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1. 1
Chapter -six
HEMOLYTIC DISEASE OF THE FETUS AND NEW
BORN (HDFN)

2. Content
2
 Overview of HDFN
 HDFN due to Rh
 HDFN due to ABO
 Assessment of HDFN
 Prevention
 Treatment

3. Learning objectives
3
 Explain the different etiologies of HDFN/HDN
 Compare and contrast the HDN caused due to ABO
and Rh incompatibility.
 Describe the prenatal treatments that are significant in
HDN caused by anti-D.
 Explain the pathogenesis and laboratory diagnosis of
HDFN .
 Describe how to prevent Rh-alloimmunization.

4. Hemolytic Disease of the Fetus and New born
(HDFN)
4
 Originally known as erythroblastosis fetalis
 Initially observed in babies from D- negative
women with D-positive mates.
 First pregnancies were not usually affected.
 Infants from subsequent pregnancies were
often still born or severely anemic and
jaundiced.

5. Overview of HDFN
5
HDFN is a condition in which the red blood cells of a fetus
or neonate are destroyed by Immunoglobulin G (IgG)
antibodies produced by the mother.
 Severity range from non- symptoms  Intrauterine death
Y
+
Fetal
RBC
Fetal RBC destruction
=

6. Overview of HDFN…
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Factors that must be present for HDFN to occur:
1. The mother must lack the Ag
following exposure to the Ag should produce Abs of
the IgG class.
Must contain Abs against Ags on Fetal cells
2. The Fetus must possess the Antigens
3. The Antigen must be well developed at birth

7. Etiology of HDFN
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 The placental barrier limits the number of fetal RBCs
entering the maternal circulation during pregnancy and
thus reduces the chances of Ab production during
pregnancy.
 Only IgG antibodies are involved because it can cross
the placenta(not IgA or IgM).

8. Etiology of HDFN……
8
Main causes of HDFN
 Delivery
At the time of delivery , a fetal RBCs escape into the
maternal circulation (known as feto maternal hemorrhage
(FMH).
 Amniocentesis
 Spontaneous or induced abortioin
 Cordocentesis
 Rupture of an ectopic pregnancy
 Trauma to the abdomen

9. Etiology of HDFN……
9
 Ags on the fetal RBC can stimulate the maternal immune
system which results in the production of IgG antibodies
.
 In a subsequent pregnancy the IgG antibodies cross the
placental barrier by active transport mechanism.
 The antibodies bind to the fetal antigens which results in
RBC destruction by macrophages in fetal liver and
spleen.
 Hemoglobin liberated from the damaged RBCs
metabolized to indirect bilirubin

10. Etiology of HDFN….
10
 Bilirubin is harmlessly excreted by the mother.
 If the RBC destruction continues, the fetus becomes increasingly
anemic
 Fetal liver and spleen enlarge as erythropoiesis increases in an effort
to compensate for the RBC distruction.
 Erythroblasts are released into the fetal circulation
 Erythroblastosis Fetalis
 If this condition is left untreated, cardiac failure can occur
accompanied by hydrops fetalis, or edema and fluid accumulation in
fetal peritoneal and pleural cavities.

11. Etiology of HDFN……
11
 Thus the greatest threat to the fetus is cardiac failure
resulting from uncompensated anemia.
 Following delivery, red blood cell destruction continues
with the release of indirect bilirubin
 The new born liver is deficient in glucuronyl transferase
 The indirect bilirubin binds to tissues results in jaundice.
 Bind with tissues of the CNS and cause permanent
damage (kernicterus)
 resulting in deafness, mental retardation, or death.

12. About 1 in 10 pregnancies involve an Rh-
negative mother and an Rh-positive father
12

13. Pathogenesis
 Maternal IgG attaches to antigens on fetal cells
 Sensitized cells are removed by macrophages in
spleen
 Destruction depends on antibody titer and number of
antigen sites
 IgG has half-life of 25 days, so the condition can range
from days to weeks
 RBC destruction and anemia cause bone marrow to
release erythroblasts, hence the name
“erythroblastosis fetalis”)

14. Increased immature RBCs

15. Pathogenesis
 When erythroblasts are
used up in the bone
marrow, erythropoiesis
in the spleen and liver
are increased
 Hepatosplenomegaly
(enlarged liver & spleen)
 Hypoproteinemia (from
decreased liver function)
leads to cardiac failure
edema, etc called
“Hydrops fetalis”

16. Bilirubin
 Hemoglobin is metabolized to bilirubin
• Before birth, “indirect” bilirubin is transported across
placenta and conjugated in maternal liver (“direct”)
where it is excreted
• After birth, the newborn liver is unable to conjugate the
bilirubin
 Unconjugated (“indirect”) bilirubin can reach toxic
levels (18-20 mg/dL)
 This is called kernicterus and can lead to permanent
brain damage

17. Etiology of HDFN….
17
 HDN is often classified into three categories
based on Antibody specificity:
 Rh
 ABO and
 Other (non-Rh) Antibodies.

