – 20) x 10 mL
appears to be well tolerated by the fetus.
For IVT
Fetoplacental volume (mL) = ultrasound estimated fetal
weight (g) X 0.14
Volume to transfuse (mL) = Fetoplacental volume X
(Hct after IVT – Hct before IVT)
Hct of donor cells
where Hct = hematocrit
31. 31
Postpartum Evaluation
A D negative mother who is not immunized
should receive an appropriate dose of RhIG,
if delivered Rh positive infant.
cord blood should be tested immediately
after delivery
32. 32
Assessment of FMHAssessment of FMH
» The Rosette Test: A qualitative test
» Kleihauer Betke Test: A quantitative test
» Flow Cytometry
34. 34
Indication (Indicated Dosea )
approximately
Postpartum (if the newborn is Rh-positive) 300 µgb
Antepartum: Prophylaxis at 26 to 28 weeks' gestation c
300 µg
Antepartum: Amniocentesis, chorionic villus sampling (CVS) and
percutaneous umbilical blood sampling (PUBS(
300 µg
Antepartum: Abdominal trauma or obstetrical manipulation 300 µg
Antepartum: Ectopic pregnancyd
300 µg
Antepartum: Abortion or threatened abortion at any stage of gestation
with continuation of pregnancyd 300 µg
Transfusion of Rh-incompatible blood or blood products d
300 µg
a
Additional doses of RhoGAM are indicated when the patient has been exposed to > 15 mL of Rh-positive red blood cells. This may
be determined by use of qualitative or quantitative tests for FMH (see below).
b
See DESCRIPTION section.
c
If antepartum prophylaxis is indicated, it is essential that the mother receive a postpartum dose if the infant is Rh-positive.
d
If abortion or termination of pregnancy occurs up to and including 12 weeks' gestation, or less than 2.5 mL of Rh-incompatible red
blood cells were administered, a single dose of (MICRhoGAM) Rh0
D Immune Globulin (Human) (approximately 50 µg)* may be
used instead of RhoGAM.
RhoGAM
35. 35
Dose CalculationDose Calculation
Fetal and maternal cells are counted separately
for a total of 2000 cells
The following formula can be used for fetal
bleeding calculation:
Fetal cells x maternal blood volume (ml) = fetal hemorrhage (ml)
total cells counted
Example:
6cells/2000 cells X 5000 ml = 15 ml fetal whole blood
300 µg of RhIG is required for 30 ml of fetal blood
= ( 0.5 of the vial), but needs 2 vials
if the calculated dose is right of the decimal point ≥ 0.5 add one more vial
36. 36
Flow Cytometry: FMH estimationFlow Cytometry: FMH estimation
Sample negative for fetal HbF. Positive fetal control. Fetal HbF positive cells show increased
fluorescent intensity staining with anti-HbF-FITC
conjugated antibodies compared to adult HbF and HbF
negative cells.
Patient sample with 0.53 per cent fetal HbF positive
cells, equating to an 11.0 ml FMH.
37. 37
RhIG dose for FMHRhIG dose for FMH
fetal cells percentage Vials to inject µg(mcg) IU
6-8 0.3-0.8 2 600 3000
9-14 0.9-1.4 3 900 4500
15-26 1.5-2.0 4 1200 6000
21-26 2.1-2.6 5 1500 7500
DoseDose
Note:Note:
1. Based on maternal blood volume of 5000 ml.1. Based on maternal blood volume of 5000 ml.
2. 1 vial of 300 µg (1500 IU) is needed for each 15 ml of fetal red cells or2. 1 vial of 300 µg (1500 IU) is needed for each 15 ml of fetal red cells or
30 ml of fetal whole blood30 ml of fetal whole blood
38. 38
ABO HDFNABO HDFN
» ABO incompatibilities are the most common cause
of HDFN but are less severe
– About 1 in 5 pregnancies are ABO-incompatible
– 65% of HDFN are due to ABO incompatibility
» Usually, the mother is type O and the child has the
A or B antigen…Why?
– Group O individuals have a high titer of IgG anti-A,B
in addition to having IgM anti-A and anti-B
39. 39
ABO HDFNABO HDFN
» ABO HDFN can occur during the FIRST
pregnancy because prior sensitization is not
necessary
» ABO HDFN is less severe than Rh HDFN because
there is less RBC destruction
– Fetal RBCs are less developed at birth, so there is less
destruction by maternal antibodies
– When delivered, infants may present with mild anemia
or normal hemoglobin levels
– Most infants will have hyperbilirubinemia and jaundice
within 12 to 48 hours after birth
40. 40
Diagnosis of ABO HDNDiagnosis of ABO HDN
» Infant presents with jaundice 12-48 hrs after birth
» Testing done after birth on cord blood samples:
– Sample is washed 3x to remove Wharton’s jelly
– Anticoagulated EDTA tube (purple or pink)
– ABO, Rh and DAT performed
– Most cases will have a positive DAT
• If DAT positive, perform elution to ID antibody
41. 41
Treatment of ABO HDFNTreatment of ABO HDFN
» Only about 10% require therapy
» Phototherapy is sufficient
» Rarely is exchange transfusion needed
» Phototherapy is exposure to artificial or
sunlight to reduce jaundice
44. 44
What type of blood to be given:What type of blood to be given:
» Exchange transfusion: Fresh Whole Blood (to
avoid Ca++
), less than 7 days old
» Irradiated
» SCT negative
» Group O, or ABO compatible with mother’s serum
with D-negative in case of Rh-D HDFN
» Group O red cells reconstituted with AB plasma
in case of ABO HDFN
» Leukoreduced
» CMV negative
