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 LECTURE NOTES
For Medical Laboratory
Technology Students

1.Immunohaematology

Misganaw Birhaneselassie

Debub University
In collaboration with the Ethiopia Public Health Training Initiative, The Carter Center,
the Ethiopia Ministry of Health, and the Ethiopia Ministry of Education

2004
 Funded under USAID Cooperative Agreement No. 663-A -00-00-0358-00.

Produced in collaboration with the Ethiopia Public Health Training Initiative, The Carter
Center, the Ethiopia Ministry of Health, and the Ethiopia Ministry of Education.

Important Guidelines for Printing and Photocopying

Limited permission is granted free of charge to print or photocopy all pages of this
publication for educational, not-for-profit use by health care workers, students or
faculty. All copies must retain all author credits and copyright notices included in
the original document. Under no circumstances is it permissible to sell or distribute
on a commercial basis, or to claim authorship of, copies of material reproduced
from this publication.

©2004 by Misganaw Birhaneselassie

All rights reserved. Except as expressly provided above, no part of this publication
may be reproduced or transmitted in any form or by any means, electronic or
mechanical, including photocopying, recording, or by any information storage and
retrieval system, without written permission of the author or authors.

This material is intended for educational use only by practicing health care workers
or students and faculty in a health care field.
Immunohaematology

Preface

This Immunohaematology Lecture Note is prepared to meet

the needs of Medical Laboratory professionals and Blood
Bank personnel for a material that comprise the theories and
laboratory techniques concerning blood transfusion service.
The Lecture Note is also important for health professionals in
other disciplines as a reference related to blood transfusion
therapy. In addition, this material alleviates the problems that
have been faced due to shortage of material on the subject
matter as it considers the actual level in most Blood Bank
laboratories in Ethiopia. It further solves the problem of
scarcity of books for the instructors.

The text consists of 10 chapters each of which begins with

specific learning objective. The end of each chapter contains
review questions that are designed to enable the evaluation of
the learner’s comprehension. The first two chapters present
the historical aspects and some background information on
Immunohaematology. Subsequent chapters, provide theories
and pre- transfusion procedures, including haemolytic
diseases. The text is concluded with two chapters that deal
with post transfusion reaction and a brief quality assurance
program in blood banking. Important terms that are used in
the text are defined in “Glossary”

At last, the author will wholeheartedly accept suggestions

from readers to improve the material.

i
Immunohaematology

Acknowledgments

I would like to extend my deepest gratitude to the Carter

Center for supporting the preparation of this Lecture Note. I
am also deeply indebted to a number of colleagues from
DCTEHS and the MLT teaching staffs from different
institutions for their valuable contribution in materialization of
the Lecture Note.

My special thanks go to Ato Gemeda Ayana for his comments

in reviewing this material.

ii
Immunohaematology

Table of Contents
Preface
Acknowledgement
Abbreviations

CHAPTER ONE: INTRODUCTION TO

IMMUNOHEAMATOLOGY
1.1 Historical Overview of Immunohematology
1.2 Blood Group Genetics
1.3 The Role of H-Gene in the Expression of ABO
Genes
1.4 Secretors and Non Secretors

CHAPTER TWO: PRINCIPLES OF ANTIGENS

AND ANTIBODIES
2.1 Antigens
2.2 Antibodies

CHAPTER THREE: THE ABO BLOOD GROUP

SYSTEM
3.1 The Discovery of ABO Blood Group
3.2 Inheritance of The ABO Groups
3.3 The ABO Blood Group
3.4 Antiserum
3.5 Manifestations and Interpretation of Ag-Ab

iii
Immunohaematology

Reaction
3.6 Techniques

CHAPTER FOUR: THE Rh-Hr BLOOD GROUP

SYSTEM
4.1 Historical Background of Rh-Hr Blood Grouping
4.2 Nomenclature & Genetic Theories
4.3 The Antigens of the Rh-Hr Blood Group System
4.4 Variants of Rh Antigen
4.5 Rhesus Antibodies
4.6 The Rh-Hr Blood Grouping Technique

CHAPTER FIVE: THE ANTI- GLOBULIN

TEST (COOMB’S TEST)
5.1 The Direct Anti- Globulin Test (DAT)
5.2 The Indirect Anti- Globulin Test (IAT)

CHAPTER SIX: HAEMOLYTIC DISEASES

6.1 Auto Immune Hemolytic Anemia (AIH)
6.2 Hemolytic Disease of the New Born (HDN)

CHAPTER SEVEN: THE CROSS- MATCH

(COMPATIBILITY TESTING)
7.1 Purpose of Cross-Match
7.2 Types of Cross-Match
7.3 Selection of Blood for Cross-Match

iv
Immunohaematology

7.4 Procedure for Cross-Match

CHAPTER EIGHT: THE DONATION OF BLOOD

8.1 Selection of Blood Donors
8.2 Collection of Blood
8.3 The Anticoagulants and Storage of Blood and
Blood Products
8.4 Potential Hazards During and after Blood
Collection

CHAPTER NINE: THE TRANSFUSION REACTION

9.1 Types of Transfusion Reaction
9.2 Laboratory Tests to be Done When Transfusion
Reaction Occurs

CHAPTER TEN: BASIC QUALITY

ASSURANCE PROGRAM IN BLOOD BANKING

Glossary
Bibliography

v
Immunohaematology

Abbreviations

ACD - Acid citrate dextrose

AHG - Anti human globulin
AIDS - Acquired immuno deficiency syndrome
AIHA - Autoimmune hemolytic anemia
Ab - Antibody
Ag - Antigen
ATP - Adenosine triphosphate
CPD - Citrate phosphate dextrose
CPDA - Citrate phosphate dextrose adenine
DAT - Direct antiglobuline test
2,3, DPG 2,3 diphosphoglycerate
EDTA - Ethyldiamine tetra acetic acid
HCT - Hematocrit
Hgb - Hemoglobin
HDN - Hemolytic disease of new born
HIV - Human immuno virus
Ig - Immunologlobulin
IAT - Indirect antiglobulin test
KB - Kleihaner- Betke
Lab - Laboratory
MW - Molecular weight
NRBC - Nucleated red blood cell
PCV - Packed cell volume
QAP - Quality assurance programme

vi
Immunohaematology

RBC - Red blood cell

Rpm - revolution per minute
Rh - Rhesus
RT - Room temperature
Sp.gr - Specific gravity

vii
 CHAPTER ONE

INTRODUCTION TO
IMMUNOHAEMATOLOGY

Learning Objectives
At the conclusion of the chapter, the student should be able
to:
- Explain a brief history of the science of
Immunohaematology
- Discuss the patterns of inheritance of A and B genes
- Describe the synthesis of H, A and B antigens
- Name the specific transferase for the A, B & H genes
- State the genotype of individuals with the Bombay
phenotype
- State the characteristic genotype of secretor and non-
secretor
- Identify the product or products found in the saliva of
persons of various ABO groups

1.1 Historical Overview of Immunohematology

Immunohematology is one of the specialized branches of

medical science. It deals with the concepts and clinical

1
techniques related to modern transfusion therapy. Efforts to
save human lives by transfusing blood have been recorded for
several centuries. The era of blood transfusion, however,
really began when William Harvey described the circulation of
blood in 1616.

In 1665, an English physiologist, Richard Lower, successfully

performed the first animal-to-animal blood transfusion that
kept ex-sanguinated dogs alive by transfusion of blood from
other dogs.

In 1667, Jean Bapiste Denys, transfused blood from the

carotid artery of a lamb into the vein of a young man, which at
first seemed successful. However, after the third transfusion
of lamb’s blood the man suffered a reaction and died. Denys
also performed subsequent transfusions using animal blood,
but most of them were unsuccessful. Later, it was found that it
is impossible to successfully transfuse the blood of one
species of animal into another species.

Due to the many disastrous consequences resulting from

blood transfusion, transfusions were prohibited from 1667 to
1818- when James Blundell of England successfully
transfused human blood to women suffering from hemorrhage
at childbirth. Such species-specific transfusions (within the

2
same species of animal) seemed to work about half the time
but mostly the result was death.

Blood transfusions continued to produce unpredictable

results, until Karl Landsteiner discovered the ABO blood
groups in 1900, which introduced the immunological era of
blood transfusion. It became clear that the incompatibility of
many transfusions was caused by the presence of certain
factors on red cells now known as antigens. Two main
postulates were also drawn by this scientific approach: 1.
Each species of animal or human has certain factor on the red
cell that is unique to that species, and 2, even each species
has some common and some uncommon factor to each other.
This landmark event initiated the era of scientific – based
transfusion therapy and was the foundation of
immunohematology as a science.

1.2 Blood Group Genetics

Blood group genetics are concerned with the way in which the
different blood groups are inherited, that is passed on from
parents to children.

Chromosomes and Genes: In the human body, the nucleus

of each body cell contains 46 small thread-like structures
called chromosomes, arranged in 23 pairs. The length of each

3
chromosome is divided in to many small units called genes,
which are important as they contain the different physical
characteristics, which can be inherited including those of the
blood groups.

Allomorphic genes (Alleles): Each gene has it own place

called its locus along the length of the chromosome. However,
a certain inherited characteristic can be represented by a
group of genes, and the place or locus can be occupied by
only one of these genes. Such genes are called alleles or
allomorphic genes.

For example, every one belongs to one or other of the

following blood groups: group A, group B, group O or group
AB. Therefore, there are three allelomorphic genes which
make up the ABO Blood group system such as gene A, gene
B, and gene O. Only one of these alleles can occupy the
special place or locus along the chromosomes for this blood
group characteristic.

Body cells and mitosis: When body cells multiply they do so

by producing identical new cells with 46 chromosomes. This
process is called mitosis.

Sex cells and meiosis: When sex cells are formed either
male or female the pairs of chromosomes do not multiply but

4
simply separate so that each of the new cells formed contains
only 23 chromosomes not 46 as in the body cells. This
process is called meiosis.

However, during fortification when the egg and sperm unite,

the fertilized ovum receives 23 chromosomes from each sex
cell half of these from the male and half from the female and
thus will contain 46 chromosomes which again arrange them
selves in pairs in the nucleus.

For example, a child who inherits gene A from its father and
also gene A from its mother would be homozygous, where as
a child who inherits gene A from its father and gene B from its
mother would be heterozygous.

Dominant and recessive genes: A dominant gene will

always show itself if it is present but a recessive gene will only
show itself if there is no dominant one, that is if both genes
are recessive.

For example, in the ABO blood group system the gene A and
B are dominant over gene O. Thus if a child receives from its
parents gene A and O it will belong to group A. In the same
way if a child receives from its parents genes B and O it will
belong to group B only if it receives gene O from both its
parents will it belong to group O.

5
Genotype and phenotype: The genetic composition from a
particular inherited characteristic is called the phenotype and
the way this can be seen is called phenotype. Thus if a person
is group A (phenotype) his phenotype could be either AA or
AO.

1.3 The Role of H-Gene in the Expression of

ABO Genes

Inheritance of A and B genes usually results in the expression

of A and B gene products (antigens) on erythrocytes, but H,A
and B antigens are not the direct products of the H,A, and B
genes, respectively. Each gene codes for the production of a
specific transferase enzyme (Table 1.1), which catalyzes the
transfer of a monosaccharide molecule from a donor
substance to the precursor substance, and enable us to
convert the basic precursor substance to the particular blood
group substance.

Table 1.1 ABH Genes and Their Enzymatic Products

Gene Enzyme
H L- fucosyltransferase
A 3 N-acetyl- D- galactosaminyl transferase
B 3-D- galactosyl transferase
O None

6
- As predicted in Fig 1.1 the H gene (HH/Hh) encodes for
an enzyme, which converts the precursor substance in
red cells in to H substance (H antigen).
- A and B genes encode specific transferase enzymes
which convert H substance in to A and B red cell antigens.
Some H substance remains unconverted (the H
substance is partly converted).
- O gene encodes for an inactive enzyme, which results in
no conversion of the substance in-group O red cells. This
indicates group O individual contains the greatest
concentration of H antigen.
- Persons who do not inherit H gene (very rare hh
genotype) are unable to produce H substance and
therefore even when A and B genes are inherited, A & B
antigens can not be formed . This rare group is referred to
as Oh (Bombay group).

7
HH or
Hh
Genes

Precursor
Substance

hh genes
 A & H antigens

H B & H antigens Substance

AB & H antigens

H antigens

ABO Genes

Unchanged
Precursor
 No ABO or H antigens (Bombay)

Fig 1.1 ABO Genetic pathway

1.4 Secretors and Non-Secretors

The term secretor and non-secretor only refer to the presence

or absence of water- soluble ABH antigen substances in body
fluids (saliva, semen, urine, sweat, tears, etc). Every individual
contains alcohol soluble antigens in body tissues and on the
red cells, whether secretor or non-secretor, but secretors, in
addition to this, possess the water soluble (glycoprotein) form
of antigen, which appears in most body fluids.

8
Majority of the population secrete water- soluble substances
in saliva and most other body fluids that have the same
specificity as the antigens on their red cells.

The production of A, B & H antigens in saliva is controlled by

a secretor gene, which is in herited independently of the ABO
and H genes. The relevant gene is called Se, and its allele
which amorphic is se. At least one Se gene (genotype SeSe
or Sese) is essential for the expression of the ABH antigens in
secretors. Individual who are homozygous for se (sese) do not
secrete H,A, or B antigens regardless of the presence of H,A
or B genes.

