This chapter discusses hemolytic diseases including hemolytic transfusion reaction, autoimmune hemolytic anemia, and hemolytic disease of the fetus and newborn. HDFN is caused by maternal IgG antibodies crossing the placenta and destroying fetal red blood cells. The major causes of HDFN are Rh and ABO incompatibility. RhIG is given to Rh-negative mothers to prevent Rh alloimmunization. Severe HDFN may be treated with exchange transfusions to reduce bilirubin levels and antibody concentrations in the infant.

1. CHPTER -SIX
Hemolytic Diseases
CH

2. 2
Content
 Hemolytic transfusion reaction
 Autoimmune Hemolytic Anemia (AIHA)
 Hemolytic Disease of the Fetus and New born
(HDFN)
 Rho (D) Immune Globulin (Human) – RhIG -mod
 Treatment of Infants Suffering from HDFN

3. 3
Learning objectives
At the end of this chapter, the student will be able to:
 Explain the cause and laboratory diagnosis of the
different hemolytic diseases .
 Explain the cause and consequence of HDN due
to ABO and Rh incompatibility.
 Name the major immunoglobulin class responsible
for hemolytic disease of the new born

4. 4
Learning..
 Explain the different etiology HDN
 Describe the prenatal treatments that are
significant in HDN caused by anti-D.
 Compare and contrast the HDN caused due to
ABO and Rh incompatibility.
 Describe how to prevent Rh-alloimmunization.

5. 5
6.1 Hemolytic Transfusion reaction (HTR)
 Is a condition in which there is intravascular
destruction of transfused RBCs
 Is mainly caused by antibodies of the ABO and
Rh system.
 Clinical consequences include
 Disseminated intravascular hemolysis(DIC)
 Hypotension
 Irreversible shock and renal failure

6. 6
6.2 Autoimmune Hemolytic Anemia (AIHA)
 Brought about through the interaction of red
cells and auto antibodies.
 Classified into four groups
 warm-reactive auto antibodies
 cold- reactive auto antibodies
 paroxysmal cold hemoglobinuria and
 drug induced hemolysis.

7. 7
AIHA…
 The diagnosis depends on the demonstration of
auto antibodies on the patient’s red cells using
the DAT.
 The autoimmune antibodies are either
 ‘warm antibody’ type or
 ‘cold antibody’ type.

8. 8
AIHA…
 The warm antibodies are active at 370C
 The cold types are optimally active at 20C but
with a temperature range which may go as high
as 320C.

9. 9
AIHA…
 A positive DAT will be found in both types;
 In the cold type , complements rather than the bound
antibody sensitizes the cells and give rise to the
positive DAT
 In the warm type, the attached antibody sensitizes the
cells, although occasionally it may be complement.

10. 10
6.3 Hemolytic Disease of the Fetus and New
born (HDFN)
 Originally known as erythroblastosisfetalis
 Initially observed in babies from D- negative
women with D-positive mates.
 Initial pregnancies were not usually affected.
 Infants from subsequent pregnancies were often
still born or severely anemic and jaundiced.

11. 11
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.

12. 6.3.1 Overview of HDFN
HDFN is a condition in which the red blood cells of
a fetus or neonate are destroyed by IgG Abs
produced by the mother.
Y
+
Fetal
RBC
Fetal RBC destruction
=

13. 13
Overview of HDFN…
Factors that must be present for HDFN to occur:
1. The mother must lack the Ag, and, following
exposure to the Ag should produce Abs of the
IgG class.
2. The Fetus must possess the Antigens
3. The Antigen must be well developed at birth

14. 14
Etiology of HDFN
 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.

15. 15
Etiology of HDFN……
 At the time of delivery , a fetal RBCs escape into
the maternal circulation (known as feto maternal
hemorrhage (FMH).
 Immunization can result from fetal RBC
exposure following:
- Abortion
- Ectopic pregnancy, or
- Abdominal trauma.

16. 16
Etiology of HDFN……
 Antigens 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.

17. 17
Etiology of HDFN……
 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.

18. 18
Etiology of HDFN….
 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
destructions.

19. 19
Etiology of HDFN……
 Erythroblasts are released into the fetal
circulation
 Erythroblastosisfetalis
 If this condition is left untreated, cardiac failure
can occur accompanied by hydropusfetalis, or
edema and fluid accumulation in fetal peritoneal
and pleural cavities.

20. 20
Etiology of HDFN……
 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

21. 21
Etiology of HDFN……
 The indirect bilirubin binds to tissues results in
jaundice.
 Bind with tissues of the CNS & cause permanent
damage (kernicterus)
 resulting in deafness, mental retardation, or death.

22. 22
Etiology of HDFN….
 HDN is often classified into three categories based
on Ab specificity:
 Rh
 ABO and
 Other (non-Rh) Antibodies.

23. 23
6.3.2.HDFN due to RH
 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.

24. 24
HDFN due to RH…
 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 .

25. 25
HDFN due to RH…
 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 inutero and develop jaundice
within hours of delivery.

26. HDFN due to RH…

27. 27
6.3.3.HDFN due to ABO
 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

28. 28
HDFN due to ABO…
 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.

