CHPTER -7

Hemolytic Diseases

CH
6.1 Hemolytic Transfusion reaction (HTR)

 Is a condition in which there is intravascular destruc-

tion 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.
 The warm antibodies are active at 370C
 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 anti-

body 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.

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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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6.3.1 Overview of HDFN

HDFN is a condition in which the red blood cells of a fe-

tus 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 expo-

sure 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 en-
tering the maternal circulation during pregnancy and thus
reduces the chances of Ab production during pregnancy.
 At the time of delivery , a fetal RBCs escape into the ma-
ternal circulation (known as feto maternal hemorrhage
(FMH).
 Immunization can result from fetal RBC exposure follow-
ing:
- Abortion
- Ectopic pregnancy, or
- Abdominal trauma.

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 Etiology of HDFN……
 Antigens on the fetal RBC can stimulate the maternal im-
mune system which results in the production of IgG anti-
bodies .
 In a second(after immunization ) pregnancy the IgG anti-
bodies cross the placental barrier by active transport mecha-
nism.
 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 metabo-
lized 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 in-

creases in an effort to compensate for the RBC de-
structions.

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Etiology of HDFN……
 Erythroblasts are released into the fetal circulation
 Erythroblastosisfetalis

 Thus the greatest threat to the fetus is cardiac failure

resulting from uncompensated anemia.

 Following delivery, red blood cell destruction contin-

ues with the release of indirect bilirubin
 The newborn liver is deficient in glucuronyl trans-
ferase enzymes
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Etiology of HDFN……

 The indirect bilirubin binds to tissues results in jaun-

dice.

 Bind with tissues of the CNS & cause permanent dam-

age (kernicterus)

 resulting in deafness, mental retardation, or death.

13
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 alloimmu-

nized (produce anti- D) after the first D- Positive preg-
nancy.

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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.
 Infants develop signs of
 jaundice,and
 corresponding elevations in bilirubin levels, during the first
few days of life
 Severely affected D- 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 necessi-
tate treatment.
 Mild red blood cell destruction, despite high levels of ma-
ternal antibody occurs because:
 1. 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…
 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.
 2. Others Poor development of ABO Ags on fetal or
infant RBCs .
 Presence of reduced number of A and B Ag sites on fe-
tal 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 of infants born to D negative

Mothers
 It is advantageous to collect a sample of cord blood
from every newborn.

 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 de-
velops signs or symptoms of HDFN.
 The cord blood should be washed free of Wharton’s
jelly.
 Infants should be tested for the D Ags, including a test
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for weak D.
Assessment…

DAT Testing
 In cases of HDFN the DAT may be positive, for Rh-
testing results.

 Its result may be weak, especially in cases of ABO

HDFN.
.

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6.4. Rho (D) Immune Globulin (Human)-RhIG
prophylaxis
 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
 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
 Mother who have an Rh-positive or Du positive new born.

 All Rh-negative women who have abortions are candi-

dates unless the father or fetus is known to be Rh-nega-
tive.

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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
28
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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Detection of Fetomaternal Hemorrhage

1. Rosette Test-Qualitative test

Purpose
 To detect the presence of Rh positive RBCs in the
circulation of Rh negative mother
 to detect FMH > 30 ml
 If FMH <30ml one vial of RHIG required.
 If FMH > 30ml greater than one vials of RHIG re-
quired
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 known 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 pat-
tern, and the suspension is examined microscopically.
Rosette test…

 A Positive test is indicated by the presence of a cer-

tain no of clumps (rosettes) in a defined no of micro-
scopic fields (eg. 7 clumps in 10 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
 Incubate a maternal 3-5% red cell suspension with IgG
anti-D at 37°C. The anti-D will bind to any infant D+
cells that are present.
 After washing to remove unbound anti-D, add indicator
red cells. Indicator cells are treated whith known O ,
R2R2 cells that will bind to the antibody-coated infant
RBCs causing agglutination (“rosettes”) that can be de-
tected microscopically.
 A specified number of agglutinates (e.g 3 clumps in 5
fields or 7 clump more in 10 fields) is designated a posi-
tive and suggests a significant FMH (>30 mL) requiring
more
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RhIG.
cont....
a)negative B)positive
2. Acid Elution Stain- Technique

To detect the presence of Hgb F Quantitative

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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 HbF/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
 ml of 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 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 from
HDFN

 For infants who develop hyperbilirubinemia and/or

anemia due to HDFN, exchange transfusion is usu-
ally carried out.
 The procedure involves slowly removing the person's
blood and replacing it with fresh donor blood.

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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 an-

tibodies

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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 jaun-
diced infants
 Usually ABO incompatibility

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Review Questions

1. Compare the causes and laboratory demonstrating meth-

ods, of AIHA with HDN.
2. What is the cause of HDN? What consequences could re-
sult 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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