The Se gene does not affect the formation of A,B or H

antigens on the red cells or in hematopoietic tissue, which are
alcohol soluble and which are not present in body secretions.
Oh (Bombay) individuals do not secrete A, B or H substance,
even when the Se gene is present.

9
Review Questions

1. Briefly outline the historical background of blood

transfusion.
2. What was the reason for the failure of attempted intra and
inter species blood transfusions (relate this with the
discovery of blood group by Karl Landsteiner).
3. Define the following terms:
A. Chromosome
B. Gene
C. Dominant gene
D. Phenotype
E. Secretors
4. Explain why group O individuals contain the greatest
concentration of H antigen.

10
 CHAPTER TWO

PRINCIPLES OF ANTIGENS AND

ANTIBODIES

Learning Objectives

At the conclusion of the chapter, the student should be able

to:
- Define an antigen
- Explain the basic essential for antigenic substances
- Define an antibody
- List the classes of immunoglobulin
- Compare the characteristics of IgG, IgM and IgA
- Contrast between the natural and immune antibodies
- Explain the non- red cell- immune antibodies

2.1 Antigens

An antigen can be defined as any substance which, when

introduced in to an individual who himself lacks the substance,
stimulates the production of an antibody, and which, when
mixed with the antibody, reacts with it in some observable
way.

11
Foreign substances, such as erythrocytes, can be
immunogenic or antigenic (capable of provoking an immune
response) if their membrane contains a number of areas
recognized as foreign. These are called antigenic
determinants or epitopes.

The immunogenicity of a substance (relative ability of a

substance to stimulate, the production of antibodies when
introduced in to a subject lacking the substance) is influenced
by a number of characteristics:

Foreignness: The substance should present, at least in part,

a configuration that is unfamiliar to the organism. The greater
the degree the antigenic determinant is recognized as non-
self by an individual’s immune system, the more antigenic it is.

Molecular weight: The antigen molecule must have a

sufficiently high molecular weight. The larger the molecule,
the greater is its likelihood of possessing unfamiliar antigenic
determinant on its surface, and hence the better the molecule
functions as an antigen.

Molecules with a molecular weight of less than 5000 fail to act

as antigen, with 14,000 are poor antigens unless conjugated
with adjuvant and with 40,000 or more are good antigens.
High MW molecules of 500,000 or more are the best antigens.

12
However, physical size of the molecule is not a controlling
factor. Since dextran (a carbohydrate) with a MW of 100,000
is not antigenic.

Structural stability: Structural stability is essential

characteristic; structurally instable molecules are poor
antigens, eg. Gelatin.

Structural complexity: The more complex an antigen is, the

more effective it will be complex proteins are better antigens
than large repeating polymers such as lipids, carbohydrates,
and nucleic acid, which are relatively poor antigens.

Route of administration: In general, intravenous (in to the

vein) and intraperitoneal (into the peritoneal cavity) routes
offer a stronger stimulus than subcutaneous (beneath the
skin) or intramuscular (in to the muscle) routes.

2.2 Antibodies

Antibodies are serum proteins produced in response to

stimulation by a foreign antigen that is capable of reacting
specifically with that antigen in an observable way. Five major
immunoglobulin (Ig) classes exist; which are called IgG, IgA,
IgM, IgD and IgE, with heavy chains gamma (γ) alpha (α), mu
(µ) delta(δ ) , and epsilon(Є) respectively. Each is unique and

13
possesses its own characteristic. Blood group antibodies are
almost exclusively IgG, IgM and IgA.

Characteristics of immunoglobulin

IgG:
- Is the predominant immunoglobulin in normal serum,
accounting for about 85% of the total immunoglobulin
- Is the only immunoglobulin to be transferred from mother
to fetus, through the placenta, a fact that explains its role
in the etiology of hemolytic disease of the new born (HDN)

- Is the smallest antibody which has a MW of 150,000

- Is capable of binding complement
- Is predominantly produced during the secondary immune
response.

Sub classes of IgG: within the major immunoglobulin classes

are variants known as sub classes. Four sub classes of IgG
have been recognized on the basis of structural and
serological differences and are known as IgG1, IgG2, IgG3 and

IgG4. They also have different characteristics as shown in

Table 2.1.

14
Table 2.1. IgG subtype characteristics

Characteristic IgG1 IgG2 IgG3 IgG4

% of total lgG in
65 25 6 4
serum
Complement
4+ 2+ 4+ +/-
fixation
Half-life in days 22 22 8 22
Placental
Yes Yes Yes Yes
passage
Some Immune Immune
specificities Anti-Rh Anti-A Anti-Rh Anti-A
Anti-B Anti-B

IgM:
- Accounts for about 10% of the immunoglobulin pool, with
a concentration of about 1.0 g/l in normal serum.
- Is the predominant antibody produced in a primary
immune response
- Is structurally composed of five basic subunit
(pentameric), and has the largest MW of 900,000.
Because of its large size IgM cannot pass the placental
barrier to the fetus
- Is complement binding

15
IgA:
- Ig A with a MW of 160,000 constitutes 10 to 15 % of the
total circulatory immunoglobulin pool.
- Is the predominant immunoglobulin in secretions such as,
tears, saliva, colostrum, breast milk, and intestinal
secretions.
- Does not fix complement and is not transported across
the human placenta.

2.2.1 Types of Antibodies

Based on their development, blood group antibodies are

classified into Natural and Immune antibodies.

Natural antibodies: are red cell antibodies in the serum of an

individual that are not provoked by previous red cell
sensitization. But, it is believed that these antibodies must be
the result of some kind of outside stimulus and the term
naturally occurring gives an inaccurate connotation, so they
are called non- red cell or non- red cell immune antibodies.

Characteristics
- Exhibit optimum in vitro agglutination when the antigen
bearing erythrocytes are suspended in physiologic saline
(0.85%) sodium chloride, sometimes referred to as
complete antibodies.

16
- Give optimum reaction at a temperature of room or lower,
and they are also called cold agglutinins.
0
These antibodies do not generally react above 37 C that
is at body
temperature, for this reason most of these do not
generally give rise
to transfusion reactions.
These antibodies are of high MW that they can’t cross the
placental barrier, eg. IgM.

Immune antibodies: are antibodies evoked by previous

antigenic stimulation either by transfusion or pregnancy, i.e.
as a result of immunization by red cells.

Characteristics
- Do not exhibit visible agglutination of saline- suspended
erythrocytes, and called incomplete antibodies
0
- React optimally at a temperature of 37 C, and are so
called warm agglutinins.
These antibodies obviously have more serious transfusion
implications than the naturally occurring ones.
- These antibodies are so small that they can cross the
placental barrier, e.g. IgG

17
Review Questions

1. Define:
A. Antigen
B. Antibody
C. Immunogenicity
2. Identify some characteristics of the IgG subtypes
3. What are the characteristic differences between Natural
and Immune antibodies?
4. Which classes of antibodies predominate during the
A. Primary immune response?
B. Secondary immune response?

18
 CHAPTER THREE

THE ABO BLOOD GROUP SYSTEM

Learning Objectives

At the end of the chapter the student should be able to:

- Describe the history of the discovery of the ABO system
- Discuss the patterns of inheritance of A and B genes
- Contrast the antigens & antibodies found in the blood in
the ABO system
- Define antiserum and its acceptance criteria for laboratory
work
-
Explain the method of grading the strength of
agglutination reactions
- Name the methods commonly used in routine blood
banking to enhance the agglutination of erythrocytes
- Prepare different percentage of red blood cells
suspensions
- Perform ABO blood grouping using different methods
- Discuss some of the result discrepancies that can be
encountered in ABO grouping

19
3.1 The Discovery of ABO Blood Group

In the 1900, a German Scientist Karl Landsteiner established

the existence of the first known blood group system, the ABO
system. Classification of the blood group was based on his
observation of the agglutination reaction between an antigen
on erythrocytes and antibodies present in the serum of
individuals directed against these antigens. Where no
agglutination had occurred, either the antigen or the antibody
was missing from the mixture.

Landsteiner recognized the presence of two separate

antigens, the A & B antigens. The antibody that reacted with
the A antigens was known as anti A, and the antibody that
reacted with the B antigen was known as anti B. Based on the
antigen present on the red cells, he proposed three separate
groups A, B & O. Shortly hereafter, von Decastello and Sturli
identified a fourth blood group AB, by demonstrating
agglutination of individuals red cells with both anti-A and anti-
B.

3.2 Inheritance of the ABO Groups

In 1908, Epstein and Ottenberg suggested that the ABO blood

groups were inherited characters. In 1924 Bernstein
postulated the existence of three allelic genes. According to

20
the theory of Bernstein the characters A,B and O are inherited
by means of three allelic genes, also called A,B and O . He
also proposed that an individual inherited two genes, one from
each parent, and that these genes determine which ABO
antigen would be present on a person’s erythrocytes. The O
gene is considered to be silent (amorphic) since it does not
appear to control the development of an antigen on the red
cell. Every individual has two chromosomes each carrying
either A, B or O, one from each parent, thus the possible ABO
genotypes are AA, AO, BB, BO, AB and OO. ABO typing
divides the population in to the four groups, group A, B, O
and, AB, where the phenotype and the genotype are both AB
(heterozygous), see Table 3.1.

Table 3.1 The ABO phenotypes and their corresponding

genotypes

Phenotypes Genotypes
A AA
AO
B BB
BO
O OO
AB AB

To illustrate the mode of inheritance, a particular mating, that

in which a group A male mates with a group B female, is

21
considered. The group A male may be of genotype AA or AO
and similarly the group B female may be of the genotype BB
or BO; therefore within this one mating four possibilities exist,
namely (a) AA with BB, (b) AA with BO, (c) AO with BB and
(d) AO with BO, see Table 3.2.

- This mating can result in children of all four ABO groups

or phenotypes although it is only in mating AO with BO
that children of all four ABO groups can occur in the same
family.
- This mating also shows that a knowledge of the groups of
relatives will sometimes disclose the genotype of group A
or group B individuals, eg. the finding of a group O child in
an AxB mating demonstrates the presence of the O gene
in both parents, and it follows that any A or B children
from this particular mating are heterozygous , i.e. AO or
BO.

22
Table 3.2 The ABO mating with possible genotype and
phenotype of children.

Mating Children
Phenotypes Genotypes Genotypes Phenotypes
AxA (1)AAxAA (1)AA A and O
(2)AAxAO (2)AA and AO
(3) AOxAO (3)AA,AO and OO
AxB (1)AAxBB (1)AB A,B AB, and O
(2)AAxBO (2)AB and AO
(3)AOxBB (3)AB and BO
(4)AOxBO (4)AB,BO, AO, and
OO
AxAB (1)AAxAB (1)AA and AB A,B and AB
(2)AOxAB (2)AB, AO,BO and
OO
AxO (1)AAxOO (1)AO A and O
(2)AOxOO (2)AO and OO
BxB (1)BBxBB (1)BB B and O
(2)BBxBO (2)BB and BO
(3)BOxBO (3)BB,BO, and BO
BxAB (1)BBxAB (1)AB and BB A,B, and AB
(2)BOxAB (2)AB,BB, AO, and
BO
BxO (1)BBxOO (1)BO B and O
(2)BOxOO (2)BO and OO
ABxAB (1)ABxAB (1)AA,AB and BB A,B, and AB
ABxO (1)ABxOO (1)AO and BO A and B
OxO (1)OOxOO (1)OO O

In1930 Thompson proposed a four allele theory of inheritance

based on the discovery of von Dungern and Hirszfeld in 1911,
which demonstrated that the A antigen could be divided in to

23
A1 and A2 sub groups. Thompson’s four-allele theory
encompassed the four allelic genes, A1, A2, B and O. This
four allelic genes give rise to six phenotypes: A1, A2, B, O,
A1B and A2B and because each individual inherits one
chromosome from each parent, two genes are inherited for
each characteristic and these four allelic gene give rise to ten
possible genotypes (table 3.3).

Table 3.3 ABO phenotypes and genotypes, including A1 and

A2

Phenotypes Genotypes
A1 A1A1
A1A2
A1O
A2 A2A2
A2O
B BB
BO
A1B A1B(or A1B/O)
A2B A2B(orA2B/O)
O OO

In group AB, the A gene is normally carried on one

chromosome and the B gene on the other, each being co-
dominant, although rare families have been described in

24
which both A and B have been shown to be inherited from one
parent, this condition is called Cis- AB . In serological testing,
individuals of this type have a weaker B antigen and possess
some kind of anti- B in the serum.

Table 3.4 shows the six possible genotype mating included in

the one phenotype mating A1 x B together with the
phenotypes which can be found among the offspring of each
mating.

Table 3.4 The mating A1xB.

Mating possible Possible

Genotypes phenotypes of
children

A1A1xBB A1B
A1A1xBO A1B,A1
A1OxBB A1B,B
A1OxBO A1,B,A1B,O
A1A2xBB A1B,A2B
A1A2xBO A1,A2,A1B,A2B

Sometimes by studying the phenotypes of the children it is

possible to say which genotype the parents belong. For
example, it can be seen that for the matings A1xB, A2 and A2
B children never occur in the same family as B or O children.

25
This follows that taking all A1xB mating together, all six
phenotypes can occur. However, the finding of, for instance, a
group O child in a family where other children are A2 and A2 B
would not be possible if they all had the same parents.

