CHPTER -SIX

Hemolytic Diseases

CH
 Acknowledgements

 Addis Ababa University

 Jimma University
 Hawassa University
 Haramaya University
 University of Gondor
 American Society for clinical Pathology
 Center for Disease Control and Prevention Ethiopia
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
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

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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.

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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

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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.

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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.

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AIHA…

 The warm antibodies are active at 37 0C

 The cold types are optimally active at 2 0C but

with a temperature range which may go as high
as 320C.

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AIHA…

 A positive DAT will be found in both types;

 Inthe cold type , complements rather than the bound

antibody sensitizes the cells and give rise to the
positive DAT

 Inthe warm type, the attached antibody sensitizes the

cells, although occasionally it may be complement.

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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.

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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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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 FetalRBC
Fetal destruction

+ RBC =
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

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 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.

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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.

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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.

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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.

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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.

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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.

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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

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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.

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Etiology of HDFN….

 HDN is often classified into three categories based

on Ab specificity:

 Rh

 ABO and

 Other (non-Rh) Antibodies.

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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.

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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 .

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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.

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HDFN due to RH…
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

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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.

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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.

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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.

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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.

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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.

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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.
 fortesting 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.

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Assessment…

Postpartum testing of infants born to D negative

Mothers

 Infants should be tested for the D Ags, including a

test for weak D.

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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

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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.

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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.

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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.

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 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.

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6.4. Rho (D) Immune Globulin (Human)-RhIG

 Administer
 tonon immunized Rho- negative mothers
 who deliver Rho- positive babies

 Use
 prevent Rh- alloimmunization.

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 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.

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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

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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.

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Fig.6-2
45
Development and prevention of HDN
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.

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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
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.
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
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

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Acid Elution Stain- Purpose

To detect the presence of Hgb F

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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

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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

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 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

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RhIG Dose
 Recommended dose (contained in one vial) is
about 300 µg

 offersprotection 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.

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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.

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Exchange transfusion
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

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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.

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

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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?

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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.

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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
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
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