29. 29
HDFN due to ABO…
 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.

30. 30
HDFN due to ABO…
 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 & bind or neutralize ABO Abs, which
reduces the amount of ABO Ab available to destroy fetal
RBCs.

31. 31
HDFN due to ABO…
 Poor development of ABO Ags on fetal or infant RBCs .
 Presence of reduced number of A and B Ag sites on
fetal or infant RBCs.
 This also explains why the DAT is only weakly
positive in most cases of ABO HDN.

32. 32
Alloantibodies 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.

33. 33
6.3.4 Assessment of HDFN
Postpartum testing
 It is 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.
 for testing if the mother is D Negative or if the new born
develops signs or symptoms of HDFN.
 Cord blood should be washed free of wharton’s
jelly.

34. 34
Assessment…
Postpartum testing of infants born to D negative
Mothers
 Infants should be tested for the D Ags, including a
test for weak D.

35. 35
Assessment…
Postpartum Testing of infants with suspected
HDFN
 Is based on medical history, physical examination
of the new born, and results of Laboratory testing
on both mother and child

36. 36
Assessment…
ABO Testing
 In cases of HDFN the DAT 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.

37. 37
Assessment…
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.

38. 38
Assessment…
 If the maternal Antibody screen is negative and
the DAT is positive, ABO HDN should be
suspected.
 ABO HDN can be confirmed by performing either
a heat or freeze thaw elutes.
 The elute should be tested against A1, B and O
cells using an antiglobulin technique.

39. 39
Assessment…
 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.

40. 40
6.4. Rho (D) Immune Globulin (Human)-RhIG
 Administer
 to non immunized Rho- negative mothers
 who deliver Rho- positive babies
 Use
 prevent Rh- alloimmunization.

41. 41
RhIG…
 Candidates for this prophylaxis are:
 mothers who are Rh-negative and
 Du - negative, and
 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.

42. 42
RhIG…
The following are not RhIG candidates:
 Rho-negative women
 Who deliver Rh-negative babies.
 Whose serum contains anti-Rho (D).
 Who are Rho- positive or Du- positive

43. 43
RhIG….
 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.

44. 44
Fig.6-2 Development and prevention of HDN

45. 45
RhIG…
Mechanism of action
 Suppresses the immune response following
exposure to D positive fetal red blood cells and
prevents the mother from producing anti-D.

46. RhIG …
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. Rosette …
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.

48. Rosette 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. 49
Rosette Test - Procedure
 Use EDTA mother’s sample drawn after delivery
 Wash cells, add chemically modified anti-D and
enhancement
 Incubate 37oC
 Wash times four with normal saline
 Add R2R2 cells
 Centrifuge, mix, read microscopically

50. 50
Acid Elution Stain- Purpose
To detect the presence of Hgb F

51. 51
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

52. 52
RhIG…
Acid Elution Stain Calculations
 Number of fetal cells/2000 adult cells x 100 = %of
fetal cells
 % of fetal cells X 50 = number of mls of fetal blood

53. 53
RhIG…
Determine number of vials RhIG
 FB/30 = number of vials needed
 300 ug RhIG will neutralize 30 ml of D+ whole blood
 If number 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

54. 54
RhIG Dose
 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.

55. 55
6.5 Treatment of Infants Suffering fr HDFN
 For infants who develop hyperbilirubinemia
and/or anemia due to HDFN, exchange
transfusion is usually carried out.

56. Exchange transfusion

57. 57
Treatment…
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

58. 58
Treatment…
 Provides the infant is with compatible donor red
cells.
 To give exchange transfusion to an infant clinical
and laboratory findings must be considered.
 Cord Haemoglobin(<10g/dl) and
 raised serum bilirubin are strong indicators for treatment.

59. 59
Treatment…
 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.

60. 60
Phototherapy
 The use of light to degrade bilirubin in mildly
jaundiced infants
 Usually ABO incompatibility

61. 61
Review Questions
1. Compare the causes and laboratory demonstrating
methods, of AIHA with HDN.
2. What is the cause of HDN? What consequences
could result from this condition?
3. What is exchange transfusion?

62. 62
Review…
4. Discuss the dose of IgG anti-D given to Rh-
pregnant women to prevent HGN?
5. When Rh HDN does occur?
6. List and discuss the postnatal laboratory
investigations to be carried out to know the
presence and extent of HDN.

63. References
1.Immunohematology for medical laboratory
science students,Yayehyirad T. and Misganaw
B., Upgraded lecture note.2008
2.Basic and applied concepts of
Immunohematology, 2nd ed. Kathy D.Blaney
and Paula R.Howard,2009
3. Blood banking and transfusion medicine: basic
principles and practice. Christopher D.Hilliyer et
al., 2nd ed.2007.
4.Safe blood donations, Module 1 WHO.2002
63

64. References…
5.Screening for HIV and other infectious agents,
Module 2, WHO. 2002
6.Blood group serology. Module 3 WHO.2002
7.Guidelines and principles for safe blood
transfusion practice, Introductory module. WHO
2002.
8.Immunohematology: Principles and Practice
Quinley. 2nd ed.1998.
9.AABB Technical Manual .15th Edition.2005
64