3.3 The ABO Blood Group

A person’s ABO blood group depends on the antigen present

on the red cells.
- Individuals who express the A antigen on their red cell i.e.
their red cells agglutinate with anti - A belong to group A.
- Individuals who express the B antigen on their red cells
i.e. their red cells agglutinate with anti-B belong to group-
B.
- Individuals who lack both the A and B antigen on their red
cells that is their red cell show no agglutination either with
anti- A or anti- B belong to group O.
- Individuals who express both A and B antigens on their
red cells that is their red cells show agglutination with both
anti- A and anti –B belong to group AB.

The distribution of ABO blood groups differ for various

population groups, different studies have provided statistics as
given in table 3.5

26
Table 3.5 Frequency of ABO blood groups in different
population

Examples A% B% AB% O%
Asian 28 27 5 40
African 26 21 4 49
Nepalese 33 27 12 28
Caucasian 40 11 4 45

Ethiopians 31 23 6 40
(Blood donors)

Whenever an antigen A and, or B is absent on the red cells,

the corresponding antibody is found in the serum (Table 3.6)
- Individuals who possess the A antigen on their red cells
possess anti- B in their serum.
- Individuals who possess the B antigen on their red cells
possess anti A in their serum.
- Individuals who possess neither A nor B antigen have
both anti A and anti- B in their serum.
- Individuals with both A and B antigens have neither anti A
nor anti B in their serum.

27
Table 3.6. Classification of the ABO blood groups

Antigen on Red Antibodies in Serum Blood Group

Cells
A Anti-B A
B Anti-A B
Neither A nor B Anti-A and Anti-B O
A and B Neither anti-A nor AB
anti-B

3.4 Antiserum

An antiserum is a purified, diluted and standardized solution

containing known antibody, which is used to know the
presence or absence of antigen on cells and to phenotype
once blood group.

Antiserum is named on the basis of the antibody it contains:

- Anti- A antiserum which contains anti- A antibody
- Anti- B antiserum which contains anti- B antibody
- Anti- AB antiserum, which contain both anti A and B
antibodies.
- Anti –D antiserum which contains anti- D antibody

28
Sources of antisera
- Animal inoculation in which animals are deliberately
inoculated by known antigen and the resulting serum
containing known antibody is standardized for use as
antiserum.
- Serum is collected from an individual who has been
synthesized to the antigen through transfusion, pregnancy
or injection.
- Serum collected from known blood groups

Antisera requirements: Antiserum must meet certain

requirements to be acceptable for use. In using antisera the
manufacturer’s instruction should always be followed. The
antiserum has two be specific: does not cross react, and only
reacts with its own corresponding antigen, avid: the ability to
agglutinate red cells quickly and strongly, stable: maintains it
specificity and avidity till the expiry date. It should also be
clear, as turbidity may indicate bacterial contamination and
free of precipitate and particles. It should be labeled and
stored properly.

3.5 Manifestation and Interpretation of

Antigen- Antibody reactions

The observable reactions resulting from the combination of a

red cell antigen with its corresponding antibody are

29
agglutination and/ or haemolysis. Agglutination is the widely
observed phenomenon in blood grouping.

Agglutination: is the clumping of particles with antigens on

their surface, such as erythrocytes by antibody molecules that
form bridges between the antigenic determinants. When
antigens are situated on the red cell membrane, mixture with
their specific antibodies causes clumping or agglutination of
the red cells.

An agglutination in which the cells are red cells synonymously

called hemagglutination. In hemagglutination the antigen is
referred to as agglutinogen and the antibody is referred to as
agglutinin.

The agglutination of red cells takes place in two stages. In the

first stage- sensitization, antibodies present in the serum
become attached to the corresponding antigen on the red cell
surface. A red cell, which has thus coated by antibodies is
said to be sensitized. In the second stage, the physical
agglutination or clumping of the sensitized red cells takes
place, which is caused by an antibody attaching to antigen on
more than one red cell producing a net or lattice that holds the
cells together. The cells form aggregates, which if large
enough, are visible to the naked eye. There are also degrees

30
of agglutination which can not be seen without the aid of a
microscope.

The strength of an agglutination reaction can be indicated by

the following grading system (Fig. 3.1 a-f), as recommended
by the American Association of Blood Banks.

(4+) one solid aggregate;

With no free cells
clear supernatant
Fig. 3.1a

(3+) several large aggregates;

Few free cells
Clear supernatant
Fig 3.1b

(2+) Medium sized

aggregate Some free cells
Clear supernatant
Fig 3.1 c

31
(1+) Small aggregates
Many free cells
Turbid reddish supernatant
Fig 3.1 d

(Weak +) Tiny aggregate

many free cells
turbid reddish supernatant
Fig 3.1e

(Negative) No aggregates,
red blood cell all intact.
Fig3.1f

Hemolysis: is the break down or rupture of the red cell

membrane by specific antibody (hemolysin) through the
activation of complement with the release of hemoglobin, and
the librated hemoglobin can easily be observed staining the
supernatant fluid.

32
3.6 Techniques:

Determination of ABO grouping is important in pretrarsfusion

studies of patients and donors as well as in cases of obstetric
patients. There are different technique to determine ABO
grouping in the laboratory: slide, test tube & microplate. In
each technique results are interpreted based on the presence
or absence of agglutination reaction. Agglutination reaction is
interpreted as a positive (+) test result and indicates, based on
the method used, the presence of specific antigen on
erythrocytes or antibody in the serum of an individual. No
agglutination reaction produces a negative (-) test indicating
the absence of specific antigens on erythrocytes or antibody
in the serum of an individual.

3.6.1 Rules for Practical Work

- Perform all tests according to the manufacturer’s direction

- Always label tubes and slides fully and clearly.
- Do not perform tests at temperature higher than room
temperature.
- Reagent antisera should be tested daily with erythrocytes
if known antigenicity. This eliminates the need to run
individual controls each time the reagents are used.
- Do not rely on colored dyes to identify reagent antisera.
- Always add serum before adding cells.

33
- Perform observations of agglutination against a well –
lighted background, and record results immediately after
observation.
- Use an optical aid to examine reactions that appear to the
naked eye to be negative.

3.6.2 The Right Conditions for RBCs to Agglutinate

The correct conditions must exist for an antibody to react with

its corresponding red cell antigen to produce sensitization and
agglutination of the red cells, or hemolysis. The following
factors affect the agglutination of RBCs:

Antibody size: normally, the forces of mutual repulsion keep

the red cells approximately 25 nanometer apart. The
maximum span of IgG molecules is 14 nanometer that they
could only attach the antigens, coating or sensitizing the red
cells and agglutination can not be effected in saline media. On
the other hand, IgM molecules are bigger and because of their
pentameric arrangement can bridge a wider gap and
overcome the repulsive forces, causing cells to agglutinate
directly in saline.

34
pH: the optimum PH for routine laboratory testing is 7.0.
Reactions are inhibited when the PH is too acid or too
alkaline.

Temperature: The optimum temperature for an antigen-

antibody reaction differs for different antibodies. Most IgG
0
antibodies react best at warm temperature(37 C) while IgM
antibodies, cold reacting antibodies react best at room
0
temperature and coldest temperature(4 to 22 C).

Ionic strength: lowering the ionic strength of the medium

increases the rate of agglutination of antibody with antigen.
Low ionic strength saline (LISS) containing 0.2% NaCl in 7%
glucose is used for this purpose rather than normal saline.

Antibody type: Antibodies differ in their ability to agglutinate.

IgM antibodies, referred to as complete antibodies, are more
efficient than IgG or IgA antibodies in exhibiting in vitro
agglutination when the antigen - bearing erythrocytes are
suspended in physiologic saline.

Number of antigen sites: Many IgG antibodies of the Rh

system fail to agglutinate red cells suspended in saline,
however IgG antibodies of the ABO system (anti-A & anti-B)
agglutinate these red cells, because there are many A&B

35
antigen sites (100 times more than the number of Rh sites)
than the D site on the cell membrane of erythrocytes.

Centrifugation: centrifugation at high speed attempts to over

come the problem of distance in sensitized cells by physically
forcing the cells together.

Enzyme treatment: treatment with a weak proteolytic

enzymes (eg. Trypsin, ficin, bromelin, papain) removes
surface sialic acid residue- by which red cells exert surface
negative charge, thereby reducing the net negative charge of
the cells, thus lowering the zeta potential, and allowing the
cells to come together for chemical linking by specific antibody
molecules. However, enzyme treatment has got a
disadvantage in that it destroys some blood group antigens.

Colloidal media: certain anti-D sera especially some IgG

antibodies of the Rh system would agglutinate Rh positive
erythrocytes suspended in colloid (bovine albumin) if the zeta
potential is carefully adjusted by the addition of the colloid.

Ratio of antibody to antigen: There must be an optimum

ratio of antibody to antigen sites for agglutination of red cells
to occur. In prozone phenomena (antibody excess), a surplus
of antigens combining site which are not bound to antigenic
determinants exist, producing false- negative reactions. These

36
can be over come by serially diluting the anti body containing
serum. It is also important to ensure that the red cell
suspension used in agglutination test must not be too week or
too strong, as heavy suspension might mask the presence of
a weak antibody.

3.6.3 Preparation of Red Cell Suspension

The concentration of erythrocytes in a saline suspension is

important to the accuracy of testing in the blood bank. Red
cell suspension can be prepared directly from anticoagulated
blood or from packed red cell (after separating the serum or
plasma). Proper concentration of suspensions can be
prepared visually as experience allows; however, as a student
you should follow the following procedures. The procedures
include a red blood cell washing step to remove certain
impurities; and when necessary you can use this formula to
prepare different red cell concentrations.

% Required = Packed cell volume x 100

Volume of suspension required

Procedure: (as an example preparation of 2% red blood cell

suspension of 10 ml volume)
1. Place 1 to 2ml of anticoagulated blood in a test tube
2. Fill the tube with saline and centrifuge the tube

37
3. Aspirate or decant the supernatant saline.
4. Repeat (steps 2 and 3) until the supernatant saline is
clear
5. Pipette 10 ml of saline in to another clean test tube.
6. Add 0.2 ml of the packed cell button to the 10 ml of saline
7. Cover the tube until time of use. Immediately before use,
mix the suspension by inverting the tube several times
until the cells are in suspension.

3.6.4 The ABO Blood Grouping

The ABO blood groups (A, B, AB, &O) represent the antigen
expressed on the erythrocytes of each group, and whenever
an antigen (A and / or B) is absent on the red cells, the
corresponding antibody is found in the serum. Based on the
above facts, an individual’s unknown ABO blood group is
performed by two methods in the laboratory: Forward (Direct)
and Reverse (Indirect) grouping. Reverse grouping is a cross
check for forward typing. Both tests should be done side by
side and the results of both methods should agree.

3.6.4.1 The Direct ABO Blood Grouping

The direct blood grouping also called cell grouping employs

known reagent anti sera to identify the antigen present or their

38
absence on an individual’s red cell.It can be performed by the
slide or test tube method.

Slide method
1. Make a ceramic ring on the slide.
2. Label one ring as anti- A and the other ring as anti-B
3. Add anti- serum to the ring labeled anti-A
4. Add anti-B serum to the ring labeled anti-B
5. Add 10% unknown cell suspension to both rings
6. Mix using a separate applicator stick.
7. Observe the reaction within 2 minutes by rotating the slide
back and forth
8. Interpreter the results: Look at Table 3.7

Test tube method:

1. Take two tubes, label one tube ‘anti- A’ and the second ‘
anti -B’
2. Add one drop of anti- A serum to the tube labeled ‘anti-A’
and one drop of anti- B to the tube labeled anti- B’
3. Put one drop of the 2-5% cell suspension to both tubes
4. Mix the antiserum and cells by gently tapping the base of
each tube with the finger or by gently shaking
5. Leave the tubes at RT for 5- minutes. Centrifuge at low
speed (2200-2800 rpm)for 30 seconds

39
6. Read the results by tapping gently the base of each tube
looking for agglutination or haemolysis against a well-
lighted white background.
7. Interpret the results as presented on Table 3.7.

Table 3.7 Reactions of patient Erythrocytes and known

Antisera

RED CELLS TESTED BLOOD GROUP

WITH INTERPRETATION
ANTI- A ANTI- B
Positive Negative A
Negative Positive B
Positive Positive AB
Negative Negative O

3.6.4.2 The Indirect ABO Blood Grouping

The indirect blood grouping, also called serum grouping

employs red cells possessing known antigen to see the type
of antibodies (anti A & -B) present, or absence of these
antibodies in serum. It usually is performed by test tube
method alone. Slide reverse grouping is not reliable as serum
antibodies agglutinate most cell samples when centrifuged,
and use of test tube enhances the agglutinated reaction.

