CHPTER -NINE

Hemolytic Diseases

CH
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

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
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.

11.1 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
 And others
11.2 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.
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.
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.
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.
11.3 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.
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.
11.3.1 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

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.
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.
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.
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.
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.
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.
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
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.

Etiology of HDFN….

 HDN is often classified into three categories based

on Ab specificity:

 Rh

 ABO and

 Other (non-Rh) Antibodies.

11.3.2.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.
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 .
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.
HDFN due to RH…
11.3.3.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
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.
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.
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.
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.
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.
11.3.4 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.
Assessment…

Postpartum testing of infants born to D negative

Mothers

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

test for weak D.
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
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.
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.
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.
 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.
11.4. Rho (D) Immune Globulin (Human)-RhIG

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

 Use
 prevent Rh- alloimmunization.
 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.
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
RhIG….
 Is a concentrate of IgG anti-D prepared from pools
of human plasma.

 Is given intramuscularly/intravascular
 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.
Fig.6-2 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.
Assessment of RhIG by lab method

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
 Acid Elution Stain
 To detect the presence of Hgb F
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
RhIG…
 The KB test is an acid-elution assay performed on
maternal blood to determine the amount of HbF
that has passed into the maternal circulation.
 The process exposes maternal blood smears to
an acid solution.
 HbF, resistant to the acid, remains intact, whereas
HbA is removed.
 Following this, the smear is stained via Shepard’s
method.
 The fetal red blood cells are left rose-pink in color,
and the maternal cells appear “ghost-like” due to
the absence of staining.
RhIG…
 Though manual detection and quantification had
been widely used, flowcytometry was more
precise and now may be utilized.
 A total of 2000 cells is counted.
 Calculating the fetal to maternal cells percentage
is used to estimate the total amount of FMH.
 The calculation is:
 percentage of fetal cells =
 number of fetal cells X 100 /
 total number of RBCs
 RhIG…
 Calculating the RhIg Dosage
 The following calculations should be used to determine an
additional number of vials of Rho(D) immune globulin needed
if the KB test is positive, indicating a large amount of FMH.
 One vial contains 300 mcg and protects against 30 mL of
fetal blood.
 Volume (mL) of fetal blood = Percentage of fetal cells x 50
 Number of Vials of 300 mcg RhIG required = Volume of fetal
blood/30 mL
 Combining equations: Number of vials* = Percentage of fetal
cells x 50 / 30
RhIG Dose
 If the number to the right of the decimal point is
less than 5, round down and add 1 vial.

 If the number to the right of the decimal point is

greater than or equal to 5, round up and add 1
vial.
 RhIG Dose
 Example 1: If 3.92% of fetal cells are reported on KB,
what dose of RhIG would be required?
 Volume of fetal blood: 3.92 x 50 = 196 mL
 Number of vials = 195 mL/30 = 6.533
 The decimal is greater than 5; therefore, round up and add
1 vial (6+1).
 Answer: 7 vials
 Example 2: If 2.6% of fetal cells are reported on KB, what
dose of RhIG would be required?
 Volume of fetal blood: 2.6% x 50 = 130 mL
 Number of vials = 130 mL/30 = 4.33
 The decimal is less than 5; therefore, round down and add
1 vial (4+0).
 Answer: 4 vials
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?
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.