18. HDFN due to RH
18
 Anti- D is responsible for the most severe cases of
HDFN.
 In most cases, D-Negative women become
alloimmunized (produce anti- D) after the first D- Positive
pregnancy.
 In rare cases, alloimmunization occurs during the first
pregnancy but rarely results in clinical signs of HDFN.
 In some cases, the maternal anti-D binds to fetal D-
positive red blood cells and causes a positive direct
antiglobulin test (DAT).

19. HDFN due to RH…
19
 Moderately affected infants develop signs of
• jaundice, and corresponding elevations in bilirubin
levels, during the first few days of life.
 Severely affected D- positive infants,
• experience anemia in utero and develop jaundice
within hours of delivery.

20. HDFN due to ABO
20
 Occurs most frequently in group A or B babies born to
group O mothers.
 Is due to the increased incidence of IgG ABO Abs in
group O individuals compared to other ABO groups.
 ABO incompatibility often affects the first pregnancy
because of the presence of non-RBC stimulated ABO
Abs

21. HDFN due to ABO…
21
 Red blood cell destruction by ABO Abs is more common
than by anti-D.
 Fortunately, most cases are sub clinical and do not
necessitate treatment.
 Some infants may experience mildly elevated bilirubin
levels and some degree of jaundice with in the first few
days of life.
 These cases can usually be treated with phototherapy.

22. HDFN due to ABO…
22
 Mild red blood cell destruction, despite high levels of
maternal antibody occurs because:
• A or B substances are present in the fetal tissues and
secretions and bind or neutralize ABO antibodies, which
reduces the amount of ABO antibody available to destroy
fetal red blood cells.
• Poor development of ABO Antigens on fetal or infant red
blood cells.
• Presence of reduced number of A and B antigen sites on
fetal or infant red blood cells.
 This also explains why the DAT is only weakly positive in

23. 23
Alloantibody causing HDN other than anti-D
 Other Rh-system antibodies are known to cause HDN
alone or in combination with anti D.
 Anti-c is the second most common cause of HDN,
followed by anti-K.
 Anti Kidd
 Anti E
 Anti e

24. Assessment of HDFN
1. Ante partum testing
2. Post partum testing
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25. 1. Ante partum testing
A. Tests for the blood type of baby’s father
 Serologic testing
 ABO typing
 Rh typing
 If positive for anitgens- molecular tests performed (
e.g DNA analysis) to identify Homozygous or
Heterozygous alleles
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26. Cont…
B. Tests for the mother
 Serologic testing on mother
 ABO and Rh testing
 Test for D antigen (test for weak D if initially
negative)
 Antibody Screen
 To detect IgG alloantibodies that react at 37°C
 If negative, repeat before RhIg therapy and/or if
patient is transfused or has history of antibodies (3rd
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27. Tests for the mother….
 Antibody ID
 Weakly reacting anti-D may be due to FMH or
passively administered anti-G (RhIg)
 If antibody is IgG, anti-D is most common followed
by anti-K and other Rh antibodies
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28. C. Tests for the fetus
Amniocentesis & Cordocentesis
 About 18-20 weeks’ gestation
 Cordocentesis takes a sample of
umbilical vessel to obtain blood
sample
 ABO & Rh testing
 Amniocentesis assesses the
status of the fetus using
amniotic fluid
 Fluid is read on a
spectrophotometer (350-700
nm)
 Change in optical density
(ΔOD) above the baseline of
450 nm is the bilirubin
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29. Analysis of amniotic fluid
(example)
29

30. Liley graph
 The ΔOD is plotted on the Liley graph according to
gestational age
 Three zones estimate the severity of HDN
 Lower: mildly or unaffected fetus (Zone 1)
 Midzone: moderate HDN, repeat testing (Zone 2)
 Upper: severe HDN and fetal death (Zone 3)
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31. Liley graph
a ΔOD of .206 at
35 weeks
correlates with
severe HDN
*