40
Test tube method
1. Take two tubes, label one tube A- Cells’ and the second
‘B cells’
2. Put one drop of the serum to be tested each tube.
3. Add one drop of 2-5% A cells to the tube labeled ‘A cells’
and one drop of 2-5% B cells to the tube labeled ‘B cells’.
4. Mix the contents of the tubes.
5. Leave the tubes at RT for 5- minutes. Centrifuge at low
speed (2200-2800 rpm) for 30 seconds.
6. Read the results by tapping gently the base of each tube
looking for agglutination or haemolysis against a well-
lighted white background.
7. Interpretation of results: look at table 3.8

Table 3.8 Reactions of patient serum and reagent

erythrocytes

SERUM TESTED WITH BLOOD GROUP

A cell B cell INTERPRETATION
Negative Positive A
Positive Negative B
Negative Negative AB
Positive Positive O

41
3.6.5 Anomalous Results in ABO Testing

Technical errors and various clinical conditions can contribute

to a discrepancy between erythrocyte and serum results in
ABO grouping. Most ABO discrepancy’s however, are
technical in nature, and can be resolved by careful repeating
of the test procedure. These include: contaminated reagents
or dirty glass ware, over centrifugation, incorrect serum: cell
ratio, under centrifugation or incorrect incubation temperature,
failure to add test specimen or reagents, and the like. If
carefully controlled repeat testing yields the same
agglutination patterns, the variation can be assigned to one of
the following four categories.

1. Missing or weak reacting antibodies

Age: testing of infants who have not begun to produce their

own antibodies, or who possess antibodies that have been
passively acquired from the mother, or during testing of
elderly persons whose antibody levels have declined.

Hypogamaglobulininemia: in conditions in which

hypogamaglobulininemia may be demonstrated, these include
lymphomas, leukemias, immunodeficiency disorders, use of

42
immunosuppressive drugs, and following bone marrow
transplantation.

Resolution: Enhancing reaction in reverse grouping by

incubating of patients serum with the red cells at room
0 0
temperature for 15 min or incubation at 16 C or 4 C for 15
min.

2. Missing weak antigens

Sub groups of A or B antigens: The A or B antigens may be

weakly expressed because of an unusual genotype (i.e, sub
groups of A&B).

Disease: In some conditions like acute leukemias, the red cell

antigens in the ABO system may be greatly depressed that
they give weak reactions.
Blood group specific substances: in conditions like ovarian
cyst & carcinomas, blood group specific substance may be of
such high concentration is that anti-A & and – B are
neutralized when unwashed cells are used.

Acquired B antigen: effect of bacterial enzymes & absorption

of bacterial polysaccharide on to the red cells of group A or O
patients results in B specificity which involve weak B antigen
reaction in the forward grouping.

43
Additives to sera: acriflavin, the yellow dye used in some
commercial anti B reagents, can produce false agglutination in
some persons, which results from antibodies against acriflavin
in the serum combining with the dye and attaching to the
erythrocytes of the individual.

Mixtures of blood: Mixture of cell types in recently transfused

patients or recipients of bone marrow transplants can produce
unexpected reactions in forward typing.

Resolution:
- Investigating the possibility of sub groups of A&B
- Investigating the diagnosis
- Washing the patient’s red cells in saline to eliminate the
problem with blood group specific substances.
- Acidifying the anti- B reagent to PH 6.0 to rule out
acquired B and then determining secretor status
- Washing the patient’s cells three times and then
regrouping if dye is suspected as the problem or using
reagents that do not contain dye.

3. Additional antibody

Autoantibody: cold autoantibodies can cause spontaneous

agglutination of the A and B cells used in reverse grouping.
Patients with warm autoimmune hemolytic anemia may have

44
red cells coated with sufficient antibody to promote
spontaneous agglutination.

Anti A1: A2 & A2 B individuals may produce naturally

occurring anti-A1, which cause discrepant ABO typing.

Irregular antibodies: Irregular antibodies in some other blood

group system may be present that react with antigens on the
A or B cells used in reverse grouping.

Resolution:
0
- Washing the patient red cells in warm (37 C) saline to
establish cold autoantibodies as the cause.
- Treating cells with chloroquire diphosphate to eliminate
bound antibodies if warm autoantibody is suspected.
- Identifying the irregular antibody, and using A & B cells,
which are negative for the corresponding antigen.

4. Plasma Abnormalities

Increased gamma globulin: elevated levels of globulin from

certain disease states such as multiple myeloma result in
rouleaux formation.

45
Abnormal proteins: Abnormal proteins, altered proportions
of globulins, and high concentration of fibirogen may cause
rouleaux formation, which could be mistaken for agglutination.

Wharton’s jelly: when cord blood is used, reverse grouping

may be affected by wharton’s jelly which causes rouleaux.

Resolution: wash the patients cells with saline or to add a

drop of saline to the test tube is sufficient to remove proteins
that cause rouleaux

46
Review Questions

1 Briefly discuss on the discovery of the ABO blood group

system
2. Classify the ABO blood group system based on the
antigen and antibodies present in an individual
3. Give a description for grade of agglutination reaction as
recommended by the America blood bank society.
4. List conditions that influence agglutination of red cells
5. Describe how to prepare a 10 ml volume of 5% red cell
suspension
6. Discuss how to perform direct & indirect method of ABO
blood typing
7. Discuss conditions that lead to anomalous results in ABO
testing

47
 CHAPTER FOUR

THE Rh-Hr BLOOD GROUP

SYSTEM

Learning Objectives

At the conclusion of the chapter the student should be able to:

- Describe the historical background of the Rh system.
- Compare the genetic inheritance and nomenclature of the
Rh antigens in Wiener and Fisher- Race theories
- List the most common Rh antigens including their
characteristics
u
- Discuss on the common variants of the D antigen (D ),
including the clinical significance.
- Discuss the characteristics and clinical significance of Rh
antibodies.

- Describe and perform the techniques used in Rh

antigen detection in routine laboratory

4.1 Historical Background of Rh-Hr Blood

Grouping

In 1940 Landsteiner & Wiener reported the discovery of a

human blood factor, which they called rhesus. They

48
immunized guinea pigs and rabbits with blood from the
Macacus rhesus monkey, and the antiserum obtained
agglutinated not only the red cells of the rhesus monkey but
also 85% of humans. They realized that this serum which they
called anti-Rh was about detecting an unknown human blood
group antigen which, independent of all other blood groups
discovered before that time. They used it to type as Rh
positive those donors whose red cells were agglutinated by
the new antibody and as Rh negative to those whose red cells
were not so agglutinated.

This discovery followed the detection of an antibody by Levine

& Stetson in 1939. This antibody occurred in the serum of a
woman delivered a stillborn fetus, who suffered a hemolytic
reaction to her husband’s ABO compatible blood transfused
shortly after delivery. The antibody was found to agglutinate
approximately 80% of randomly selected ABO compatible
donor’s and latter was shown to be anti-Rh in specificity.
Levine and Stetson also postulated that the antibody had
arisen as the result of immunization of the mother by a fetal
antigen which had been inherited from the father.

In 1940, Wiener & Peters showed that the antibody anti- Rh

could be found in the serum of certain individuals who had
had transfusion reaction following ABO group- compatible
transfusions. In 1941 Levine & his Co- workers showed that

49
not only could an Rh negative mother become immunized to
an Rh positive fetus in utero but also that the antibody could
then traverse the placenta and give rise to erythroblastosis
fetalis (HDN).

Later work demonstrated that the animal or rabbit anti-Rhesus

and human anti-Rh are not the same, and were not detecting
the same antigen but the system had already named the
human antibody as anti-Rh. The animal anti-hesus was
detecting another antigen possessed by Rh positive & Rh
negative persons but in much greater amount in Rh positives.
Therefore the animal antibody was renamed anti- LW after
Landsteiner & Wiener who discovered it, and the human
antibody retained the title anti Rh.

4.2. Nomenclature & Genetic Theories

Fisher- Rase Nomenclature

The Fisher- Race theory states that there are three closely
linked loci, each with one of the set of allelic gene (D& d, C &
c, E &e) and these three genes are inherited as a complex.
These three loci are believed to be so closely linked that
crossing over occurs only very rarely.

Complex Rh genes control the Rh antigens, these genes are

C, D, E, c, d & e. The Rh antigens are therefore named C, D,
E, c, d &e. The antigen d (and anti- d) do not exist, the symbol

50
“d” is used to express the absence of D.The Rh gene complex
possesses closely linked genes (antigens) which could be
assembled in eight different ways: CDe, cDE, cde, cDe, cdE,
Cde, CDE & CdE. Because of the strong antigenic characters
of D, all individuals who lack the D antigen are said to be Rh
negative regardless of whether the C or E antigen or both are
present.

Wiener nomenclature
Wiener’s theory states one gene instead of three closely
linked ones, produces one complex antigen which is made up
of three factors found on the red cells. There are eight genes
1 2 0 z y
called R ,R ,r,R ,r’,r”,R &r . Comparison of nomenclature is
presented in Table 4.1 and 4.2.

Eg. Gene Antigen factor on the red cell

’ ”
R’ Rh1 rh , Rho & hr
Table 4.1 Comparison of Nomenclature of antigens of the Rh
system

Wiener Fisher- Race Rosenfield

Rho D Rh1
’
rh C Rh2
’’
rh E Rh3
’
hr c Rh4
’
hr e Rh5

51
Table 4.2 Comparison of Fisher- Race & Wiener
nomenclature

Fisher- Race Wiener

’, ’’
CDe Rh1 (rh Rho,hr )
’, ’’
c DE Rh2 (hr Rho,rh )
’’, ’’
c de rh (hr hr )
1 ’, ’’
Cde rh (rh hr )
’’ ’ ’’
c dE Rh (hr ,rh )
y ’ ’’
CdE rh (rh ,rh )
z ’ ’’
CDE Rh (rh ,Rho,rh )
’ ’’
c De Rho (hr ,Rho,hr )

The Rh gene that determine the Rh antigens are inherited as

a single gene (wiener) or gene complex (Fisher- Race) from
each parent. According to Fisher – Race, three pairs of allelic
genes on the same chromosome (haplotype) will determine
the production or non- production of D with C or c, E or e. The
inheritance of the Rh genes through haplotype gene is shown
in Fig 4.1

52
 Parents

Offspring

Fig 4.1 Rh Inheritance with Fisher Race nomenclature

4.3 The antigens of the Rh-Hr blood group

system

The Rh antigens can be demonstrated on fetal red cells as

early as 38 days after conception, and are well developed at
birth. There are five rhesus antigens, D, C, c, E &e which are
only expressed on red cells. They are not found in body fluids
(like saliva, amniotic fluid) and not detected on leucocytes or
platelets. The ‘d’ gene is not expressed and there is no ’d’
antigen, it only implies the absence of ’D’. Individuals who lack
any of these antigens may be stimulated to produce the
corresponding antibodies (anti-D, anti- C, ant -c, anti-E, anti-e)
by transfusion or pregnancy.

Antigen D, having antigen site between 110,000 and 202,000

per erythrocyte, is the most important of the rhesus antigens

53
medically, because it is highly antigenic than the other Rhesus
antigens.

A person is grouped as Rhesus (Rh) positive or negative

based on the presence or absence of antigen D:
- Rh positive: a person who inherits gene D and the red cell
express antigen D.
- Rh negative: a person who does not inherit gene D and
the red cells do not express antigen D
For transfusion purpose, Rh positive blood can be given to Rh
positive individuals and Rh negative blood can be given to
+ - + -
both Rh & Rh individuals. Never give Rh blood to Rh
individuals especially to women of child bearing age.

4.4 Variants of antigen

u
Weak antigen D (D )
Weak forms of antigen D where the number of D sites on the
red cells is reduced. Such weak D cells react less strongly
than red cells with normal numbers of D receptors. There are
u u
two grades of D : High grade D red cells, which are
u
agglutinated by certain anti-D sera and lower grade D red
cells, which are agglutinated only by the Indirect Antiglobulin
(IAG )test.

54
 u
In case of blood transfusion, donors with D + red cells are
regarded as Rh+ because, a severe hemolytic transfusion
u
reaction may result from the transfusion of D + red cell to a
recipient whose serum contains anti D. As a recipient
u
individuals with D + red cells regarded as Rh negative,
u
because of the risk of provoking the formation anti-D in a D +
subject through the transfusion of D+ blood.

In addition, Du + red cells are clinically important in that, they

u
may be destroyed at a higher rate by anti-D, and a D infant
can suffer from HDN if the mother possesses anti –D.
u
- As a donor individuals with D positive antigen regarded
as Rh positive.
u
- As a recipient individuals with D positive antigen
regarded as Rh negative.

4.5. Rhesus Antibodies

The common Rh antibodies are anti –E, anti -e, anti -C,anti-c
and anti –D. Rh antibodies occur in individuals who lack the
corresponding antigens, and as a consequence of transfusion
or pregnancy (i.e as a result of immunization by red cells).
However, some exception, in a few percentage found to be
naturally occurring, as example anti- E & anti- C are non red
cell immune antibodies, and agglutinate red cells suspended
in saline at room temperature.

55
Rh antibodies generally develop from 2 to 6 months after the
initial immunization by red cells. Their production is consistent
with the classical immune response in that the earliest
antibody to appear is IgM , followed by IgG, some IgA have
also bean identified. The predominant Rh antibodies however,
are immunoglobulin class IgG, which most of them are IgG 1 or

IgG3 subclasses.

Following transfusion of one or more units of Rh positive

blood, 50 to 75% of D negative recipients develop anti- D; but
25 to 30% of D negative individuals are non responders,
unable to produce anti-D inspite of repeated stimulation with
Rh+ blood. Secondary immunization in subjects who are
primarily immunized to Rh (D) may result in maximal increase
in antibody concentration in 3 weeks.

Rh antibodies cause severe hemolytic transfusion reaction in

a recipient if transfused with blood possessing the offending
antigen. In addition, Rh antibodies being IgG, are capable of
crossing the placenta and are associated with HDN.

4.6 The Rh- Hr Blood Grouping Technique

4.6.1 Methods of Rh Technique

Rh grouping can be performed in the routine laboratory by the

direct slide and tube methods. In the Rh blood group system,

56
naturally occurring Rh antibodies are not found in the serum
of persons lacking the corresponding Rh antigens. Therefore
‘reverse grouping’ cannot be done in Rh blood group system.
In performing Rh grouping the number of drops, time and
speed of centrifugation shall be determined by manufactures
directions.

4.6.2 Slide Test Method

1. Place a drop of anti- D on a labeled slide

2. Place a drop of Rh control (albumin or other control
medium) or another labeled slide.
3. Add two drops of 40-50% suspension of cells to each
slides.
4. Mix the mixtures on each slide using an applicators stick,
spreading the mixture evenly over most of the slide.
Interpretation or results:
Agglutination of red cells- Rh positive.
No red cell agglutination- Rh negative.
A smooth suspension of cell must be observed in the control.
Note: Check negative reactions microscopically.

4.6.3 Modified Tube Test Method

1. Make a 2-5% red cell suspension .

2. Mark ”D” on a test tube and add two drops of anti-D

57
3. Place a drop of Rh control (albumin or other control
medium) on another labeled slide.
4. Add one drop of a 2-5% cell suspension to each tube
5. Mix well and centrifuge at 2200-2800 rpm for 60 seconds
6. Gently re suspend the cell button and look for
agglutination and grade the results (a reaction of any
grade is interpreted as Rh positive) a smooth suspension
of cells must be observed in the control.
+
7. Collect a weakly positive ( ) and negative sample to
u
perform the D test.

4.6.4 Du Typing Using Indirect Anti- Globulin Test

(IAT)

1. Use the intial Rh D typing tube and control in procedure

4.6.3 and incubate the Rh . Negative or weakly reactive
+ 0
( ) samples and the control at 37 C for 30 minutes.
2. Wash cells in both test and control tube 3-4 times with
normal saline.
3. Add one drop of the poly specific anti- human globulin
(coombs) to each tube and mix well.
4. Centrifuge at 2200-2800 rpm for 10 second.
5. Gently re suspended the cell button and observe for
agglutination

58
6. Interpretation: the positive result is agglutination in the
tube containing anti–D and the control is negative. A
negative result is absence of agglutination in both the test
& control.

59
Review Questions

1. Discuss the history of the development of the Rh system

2. Contrast the Fisher- Race & Wiener’s genetic theories on
the Rh antigens.
3. List the common Rh antigens and their characteristics.
4. Discuss the clinical significance of the Du antigen.
5. How do the Rh antibodies are produced?
6. Discuss the clinical significance of the Rh antibodies
7. Out line a brief lab procedure to classify an individual as
Rh positive or Rh negative

60
 CHAPTER FIVE

THE ANTI- GLOBULIN TEST

(COOMB’S TEST)

Learning objective

At the conclusion of the chapter, the student should be able

to:
- Describe the purpose of the antihuman globulin (AHG)
test.
- Name and contrast the Anti-human globulin (coomb’s)
reagents.
- Know the principle and carryout the AHG procedure.

THE ANTI- GLOBULIN TEST is introduced in to clinical

medicine by Coomb’s in 1945. It is a sensitive technique in the
detection of incomplete antibodies, anti bodies that can
sensitize but which fail to agglutinate red cells suspended in
saline at room temperature, mainly IgG. These antibodies are
agglutinated by the anti- IgG in antiglobulin serum through the
linking of the IgG molecules on neighboring red cells,as
shown in Fig 5.1.

61
Fig 5.1. A representation of the Antiimmunoglobulin (Coombs) Test

Red cells can also be agglutinated by a reaction of

complement components on their surface with anti-
complement serum, in the antiglobulin reagent, since the anti-
C3 in the AHG reagent binds to the C3 on the sensitized red

cells and bridge the gap between the cell- bound human C3
on adjacent red cells. The anti complement helps in detecting
IgM antibodies, which bind complement but may elute off the
red cells with increase in temperature, leaving complement
components alone on the red blood cells. They also enhance
the reactions seen with complement binding antibodies.

The anti globulin reagent is prepared by immunizing animals,

often rabbits with human gamma globulin (antibody) and, or
beta globulin (components of complement). There are two
types of anti globulin reagents that can be used in laboratory
procedure: broad spectrum (polyspecific sera) and
monospecfic sera.

62
Broad spectrum (polyspecific): prepared by combining anti-
IgG & anti complement. The reagent may also contain
antibodies of other specificities such as anti- IgM, anti-IgA,
anti C-3, or anti C-4.

Monospecific: contains only a sigle antibody: anti-IgG or only

anti-
complement.

Two kinds of antiglobulin tests are known: the direct

antiglobulin test (DAT) & the indirect antiglobulin test (IAT).

5.1. The Direct Antiglobulin Test (DAT)

It is used to demonstrate whether red cells have been

sensitized (coated) with antibody or complement in vivo, as in
case of HDN, Autoimmune haemolytic anemia, and drug
induced haemolytic anemia, and transfusion reactions.

Principle: Patients erythrocytes are washed to remove free

plasma proteins and directly mixed with AHG, and if
incomplete antibodies are present, agglutination occurs.

63
Procedure
1. Place one drop of 5% saline red cell suspension in a test
tube.
2. Wash the red cells 3-4 times using normal saline,
ensuring adequate removal of the supernatant after each
wash.
3. Add one or two drops of antiglobulin reagent to the tube.
4. Centrifuge at 3,400 rpm, for 15 sec.
5. Gently re suspend the red cells and examine
macroscopically and microscopically for agglutination or
hemolysis.
6. Add IgG sensitized red cells as a control, centrifuge and
read. If a negative result is obtained, the test result is
invalid if mono anti complement reagents are used
complement sensitized red cells should be for substituted
for IgG sensitized red cells.

5.2 The Indirect Antiglobulin Test (IAT)

It is used for the detection of antibodies that may cause red

cells sensitization in vitro. The sensitizing antibody or
complement acts as the antigen for the antiglobulin reagent.
IAT used in cross- matching, to detect antibodies that might
u
reduce the survival of transfused red cells and D technique,
u
in the detection of D antigen.

64
Principle: The serum containing antibodies is incubated with
erythrocytes containing antigens that adsorb the incomplete
antibodies. After washing to dilute the excess antibody in the
serum, the addition anti globulin serum produces agglutination
in the presence of incomplete antibodies.

Procedure (IAT)
1. Put 2-4 drops of serum in a test tube
2. Add a drop of 5% red cell suspension
0
3. Mix & incubate at 37 C for 15-30 minute
4. Centrifuge at 3400 rpm, for 15 sec and examine for
agglutination or haemolysis.
5. Wash 3-4 times decanting the supernatant.
6. Add 1 or 2 drops of antiglobulin reagent.
7. Mix and centrifuge at 3400rpm, for 15 sec.
8. Examine for agglutination or haemolysis

65
Review Questions

1. What is the purpose of the AHG test?

2. How can the AHG reagent make agglutination of
sensitized erythrocytes?
3. Write the principle of the DAT and IAT.
4. What conditions can be diagnosed by the DAT.

66
 CHAPTER SIX

HAEMOLYTIC DISEASES

Learning Objective

At the conclusion of this chapter the student should be able to:

- Understand the cause of and the laboratory method to

demonstrate Auto Immune Hemolytic Anemia (AIHA).
- Briefly explain the cause and consequences of hemolytic
disease of the newborn (HDN).
- Name the major immunoglobulin class and sub classes
responsible for HDN.
- Describe the laboratory diagnosis & treatment of HDN
caused by ABO and Rh incompatibility.
- Compare the relationship of HDN caused by ABO
incompatibility to HDN caused by Rh incompatibility.
- Describe the prenatal treatment that is significant in HDN
caused by antiD.

6.1. Autoimmune hemolytic anemia (AIHA)

AIHA is brought about through the interaction of red cells and

autoantibodies. It is

67
classified into four groups namely: warm- reactive
autoantibodies, cold – reactive auto antibodies paroxysmal
cold hemoglobinuria and drug induced hemolysis

The diagnosis of AIHA depends on the demonstration of

autoantibodies on the patient’s red cells using the DAT. The
autoimmune antibodies will be either of the ‘warm antibody‘
types or the ‘cold antibody’ type. The warm types have
0
antibodies active at 37 C but no abnormal cold antibodies. In
the cold type the patient’s serum contains high titer cold
0
agglutinins, optimally active at 2 C but with a temperature
0
range which may go as high as 32 C.

A positive DAT will be found in both types; in the cold type it is

complement which sensitizes the cells and give rise to the
positive DAT rather than specifically bound antibody, whereas
in the warm type it is more usually the attached antibody,
although occasionally it may be due to complement.

6.2. Hemolytic Disease of the New Born (HDN)

HDN, originally known as erythroblastosis fetalis, results from

blood group incompatibility in which maternal antibodies
destruct fetal red cells. An infant having inherited an antigen
from the father, which is absent in the mother, causes her to
form the corresponding antibodies. These antibodies pass

68
through the placenta by active transport mechanism, coat the
fetal erythrocytes and cause damage to them.

Every blood group antibody that can occur as IgG can cause
HDN. It is only IgG immunoglobulin that is capable of passing
the placental barrier and which is found in cord blood in a
concentration equivalent to that found in maternal blood. IgM
agglutinin though produced in response to fetal red cells in
utero, plays no part in the cause of HDN, and are either
present in much lower concentration in the new born than the
mother or entirely absent.

Fetal hematopoietic tissue (liver, spleen and bone marrow)

respond to hemolysis by increased production of RBCs ,
predominantly NRBCs . Increased destruction of red cells
leads the fetus to develop anemia and jaundice from the
hemoglobin breakdown product, bilirubin. If this blirubin
reaches excessive levels in the newborn or infants circulation
it causes mental retardation or death.

6.2.1 HDN Due to Rh Blood Group Incompatibility

HDN due to anti- Rh(D) occurs when mother and infant are
always incompatible with respect to the Rh factor: The mother
Rh(D) negative, and the infant Rh (D) positive (inherited the D
factor from the father). ABO incompatibility between the

69
mother and fetus reduces the chance of maternal
immunization to the D Ag. This is probably because the fetal
cells, which are incompatible with the maternal ABO
antibodies are destroyed by existing ABO antibodies before
they have a chance to act as an antigenic stimulus.

The first Rh- incompatible infant is usually unaffected because

the number of fetal cells that cross the placenta during
pregnancy (after 24 weeks gestation) is small and insufficient
to cause IgG anti D production, unless a prior transfusion of D
positive blood has been given.

During transplacental hemorrhage, the amount of fetal blood

that enters the maternal circulation increases, and in 6 months
time after delivery only 10% of these Rh negative women
could produce detectable antibodies. The actual production of
anti-D antibodies depends on the dosage and antigenecity of
the D antigen, and the mother’s ability to respond to these
foreign antigens. About one third of mothers are non-
responders, they fail to form anti- D despite intentional
repeated injections of Rh (D) positive erythrocytes.

During a second pregnancy with a Rh positive fetus, small

0
number of fetal cells cross the placenta (2 doses of antigen)
stimulating the antibody to high concentration, mainly Ig G
anti- D that passes in to the fetal circulation destroying fetal

70
red cells. The severity of the disease increases with each Rh
positive pregnancy. IgG anti-D is found predominantly in sub
classes IgG1 & IgG3 and these subclasses possess properties
that play and effective role in erythrocytolysis in vivo.

6.2.2 HDN due to ABO Blood Group Incompatibility

HDN due to ABO blood grouping usually occurs when the

mother is invariably group O (posses Ig G anti- A&B), the
infant: group A or B (usually group A) and when the mother &
infant are Rh compatible.

The fetal red cells cross the placenta in to the maternal

circulation stimulating the existing anti A& B to high titers; the
“immune” anti A& B stimulated is largely IgG. ABO HDN
occurs in the first pregnancy because anti A & anti- B are
always present (naturally occurring) and therefore readily
stimulated. Although ABO incompatibilities between mother
and baby occur frequently and represent a common form of
HDN, the clinical course of ABO HDN is relatively mild,
probably because of the antigenic development of the fetal
red cells and may also be due to the presence of A&B
substances in the fetal tissues and fluids that will neutralize
the anti A & anti – B antibodies before they can attack the
fetal red cells.

71
6.2.3 Assessment of HDN

Prenatal
Some investigations are carried out on blood of the mother to
identify women at risk of having a child affected with HDN. It is
recommended that all pregnant women at their first
attendance at a clinic need to have ABO grouping, Rh typing
u
for D & D , alloantibody screening test and amniocentesis.

Postnatal
After birth different laboratory procedures are helpful in
determining the presence and assessing the severity of HDN.
1. ABO & Rh Typing: the ABO group of the infant is based on
forward (cell) grouping as anti- A and anti B
agglutinins do not develop until a few months after birth.
- ABO grouping most commonly reveals the mother to be
group O and the baby to be group A or possible group B.
U
- Rh typing shows the baby to be D or D positive and the
U
mother D or D negative 2.
DAT on cord or infants blood:
- In ABO HDN, DAT is usually negative or weakly positive ,
as weak Ag-Ab interaction which cause the antigen to be
removed during the washing phase of the DAT. Also, due
to too low antibody titer to be detected.
- In Rh HDN, DAT gives positive result.