32. 2. Post partum testing
 Testing of infants Born to D-Negative Mothers
 Based on medical history, physical examination of the
new born, and results of Laboratory testing on both
mother and child
 Advantageous to collect a sample of cord blood from
every new born.
 The specimen should be properly labeled and stored
for up to 7 days
 Cord blood should be washed free of Wharton's jelly.
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33. Post partum testing …
ABO/Rh Testing
 Infants should be tested for the D antigen, including a test for
weak D.
 In cases of HDFN, the DAT for ABO testing may be positive,
which can lead to false positive or false negative Rh- testing
results.
 False Rh- negative results may be due to a blocking
phenomenon requiring gentle elution to resolve.
 To ensure the validity of an Rh- positive test result, it is
essential to include appropriate Rh controls.
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34. DAT
 Its result may be weak especially in cases of ABO
HDFN.
 When positive, performing an elution is optional if
Ab identification test were performed on the mother
at the time of admission.
 If a maternal sample is unavailable, testing the elute
may be useful to confirm HDFN.
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35. DAT.....
 Positive results with A1 and/or B cells and
negative results with the O cells is indicative
and /or ABO HDN.
 If the elute is negative with all cells, an
antibody to a low frequency antigen should be
suspected and maternal serum tested against
the paternal cells.
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36. Management of HDFN
1.Intrauterine transfusion
2.RhIG
 Rosette test
 Acid elution test
 FC
3.Exchange transfusion
4.Photo therapy
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37. Management of HDFN…
1. Intrauterine transfusion
is done if:
 Amniotic fluid ΔOD is in high
zone II or zone III
 Cordocentesis has hemoblobin
<10 g/dL
 Hydrops is noticed on ultrasound
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38. Management of HDFN…
2. Rho (D) Immune Globulin (Human)-
RhIG administered:
 To non immunized Rho- negative mothers
 Who deliver Rho- positive babies
 Use
 prevent Rh- alloimmunization.
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39. Management of HDFN…
Rh immune globulin (RhIG)
 Protects D- negative mothers against the
production of anti- D following delivery.
 Anti-C, anti-c, anti-E, anti-e are not protected
by RhIG and can cause HDN.
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40. Management of HDFN…
 Candidates for this prophylaxis are:
 mothers who are Rh-negative
 Du - negative
 have an Rh-positive or Du positive new born.
 All Rh-negative women who have abortions are
candidates unless the father or fetus is known to be
Rh-negative.
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41. Management of HDFN…
The following are not RhIG candidates:
 Rho-negative women
 Who deliver Rh-negative babies.
 Whose serum contains anti-Rho (D).
 Who are weak Rho- positive or Du- positive
41

42. Rh immune globulin (RhIG)
42
 Is a concentrate of IgG anti-D prepared from pools of
human plasma.
 Is given intramuscularly
 to non-sensitized D-negative women at 28 weeks of
gestation (ante partum) and
 again within 72 hours of delivery (post partum) of a D
positive infant.

43. RhIG…
43
Mechanism of action
 Suppresses the immune response following
exposure to D positive fetal red blood cells
and prevents the mother from producing anti-
D.

44. Development and prevention of HDN
44

45. 45
 Rh Immune globulin is a concentrate of
predominantly IgG anti-D prepared from
pooled human plasma.
 Two different preparations are available
 IM preparation, which is most commonly used, [is
available in 50- and 300µg doses.]
 IM or IV preparation, available in 300 – and 120 -
µg doses.
 A 300 - µg dose of either preparation is
protective up to 15ml of Rh+ve RBCS

46. Tests to administer Rh immune globulin (RhIG)
A. Rosette test
B. Acid elution test
C. Flowcytometry
46

47. A. Rosette Test-Qualitative test
Purpose
 To detect the presence of Rh positive RBCs in
the circulation of Rh negative person
 To detect FMH > 30 ml
47

48. A. Rosette Test-Qualitative
test…
 Principle
Fetal Rh+ve RBCs in the maternal sample react with the
anti-D. The unbound antibody is washed away and a
suspension of group O, Rh+ve cells is added.
The anti-D reacts with both the Rh+ve and the fetal Rh+ve
RBCs. The RBCs agglutinate in a rosette pattern, and the
suspension is examined microscopically.
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49. A. Rosette Test-Qualitative
test…
 A Positive test is indicated by the presence of a certain no
of clumps (rosettes) in a defined no of microscopic fields
(eg. 7 clumps in 5 fields)
 A negative test means that the fetal bleed was less than
30ml. The administration of RhIG, however is still indicated
in these cases
49

50. A. Rosette Test-Qualitative
test…
Rosette Test – Procedure
 Use EDTA mother’s sample drawn after delivery
 Wash cells, add chemically modified anti-D and
enhancement
 Incubate 37oC
 Wash four times with normal saline
 Add R2R2 cells
 Centrifuge, mix, read microscopically
50

51. B.Kleihauer-Betke Acid Elution
 To detect the presence of Hgb F
51

52.  The fetus has a high amount of hemoglobin F in their red
blood cells.
 While adults have mostly Hgb A.
 Therefore the purpose of this test is to detect the presence
of Hgb F cells by reducing the hgb A cells to ghosts and
staining the Hgb F left behind.
 Acid will eluate Hgb A but will not eluate Hgb F. So you can
stain the hgb F cells.
 Note that the mother does not have to be Rh negative to do
this test. Some physicians will use this test after the mother
has experienced a trauma to the abdomen to determine if
there has been a fetal bleed.
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53. B. Acid Elution test …
Acid Elution Stain Procedure
 EDTA sample
 Make a slide
 Fix smear
 Treat with acid
 Stain with Eosin
 Count number of stained HbF cells within 2000
HbA cells
53

54. Acid Elution Stain- Purpose
54
 To detect the presence of Hgb F
Adult RBC appear as ghost cells, stroma only, while fetal
cells appear bright pink and refractile.
Count number of fetal cells in 2000 adult cells.
Precision of test poor even in experienced hands.