72
3. Antibody elution test of cord blood: done if DAT is
positive, may reveal the presence of immune anti- A or
anti-B in ABO HDN and anti-D in Rh HDN.
4. Hgb level of cord blood: may be slightly to severely
decreased
5. Serum bilirubin level on cord serum: may exceed the
normal values of cord total serum bilirubin of 1 to 3 mg/ml.
6. Peripheral blood smears on cord blood: blood smear
evaluation shows anemia with RBC morphology
abnormalities: hypochromia, microspherocytosis with the
demonstration of reticulocytes and immature nucleated
RBCs.
7. Kleihauer- Betke acid elution test: is a test to be
performed for quantitating the extent of fetal maternal
hemorrhage (number of fetal cells in the maternal
circulation). It is an indicator for treatment of the mother
with anti-D immunoglobulin, more importantly used to
determine the size of dose to be given.

Reagents: 80% ethanol

Solution A: 0.75 haematoxylin in 96% ethanol
Solution B : 2.4 g FeCl3 & 2 ml of 25% HCl in 100 ml distilled
water
Elution solution: 2 parts of A mixed with 1 part of B & 9 part of
80% ethanol
Counter stain: 0.1% erythrosin or 0.5% aqueous eosin.

73
Method: Mother’s whole blood is diluted with an equal
quantity of saline and used for making films. The slides are
fixed in 80% ethanol for 5 minutes at RT. They are thoroughly
dried. They are then placed for 20 seconds in the elution
solution, rinsed in distilled water and counterstained for 2
minutes. At least 50 fields are examined using a magnification
x 400. The white cells, including lymphocytes, stain gray, the
adult red cells, which have soluble hemoglobin appear as
‘ghost’ cells, while the fetal cells stain bright red. Report the
percent & fetal cells after counting 2000 cells in the acid
elution smear.

6.2.4 Prevention of HDN

Fetal red cells in the maternal circulation might be destroyed

by administration of suitable quantity of IgG anti- D to prevent
Rh immunization of the mother, given to Rh-negative women
within 72 hours of delivery. This dramatically decreases the
incidence of anti-D HDN. Combined prenatal-postnatal
treatment is more effective than postnatal treatment alone in
-
suppressing Rh immunization. All pregnant Rh women should
receive Rh IG even if the Rh status of the fetus is unknown
because fetal D antigen is present in fetal erythrocytes as
early as 38 days of conception.

74
Dose: The usual recommended dose (contained in one vial) is
about 300 µg which is believed to offer protection against a
fetomaternal hemorrhage of 30 ml (15 ml packed cells) or
less.

If a massive fetomaternal hemorrhage has occurred, the

volume of the hemorrhege must be determined to calculate
the number of vials of Rh (D) immune globulin to administer.
Calculated as follows:

Volume of fetomaternal Percentage of fetal cells

hemorrhage = (seen in the acid elution stain) x*50

No of vials of Rh IG = volume of fetomaternal hemorrhagex*2

30
NB: Factor of *50 = 5000 ml (estimated maternal volume) x
1/100%
*2 is a common factor, because the actual fetomaternal
hemorrhage may be twice the estimate.
Example:
- KB reported as 1.2% fetal cells
- 1.2 x 50= 60ml fetomaternal hemorrhage
- 60 x2 = 4 vials
30

75
Candidates for the administration of RhIG: Anti- D
prophylaxis should be given as soon as possible If the women
is Rh D negative and has not already developed anti D within
72 hrs after delivery of a D positive infant, or after obstetric
intervention such as amniocentesis, abortion, miscarriage, or
pregnancy.

The laboratory criteria are:

U
- The mother is D and D negative.
- The screening test for alloantibodies is negative for anti D
antibody.
u
- The infant is D or D positive.
- DAT on cord or infant’s cells is negative. If DAT is positive
perform elution test to establish anti – D is not the coating
antibody.

6.2.5 Treatment of Infants Suffering from HDN

For infants who develop hyperbilirubinemia and/or anemia

due to HDN, exchange transfusion is usually carried out.

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. By exchange transfusion the
concentration of bilirubin and incomplete antibodies decrease,

76
simultaneously the infant is provided with compatible donor
red cells. To give exchange transfusion for an infant clinical &
laboratory findings must be considered. Cord Hgb (<10g/dl)
and raised serum bilirubin are strong indicator for treatment.

For compatible exchange transfusion, donor’s blood should be

cross- matched with the maternal serum and this blood should
lack the red cell antigen corresponding to the maternal
antibodies. It must also be ABO group & 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.

77
Review Questions

1. Compare the causes and lab 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?
4. Discuss on the dose of IgG anti-D given to Rh- pregnant
women to prevent HDN?
5. When does Rh HDN occurs?
6. List & discuss the postnatal lab investigations to be
carried out to know the presence & extent of HDN.

78
 CHAPTER SEVEN

THE CROSS- MATCH

(COMPATIBILITY TESTING)

Learning Objective

At the conclusion of this chapter, the student should be able

to:
- Understand the cross match and its primary purpose.
- Explain the constituents of the major and minor cross
match.
- Select appropriate blood for cross match.
- Describe the types of antibodies that can be encountered
at various phases of a cross match.

7.1. Purpose of Cross-Match

The cross- match (compatibility testing) is a procedure

performed before transfusion to select donor’s blood that will
not cause any adverse reaction like hemolysis or agglutination
in the recipient. In addition it helps the patient to receive
maximum benefit from transfusion of red cells, which will
survive maximum in his circulation. This is done by ensuring

79
the ABO and Rh group of the blood to be transfused is
compatible with patient’s ABO and Rh group and by detecting
most unexpected (irregular) antibodies in the patient’s serum
that will react with the donor’s red cells causing their
destruction or reducing their normal survival.

However, a cross match will not prevent immunization of the

patient, and will not guarantee normal survival of transfused
erythrocytes or detect all unexpected antibodies in a patient’s
serum.

7.2. Types of Cross Match

There are two types of cross- matches major & minor curse
match.

Major cross- match: includes mixing recipient’s serum with

the donor’s red cells. It is much more critical for assuring safe
transfusion than the minor compatibility test. It is called major
because the antibody with the recipient’s serum is most likely
to destroy the donor’s red cells and that is why it is called
major cross match.

Minor cross match: involves mixing the donor’s serum with

patient’s red cells. It is usually thought that any antibody in the
donor’s serum will be diluted by the large volume of the

80
recipient’s blood, so it causes relatively less problem and so
called minor cross match.

7.3 Selection of Blood for Cross Match

Generally, when whole blood is to be transfused, the blood

selected for cross- match should be of the same ABO and Rh
(D) group as that of the recipient. However, Rh positive
recipients may receive either Rh positive or Rh negative
blood.

Table 7.1 Selection of ABO blood for cross-match

Group of Choice of blood

patient 1st 2nd 3rd 4th
Group A Gp A Gp O - -
Group B Gp B Gp O - -
Group O Gp O - - -
Group Gp AB GP A* Gp B Gp O
AB
* Group A is the second choice of blood because anti-B in
Gp A blood is likely to be weaker than anti-A in Gp B blood.

81
7.4 Procedure for Cross-Match

For a full cross match for non- emergency transfusions the

following procedure which include different phases (the saline,
protein, AHG and enzyme) is recommended:
1. Saline tube technique at RT: provides the optimum
temperature and medium for the detection of IgM
antibodies of ABO system and other potent cold
agglutinins.
o
2. Saline 37 C: is the optimum for the detection of warm
agglutinin, of which are saline reactive IgG antibodies of
the Rh/ Hr system.
3. AHG: is highly efficient for the detection of most kinds of
incomplete antibodies.
4. Enzyme technique- is a very sensitive one for the
detection of some low affinity Rh antibodies, which are not
detected by other methods including the antiglobulin
technique.

Procedure:
1. Put 3 drops of patient’s serum in to a test tube.
2. Put one drop of donor’s 3% red cells suspension.
3. Mix and centrifuge at 3400 rpm for 15 seconds.
4. Examine for agglutination or haemolysis, if compatible
proceed with the next phase.
0
5. Mix the contents of the tube and incubate at 37 C for 20-
30 min.

82
Note: potentiators such as a drop of 22% albumin may be
added at this phase to increase the sensitivity of the test.
6. Centrifuge at 3400 rpm for 15 seconds and examine for
agglutination or hemolysis. If there is no hemolysis or
agglutination proceed with the next phase.
7. Wash the contents of the tube 3-4 times with normal
saline.
8. After the last wash, decant all saline and add two drops of
AHG reagent and mix.
9. Centrifuge at 3400 rpm for 15 seconds.
10. Gently re suspend the cells button and examine
macroscopically and microscopically for agglutination or
hemolysis .
Enzyme cross match can be performed by using different
enzymes: bromelin, ficin, papain & trypsin. Two methods are
available to carry out enzyme cross mactch - One stage & two
stage methods. The one-stege technique involves enzyme,
patient’s serum and donor’s red cell incubated together. The
two-stage technique involves red cells pretreated with enzyme
and then tested with the patient’s serum.

83
Review Questions

1. What is cross match?

2. What is the purpose of cross-match?
3. List the types of cross-match with their constituents.
4. List the phases of cross- match and their respective
importance in antibody detection.

84
 CHAPTER EIGHT

THE DONATION OF BLOD

Learning objective:

At the end of this chapter the student should be able to:

- Discuss the medical and physical requirements that would
exclude an allogeneic donor
- Describe the proper procedure for collecting blood from
donors
- Name the commonly used anticoagulants for donated
blood and their respective approved maximum storage
time
- Name the common blood components with their storage
temperature and shelf life
- Explain the possible donor reactions

8.1 Selection of blood donors

A blood transfusion service aims to prepare safe blood from a

safe donor to a recipient who needs blood. The medical
person who screens donors should identify conditions which

85
can harm both the donor who gives his blood on one hand
and the recipient who receives blood and blood products on
the other hand. Therefore, to ensure the well being of both
donors and patients the screening person should understand
the requirements that make a donor acceptable and not
acceptable to donate blood.

8.1.1 Selection Criteria:

Age:
If between 17-65 years acceptable.
If less than 17 years after guardian’s consent or depending on
the local law
If more than 65 years after consulting a medical doctor.

Hemoglobin:
Females should not be less than 12.5 g/dl (PCV 38%)
Males should not be less than 13.5 g/dl (PCV 41%)
In both sexes Hgb above 19g% (Hct above 57%) are not
acceptable.

Pulse, Blood pressure & Temperature:

Pulse between 60-100 per minute acceptable.
Systolic pressure between 90 and 180 mmHg acceptable
Diastolic pressure between 50 and 100 mmHg acceptable A
o
donor’s temperature must not exceed 37.5 C.

86
Weight:
If between 45-50 kgs can donate 350 ml of
blood If above 50 kg can donate 450 ml of blood
- Obese donors who are unable to climb the coach are not
acceptable.
- If weight is very low compared to the height of the donor
do not accept.
- Donors with unexplained weight loss of a significant
degree (more than kg) are not acceptable to donate.
If a prospective donor weighs less than 50 kg, a lesser
amount of blood may be collected, and the amount of
anticoagulant in the collecting bag must be reduced
proportionally, calculated as follows:

Volume of blood to draw = Donor’s weight in kg x 450 ml

50
Amount of anticoagulant to 63ml – Donors weight x 63 m
remove from a 450 ml bag = 50 kg

Pregnancy:
pregnant women excluded from donating for 1 year after the
conclusion of their pregnancy.

Medication:
In general, medications taken by a donor are not harmful to a
recipient. Deferral of a donor because of drug depends on the

87
nature of the disease for which the drug was ordered. Consult
medical doctor for donor’s on long term treatment.

Illness: prospective donors with disease of the heart, liver,

lungs, or individuals with a history of cancer, or those with
bleeding problems should be excluded subject to evaluation
by a physician.
- Donors who have had leukemia must be permanently
deferred.
- Donors with previous history of tuberculosis are
acceptable after completion of therapy and if no longer
active.

Infectious diseases: A donor must be free from infectious

diseases that can be transmitted by blood such as hepatitis,
HIV & malaria.

- Recipients of blood or blood products known to be

possible sources of hepatitis and donors having had close
contact with an individual with viral hepatitis must be
deferred for 1 year.
- Persons at high risk for acquiring or transmitting AIDS
should not donate blood.
- Donors who have a history of malaria, or were previously
resident in an endemic area, should be deferred for 3

88
 years after becoming symptomatic or after leaving the
endemic area.

Previous donation: If a person has donated blood, an

interval of at least four months for men and six months for
women is required before the next donation.

Surgery: If the surgery is minor (such as tooth extraction) a

donor is excluded until healing is complete and full activity has
been resumed.

Vaccinations: Persons recently immunized with toxoids and

killed viral, bacterial and rickettsial vaccines (such as for
anthrax, cholera, diphtheria, influenza, polio, tetanus, typhoid,
typhus) are acceptable, if they are symptom free and a febrile.

- After small pox vaccination, a donor is acceptable when

the scab has fallen off, or 2 weeks after an immune
reaction.
- A donor who has received an attenuated live virus vaccine
such as mumps or yellow fever is deferred for 2 weeks
after the last immunization.
- If rabies vaccination has been given following a bite by a
rabid animal, the donor must be deferred for 1 year after
the bite.

89
8.2 Collection of Blood

Trained personnel must collect donation of blood. Basic

information from the donor: date of donation, full name,
address, sex, age and the ABO & Rh blood group including
the prospective donor’s medical history must be obtained and
signed by the phlebotomist who perform the procedure. A
form similar to Figure 8.1, which is taken from Ethiopian Red
Cross Society, is the basic guideline for donor registration and
the medical history interview.

90
Figure 8.1 The front and back view of a form for donor registration and
medical history interview.

Patient identification also is an important step in blood

collection. Forms accompanying blood samples from the
recipient must contain sufficient information: full name,
identification number of patient, sex, age, clinical diagnosis
and the like for identification of the recipient.

91
8.2.1 Micro Sampling

Micro sampling is the collecting of small quantity of blood from

capillaries of the fingertip. Capillary blood collection is
performed using a sterile, disposable lancet. In
immunohematology laboratory, this blood is used for blood
grouping, hemoglobin or hematocrit determination.

Copper sulphate method of hemoglobin determination:

CuSO4 method is based on the relationship of specific gravity
to hemoglobin concentration. It is used to check that a donor
has sufficiently high hemoglobin level to be eligible to give
blood. Two strengths of CuSO4 solution are normally used,
each of which has a different specific gravity: one for male
donor with a sp.gr. of 1.055 (equivalent to 13.5g/dl of
hemoglobin)and one for female donors with a sp.gr of
1.053(equivalent to 12.5g/dl of hemoglobin ).

In this method, a drop of blood is allowed to fell gently at a

height of about 1 cm above the surface of the CuSO 4 solution.
If the drop of blood has a satisfactory hemologin
concentration, it will sink in the solution within 15 seconds. An
unacceptable specimen will either remain suspended or will
sink slightly and then rise to the top of the solution within 15
seconds.

92
8.2.2 Macro Sampling

Macro sampling is the collection of large volume of blood from

the veins by venipuncture. In Blood Bank laboratory, this
blood is usually collected for transfusion purpose from
volunteer blood donors, and for serologic tests of diseases
that are transmittable by blood transfusion: hepatitis, HIV and
syphilis.
- Before starting the phlebotomy, the phlebotomist should
introduce himself/ herself pleasantly to the patient and
briefly explain the phlebotomy procedure in early- to-
understand terms.

1. Visually inspect both arms, and choose the arm that is

free of bruises and brasions. In the arm, three veins can
be used for venepuncture the cephalic, basilic, and
median cubital veins. (Fig. 8.2).

Fig. 8.2 Veins commonly used for venipuncture (left arm shown)

93
2. Apply the tourniquet, and ask the patient to make a fist
(sometimes a roll of gauze is placed is the patient’s hand).
This usually makes the veins more prominent. Using the
left index finger, palpate for an appropriate vein. The ideal
vein is generally near, or slightly below, the bend in the
arm. Do not leave the tourniquet on for more than 2
minutes (After an appropriate site has been chosen,
release the tourniquet).

Using 70% alcohol swab cleanse the intended site of

venepuncture in a circular motion from the center outward.
Allow the site to dry.

The phlebotomy procedure

1. Inspect the anticoagulant donor bag for leaks, and make
sure that the anticoagulant solution is clear.
2. Position the bag below the level of the donor- arm balance
system, making sure that the counterbalance is level and
adjusted for the amount of blood to be drawn. Make a
loose knot of the blood bag tubing.
3. Reapply the tourniquet or blood- pressure cuff (inflated to
40-60 mm Hg) and have the donor open and close the
hand until the selected vein is again prominent.
4. Apply the hemostat clamp to the tubing atleast 5 cm
above the needle, uncover the sterile needle, and perform

94
 the venipuncture immediately. A clean venepuncture will
guarantee a full, clot free unit
5. Open the hemostat clamp and check that the blood flow is
adequate. Carefully tape the tubing to hold the needle in
place and cover the venipuncture site with a sterile gauze
pad. Have the donor squeeze a rubber ball or other soft
object every 10 to 12 seconds during collection.
6. Keep the donor under observation throughout the
phlebotomy. A person should never be left unattended
during or immediately after donation.
7. Mix the unit of blood periodically (every 30 seconds). Time
limits for collecting a unit are not fixed, so long as the
blood flow is continuous However, it usually takes 8-10
minutes. A unit containing 450-495 mL should weigh 425-
520 g plus the weight of the container with its
anticoagulant.
8. Remove the tourniquet & hold a sterile gauze lightly over
the venipuncture site and remove the needle from the
donor’s arm. Apply pressure on the gauze. Have the
donor raise the arm (elbow straight) and hold the gauze
firmly over the phlebotomy site with the opposite hand.
9. Strip the donor tubing from the end of the tube towards
the bag as completely as possible in order to mix well with
the anti coagulant. Invert the donor unit and allow the line
to refill. Then strip again. Seal the tubing attached to bag
into segments suitable for subsequent tests with either a

95
 heat sealer or metal clips.(see fig 8.3 for appropriately
collected donor blood).
10. Place blood at appropriate temperature.

Fig. 8.3 Donor blood bag with segmented tubing

Note: immediately after collection, blood must be placed in

o
storage at a temperature between 1 C &
0
6 C.However, if the blood is to be used as a source of
components, up to 8 hours may elapse before
storage.

96
8.3 The Anticoagulants and Storage of Blood
and Blood Products

Anticoagulant is a substance that prevents the clotting of

blood some anticoagulants contain preservatives that provide
proper nutrients for metabolism in the red cell during storage.
Anticoagulants maintain red blood cells hemoglobin function
and viability and the biochemical balance of certain elements:
glucose, ATP, 2,3 diphosphoglycerate (2,3DPG) and PH, so
that the red cells will maintain the means of delivering oxygen
to the tissues of the recipient.

Anticoagulants and/or anticoagulant preservation for whole blood

and red cell concentrate storage include: ACD(acid-citrate-
dextrose),CPD(citrate- phosphate dextrose).CPD-A1 and CPD-

A2(citrate phospate dextrose adenine) and heparine.

1. ACD
- Acts as an anticoagulant by binding Ca
- Composition: Trisodium citrate- binds Ca
H
Citric acid:- maintains P
Dextrose: acts as a nutrient & preservative
- To prevent the clotting of 100 ml of blood 15 ml ACD is
required.
- Shelf life: 75% survival after 21 days of storage.

97
2. CPD
- Acts by binding Ca.
- Composition: In addition to the composition of ACD, CPD
contains sodium phosphate, which maintains ATP levels
in the red cells.
- Advantages of CPD over ACD.
• Contains less acid.
• Gives less hemolysis.
• Smaller leak of K from the red cells.
• Prolonged post- transfusion survival of red cells.
- To prevent the clotting of 100 ml blood 14 ml of CPD is
required.
- Shelf life: survival of red cells in CPD 24hrs post
transfusion is 80 to 85% after 21 days

The composition of two anticoagulant preservative

solutions(CPD and CPDA1) is presented comparison purpose
in Table 8.1

98
Table 8.1 Two anticoagulant preservative solutions in general
use
CPD CPDA-1
Na3 citrate 26.3gm 26.3 gm
Citric acid 3.27gm 3.27 gm
Dextrose 25.2 gm 31.9 gm
NaH2PO4.H2O 2.22gm 2.22 gm
Adenine - 0.275 gm
Water 1000 ml 1000 ml
Volume per 100 mL blood 14 ml 14 ml
Storage limit 21 days 35 days

3. CPD-A: It is a combination of CPD and adenine. Adenine

provides a substrate from which red cells can
synthesize ATP during storage.
- Survival of red cells is 80% after 28 days storage and 75-
80% after 35-days storage.

4. EDTA and Heparin: are not commonly used in Blood

Banking. Whole blood or red cell collected and stored in
heparin solution must be used within 48 hours of
phlebotomy.

Avariety of blood components can be harvested from a single

unit of whole blood (Fig –8.4). Each component can be

99
collected, processed and stored under conditions, which
maximize its storage capacity.

Fig.8.4 Blood component preparation

These components can effectively meet patient transfusion

needs while keeping the risk of transfusion to a minimum. By
using a single unit one can treat anemia with the packed cells,

100
platelate deficiency with platelate preparations, clotting factor
and other plasma deficiencies with plasma preparation.

8.4 Potential Hazards During & after Blood

Collection

Donor reactions though are rare, may include fainting,

nausea, vomiting, development of hematoma and convulsion.
At the first sign of reaction, the phlebotomist should stop the
phlebotomy, give initial first aid procedures and call the blood
bank physician.

101
Review Questions

1. List some clinical conditions that exclude a donor.

2. What are the steps in performing a venipuncture?
3. Where is the most common venipuncture site?
4. Write the composition of CPDA with their respective
importance.
5. Name the common blood components that can be
prepared from a unit of donated blood.
6. List some potential hazards that occur during or after
blood donation.

102
 CHAPTER NINE

THE TRANSFUSION REACTION

Learning ObjectiveS

At the conclusion of this chapter the student should be able to:

- Define the term “transfusion reaction”
- Classify transfusion reaction
- Carryout laboratory tests during transfusion reaction

9.1 Types of Transfusion Reaction

Any unfavorable response by a patient that occurs as a result

of the transfusion of blood or blood products is termed as the
transfusion reaction. Transfusion reactions can be divided into
hemolytic and non-hemolytic types. Hemolytic reactions may
be defined as the occurrence of abnormal destruction of red
cells of either the donor or recipient following the transfusion
of incompatible blood. Nonhemolytic reactions on the other
hand are not usually associated with erythrocyte hemolysis,
constitute conditions such as shortened post transfusion
survival of erythrocytes, febrile reactions, allergic response,
and disease transmission.

103
Febrile reactions are the most prevalent type of immediate
nonhemolytic reaction and are commonly caused by
leukocytes or platelate antibodies present in the recipient’s
plasma, a reaction occurs between these antibodies and the
antigen present on the cell membrane of transfused
leukocytes or platelates.

Transfusion reactions can be further classified in to acute

(immediate) or delayed in their manifestations. Factors such
as antibody concentration, class or subclass, ability to fix
complement, temperature of activitiy and concentration of red
cell antigen infused also influence whether a transfusion
reaction will be acute or delayed.

Acute hemolytic reactions, which are the most serious and

potentially lethal, occur during or immediately after blood has
been transfused. Most commonly are caused by Ag-Ab
reaction between the patient’s serum and the donor’s red cells
and vice versa, of transfusing ABO incompatible blood.

Delayed hemolytic reactions, as the name implies the

transfusion reaction is delayed due to weak antibody in the
recipient 7 to 10 days of post transfusion.

In most cases of delayed hemolytic reactions, the patient has

been primarily immunized by previous transfusion or

104
pregnancy. The antibody is too weak to be detected in routine
cross-match, but becomes detectable 3 to 7 days after
transfusion, eg. Antibodies of the Rh system & kidd system.

9.2 Laboratory Tests to be done When

Transfusion Reaction Occurs

Most of fatal transfusion reactions result from misidentification

or clerical error such as misidentification of patients,
mislabeling of blood sample, error in laboratory records,
mistake in blood typing and inaccurate crossmatching.

Whenever adverse reaction experienced by a patient in

association with a transfusion it should be regarded as a
suspected transfusion reaction, and the following lab.
investigations must be performed.
- Check the identification of the patient and transfused unit
- Obtain a post transfusion specimen from the patient and
visually examine it for hemolysis
- Direct Antiglobulin test from post transfusion sample,
taken as soon as possible after the reaction has taken
place.
- Re-type the red cells of both donor and recipient for ABO
and Rh grouping.
- Re-cross match blood from each unit transfused using
serum from both pre-and post-transfusion specimens from
the patient.

105
Review Questions

1. What is a transfusion reaction?

2. On what basis do the transfusion reaction classified?
3. List laboratory investigations to be carried out when
incompatible transfused reactions are suspected?

106
 CHAPTER TEN

BASIC QUALITY ASSURANCE

PROGRAM IN BLOOD BANKING

Learning objectives:

At the conclusion of the chapter the students should be able

to:
- Understand the purpose of quality assurance program
(QAP)
- Understand the areas to be focused in QAP

- Describe how to evaluate the quality of reagents,

equipment and personnel

Quality Assurance is employed in the blood bank to support

error- free performance to ensure the highest quality of patient
care. Important factors in a routine quality assurance program
include evaluation of reagents, equipment, and personnel
qualification.

Quality control of reagents: commercial reagents in blood

bank such as ABO and Rh antisera, Red blood cell products
and Anti human globulin (AHG) reagent must meet the
required specificity and potency. Each reagent on each day of

107
use must be inspected visually for color, cloudiness and other
characteristics, and the manufactures procedure should
strictly be followed to confirm its reactivity.

Quality control of equipment: Instruments and equipments

in blood bank laboratory such as centrifuge and water bath
must be properly maintained and monitored to ensure they
are working accurately. Check centrifuge speed and the
actual revolution perminute (rpm) by a device
(TACHOMETER), and check the timing with the actual time of
centrifugation with a stop watch. Water baths temperature
should be constantly monitored by using thermometer to
0
achieve a temperature of 37 C for the detection of warm
reacting antibodies.