55. Kleihauer-Betke Acid Elution
 To calculate volume of fetomaternal
hemorrhage:
 Determine percentage of fetal rbcs.
 Multiply x 50 (represents 5000 mL, arbitrary
blood volume of mother).
 Divide by 30, volume of whole blood
covered by 1 vial of RhIg, to determine
number of vials required.

56. RhIG…
56
Acid Elution Stain Calculations
 # fetal cells/2000 adult cells x 100 = %of fetal
cells
 % of fetal cells X 50 = number of mLs of fetal
blood
 EXAMPLE
 26 fetal cells counted in 2000 total.
 26/2000= 1.3%
 1.3 x 50 = 65 mL of fetal whole blood

57. RhIG…
57
Determine # vials RhIG
 FB/30 = # vials needed
 Recommended dose (contained in one vial) 300 ug
RhIG will neutralize 30 mL of D+ whole blood
 If # calculated is <0.5 round down, > or =0.5 round
up
 1.4 round down to 1 and add 1 vial=give 2 vials
 1.6 round up to 2 and add 1 vial – give 3 vial
 For the above example
 65/30 = 2.2 doses of RhIg
 2.2+ 1(safety dose) = 3 vials

58. Cont….
 Notes: there are inherent errors in this
procedure therefore the extra vial is always
added.
1. assuming all the Hgb F cells are from the
neonate. Adults are known to have some Hgb
F cells and some people can higher amounts
than others.
 fetus could have bled some Hgb A cells and these
would not have been detected.
2. The 5000 mls of blood used for the average
female does not account for larger women with
more blood cells.
3. Counting of the ghost cells is not easy and this
test is hard to get duplicate answers.
58

59. Giving RhIg
 Given intramuscularly.
 No more than 5 doses should be injected at one time.
 If more than 5 doses needed space over 72 hours.
 Recommended dose (contained in one vial) is about 300 µg
 offers protection against a feto-maternal hemorrhage
(FMH) of 30 ml (15 ml packed cells) or less.
 If a massive FMH has occurred, the volume of the
hemorrhage must be determined to calculate the number of
vials to administer.

60. Cont…
Determine # vials RhIG
 FB/30 = # vials needed
 300 ug RhIG will neutralize 30 mL of D+ whole
blood
 If # calculated is <0.5 round down, > or =0.5 round
up
 1.4 round down to 1 and add 1 vial=give 2 vials
 1.6 round up to 2 and add 1 vial – give 3 vial
60

61. Treatment of Infants Suffering from HDFN
61
For infants who develop hyperbilirubinemia and/or
anemia due to HDFN, exchange transfusion is
usually carried out.
Exchange transfusion

62. Treatment…
62
Exchange transfusion:
 is a continuous removal of small amounts of blood
from the neonate with simultaneous continuous
infusion of donor blood until a one or two-volume
exchange is accomplished.
 Helps to reduce the concentration of bilirubin and
incomplete antibodies
 Provides the infant is with compatible donor red
cells.

63. Treatment…
63
 To give exchange transfusion to an infant
clinical and laboratory findings must be
considered.
 Cord Hemoglobin (<10g/dl) and
 raised serum bilirubin are strong indicators for
treatment.

64. Treatment…
64
 For compatible exchange transfusion, donor’s
blood should be:
 Cross-matched with the maternal serum and
 Lack the RBC Ag corresponding to the maternal
Abs
 ABO group and Rh type compatible with the infant’s
blood group.
 If the mother’s antibody is not available,
group
O Rh negative red blood cells must be
selected.

65. Blood for exchange
Transfusion
 ABO compatible with both mother and baby,
Rh(D) compatible with mother
 Antigen negative for the antibody identified in
mother
 Crossmatch negative with mother’s serum
 CMV negative
 Irradiated
 HbS negative
 < 5 (7) days old
 High Hct (semi-packed)
65

66. Phototherapy
 The use of light to degrade bilirubin in mildly
jaundiced infants
 Usually ABO incompatibility
66

67. HDFNB.
 Causes of severe HDFNB in order of incidence:
 Anti-D
 Anti-c
 Anti-K
 ABO antibodies (common, but not usually severe).
 Anti-E
 Anti-Fya
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68. THANK YOU!
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