Quality control of personnel: Though it is the most difficult

to control, the maintenance of high personnel standards is
one of the most important functions of a quality assurance
program.

Evaluate person’s employment in the laboratory for

competency: proper qualification, dedication, trust and ability
to work in stressful conditions. It is also essential to maintain
competence of personnel by participation in continuing
education activities. This helps them to acquire new
knowledge to practice it in the field, and to maintain their
motivation as well.

108
Review Questions

1. What is the purpose of quality assurance program in

Blood Banking.
2. List the areas to be focused in QAP in Blood Banking
3. How do you evaluate the competency of Blood Bank
personnel?

109
Glossary

AB cis gene. A condition in which both the A and B genes

seem to be inherited on a single chromosome.
ACD (Abbr) Acid-citrate-dextrose. An anticoagulant composed
of citric acid, sodium citrate, and dextrose.
Acriflavin The yellow dye used in some commercial anti-B
reagents. This additive can produce false
agglutination in some individuals but this is rare.
Acquired antigen An antigen that is not genetically
determined and is sometimes transient.
Adenine An agent that, when added to ACD or CPD blood,
prolongs the maintenance of red cell viability .
Adenosine An agent that improves the maintenance of red
cell viability and is capable of restoring the adenosine
triphosphate content of stored red cells.
Agammaglobulinemia The absence of plasma gamma
globulin due to either congenital or acquired states.
Alleles Alternate forms of genes that code for trains of the
a B
same type; for example, the genes Fy and Fy are
alleles.
Aminiocentesis The process of removing fluid from the
amniotic sac for study, for example, chromosome
analysis or biochemical studies.
Anamnestic antibody response An antibody”memory”
response. This secondary response occurs on

110
 subsequent exposure to a previously encountered
and recognized foreign antigen. An anamnestic
response is characterized by rapid production of IgG
antibodies.
Atypical antibody An antibody that occurs as an irregular
feature of the serum.
Autologous donation Donation of blood for one’s self.
Autologous donation may take the form of predeposit
or autotransfusion, for example, intraoperative
autotransfusion, hemodilution, or postoperative auto
transfusion.
Avidity (of an antiserum) A measure of the ability and speed
with which an antiserum agglutinates red cells as a
property of the combining constrant (K)
Bombay phenotype The failure of an individual to express
inherited A or B genes because of the lack of at least
one H gene and the subsequent lack of the resulting
H precursor substance.
Bromelin A proteolytic enzyme prepared from the pineapple
Ananas sativus.
Co-dominant genes Tow or more allelic genes, each capable
of expressing in single dose.
Compatibility test A series of procedures used to give an
indication of blood group compatibility between the
donor and the recipient and to detect irregular
antibodies in the recipient’s serum.

111
Coombs’test: The older term for the antiglobulin test.
Cord blood Blood taken from the umbilical vein or the
umbilical cord of a newborn
Delayed hemolytic transfusion reaction A rapid increase in
antibody concentration and destruction of transfused
red cells a few days after transfusion usually due to
low amount of antibody undetectable in pretransfusion
tests on the recipient, which are stimulated to high
titers by the transfusion of red cells possessing the
offending antigen.
Eluate In blood banking, the term denotes an antibody
solution made by recovery into a fluid medium of
antibodies that have been taken up by red cells (i.e.,
the removal of antibody from the red cells).
Immune Response Any reaction demonstrating specific
antibody response to antigenic stimulus.
Immunoglobulin antibody containing globulins including
those proteins without apparent anti body activity that
have the same antigen specificity and are produced
by similar cells.
In vitro. Outside the body, for example, in the test tube
In vivo. In a living organism.
Incompatible transfusion Any transfusion that results in an
adverse reaction in the patient (including reduced red
cell survival).

112
Incomplete antibody Any antibody that sensitizes red cell
suspended in saline but fails to agglutinate them.
Inheritance The acquisition of characteristics by transmission
of chromosomes and genes from ancestor to
descendant.
Kleinhauer- Betke test. A procedure based on the
differences in solubility between adult and fetal
hemoglobin. The test is performed on a maternal
blood specimen to deterct fetal-maternal hemorrhage.
Naturally occurring antibody antibodies that occur without
apparent stimulus. Also known as non-red cell-
immune antibodies
Non-red cell-immune see Naturally occurring antibody.The
observed or discernible characteristics of an individual
Phenotype as determined by his or her genotype and the
environment in which he or she develops. With respect to
blood groups, the out ward expression of genes(i.e., the
product that is detectable on the red
cells)
Nonsecretor. The ansenceo f water- soluble antigens in body
fluids.
Paroxysmal cold hemoglobinuria (PCH). This form of
destruction of erythrocytes is due to an IgG protein
that reacts with the red blood cells in colder parts of
the body and subsequently causes complement
components to bin irreversibly to erythrocytes. It is

113
 commonly seen as an acute transient condition
secondary to viral infection.
Phenotype. The detectable or expressed characteristics of
genes.
Postpartum. After birth.
Postnatal subsequent to birth
Post- transfusion viability. The length of survival of blood
ce4lls after infusion into the human body, believe to
be related to the structural and metabolic status of the
cell membran
Prenatal. Before birth.
Primary antibody response. An immunologic (IgM antibody)
response following a foreign antigen challenge.
Prozone phenomenon. A possible cause of false- negative
antigen- antibody reactions due to an excessive
amount of antibody.
Quality control A control of all facets of daily work to ensure
a high level of performance.
Reagent red cells red cells used in laboratory testing
Recessive gene A gene that gives rise only to its
corresponding character when present in “double
dose”(i.e., in the homozygote).
Secondary response A second response to exposure to a
foreign antigen, resulting in the production of large
amounts of antibody.

114
Rouleaux. Pseudoagglutination or the false cluming of
erythrocytes when the cells are suspended in their
own serum. This phenomenon resembles
agglutination and is due to the presence of an
abnormal protein in the serum, plasma expanders,
such as dextran, or wharton’s jelly from cord blood
samples.
Specificity. The complementary relationship between the
binding sites of anibodies directed against
determinants of a similar- type antigen.
Sensitization(of red cells) The specific attachment of
antibody to its antigenic receptors on red cells without
agglutination or lysis.
Sialic acid Any of a family of amino sugars containing nine or
more carbon atoms that are nitrogen- and oxygen-
substituted acylderivatives of neuraminic acid. It is a
component of lipids, polysaccharides, mucoproteins
and it is the main substance removed from the red
cells by enzyme treatment.
Species- Specific Antigens restricted to members of a
particular species.
Subgroups subdivisions of antigens; often weakened forms.
Specificity. The complementary relationship between the
binding sites of anibodies directed against
determinants of a similar- type antigen.

115
Transferase enzyme. A type of enzyme that catalyzes the
transfer of a monosaccharide molecule from a donor
substrate to the precusrsor substance. This type of
biochemical activity is related to the development of
A,B, and H antigens
Transplacental hemorrhage. The entrance of fetal blood cells
into the maternal circulation.
Universal donor. A minomer often used for group O Rh
negative blood.
Universal recipient. A general term used to refer to a group
AB patient.
WAIHA. Warm autoimmune hemolytic anemia. This form of
autoimmune anemia is associated with antibodies
reactive at warm temperatures.
Wharton’s jelly A mucoid connective tissue that makes up
the matrix of the umbilical cord
Zeta potenitial The difference in electrostatic potential
between the net charge at the cell membrane and the
charge at the surface of shear.

116
Bibliograph
y
Benjamini E,
Coico R,
sunshine G.
Immunology
A short
th
Course, 4
ed. Wiley
Liss,2000.

1. Boorman
Kathleen
E, Dodd
Barbara
E,
Lincolon
P.J.

Blood
Group
Serolog
th
y, 6 ed.
Churchill
Livingsto
ne,UK,
1988.

2. Bryant
Neville J
An
Introduc
tion to

Immuno
hematol
 rd
ogy, 3
ed.
Philedelp
hia, WB
saunders
, 1994.

3. Cheesbr
ough
Monica.
District
Laborat
ory
practice
in
Tropical
countrie
s, Part 2.
Cambrid
ge, UK,
2000.
4. Estridge
BH,
Reynolds
AP,
Walters
NJ.
Basic
Medical
Laborat
ory
Techniq
ues, 4th
ed.
Delmar,
2000.
5. Linne JJ,
Ringsrud
 KM.
Clinical
Laborat
ory
science:

The
Basics
and
Routine
Techniq
th
ues, 4
ed.
Mosby,
1999.

6. Louise
Mary
Turgeon.
Fundam
entals of

Immuno
hematol
ogy,
Theory
and
Techniq
nd
ue. 2
ed.
Williams
and
Wilkins,
New
York,
1995.

7. Mc
 Clelland
DBL.
Hand
book of
transfus
ion
medicin
rd
e, 3 ed.
The
stationer
y office,
London,
2001.
8.Tibebu M,
The Blood
Bank
Manual,
Ethiopian
Red cross
society,
National
Blood
Transfusion
Service,
Addis Ababa,
1998.

1
1
7
 MODULE

2.Harmful
Traditional
Practices
For the Ethiopian Health Center Team

Dawit Assefa, Eshetu Wassie, Masresha Getahun,

Misganaw Berhaneselassie, and Atsinaf Melaku

Awassa College
In collaboration with the Ethiopia Public Health Training Initiative, The Carter Center,
the Ethiopia Ministry of Health, and the Ethiopia Ministry of Education

2005
 Funded under USAID Cooperative Agreement No. 663-A -00-00-0358-00.

Produced in collaboration with the Ethiopia Public Health Training Initiative, The Carter
Center, the Ethiopia Ministry of Health, and the Ethiopia Ministry of Education.

Important Guidelines for Printing and Photocopying

Limited permission is granted free of charge to print or photocopy all pages of this
publication for educational, not-for-profit use by health care workers, students or
faculty. All copies must retain all author credits and copyright notices included in
the original document. Under no circumstances is it permissible to sell or distribute
on a commercial basis, or to claim authorship of, copies of material reproduced
from this publication.

©2005 by Dawit Assefa, Eshetu Wassie, Masresha Getahun,

Misganaw Berhaneselassie, and Atsinaf Melaku

All rights reserved. Except as expressly provided above, no part of this publication
may be reproduced or transmitted in any form or by any means, electronic or
mechanical, including photocopying, recording, or by any information storage and
retrieval system, without written permission of the author or authors.

This material is intended for educational use only by practicing health care workers
or students and faculty in a health care field.
 TABLE OF CONTENTS

Topic Page
Table of Contents ............................................................................................................ i
Acknowledgment ............................................................................................................ii
Preface ..............................................................................................................................iii
About the Authors ......................................................................................................... v
Abbreviations ................................................................................................................. vi
Core Module ...................................................................................................................... 1
Introduction to the core module ........................................................................... 1
Pre-test and post-test questions .......................................................................... 3
Case Studies 1 ....................................................................................................... 8
Case Studies 2 ....................................................................................................... 10
Objectives of the core module.............................................................................. 11
Major Harmful Traditional Practices ........................................................................... 12
1. Female Genital Mutilation ................................................................................. 12
2. Uvulectomy ......................................................................................................... 20
3. Milk teeth extraction .......................................................................................... 23
4. Early marriage/ Arranged marriage ................................................................ 25
5. Marriage by abduction....................................................................................... 30
6. Food taboos ........................................................................................................ 32
7. Other HTPs ......................................................................................................... 34
Conclusion of the core module ............................................................................ 37
Satellite Modules .............................................................................................................. 38
Introduction to the satellite modules.................................................................... 38
1. Satellite module for Public Health Officers .................................................... 39
2. Satellite module for Public Health Nurses ..................................................... 49
3. Satellite module for Medical Laboratory Technicians .................................. 58
4. Satellite module for Environmental Health Technicians .............................. 66
References…………………………………………………………………..……………....75
Key Answer…………………………………………………………………………………77

i
 Acknowledgments

First, we would like to thank the Ethiopian Public Health Training Initiative (The
Carter Centre) for giving us the opportunity to develop this module and organize
the process of preparation. Next, we also acknowledge the members of staff of
Dilla College involved in reviewing this material at the workshop held at Furra
College in Yirgalem town. Our thanks also go to both the local and international
reviewers who gave valuable comments and suggestions. Our heart felt thank
goes to Dr. Fran Weinger for her valuable comments and Mr. Tariku Lambiyo for
incorporating the comments to the module. Last but not least, the secretaries of
the Health Science Faculty of Dilla College who helped us in typing this
manuscript are also appreciated. Our special thanks goes to W/o Berhane
Abebe, secretary of the department of Biomedical and Behavioral Sciences, for
compiling and being involved in all types of activities needed for the module.

ii
 Preface

The module is prepared for the health center teams of Ethiopia to be used as a
quick reference on HTPs. Though HTPs are very prevalent in the country, they
are not incorporated in school curricula nor has enough research materials been
developed in this area. Therefore, this module helps the learner to understand
the most common HTPs prevailing in the country, how and why they are done.
Emphasis is also given to the impacts of these practices on communities and the
victim him/herself. These facts will provide base line knowledge to design
appropriate management approaches. This means that you are expected to
understand these deep-rooted problems and to be involved in the treatment of
victim cases as well as preventing them happening to the new coming generation
through the implementation of appropriate measures.

Harmful traditional practices that affect certain specific population groups such as
omen and children are very rampant in Ethiopia. It is said that there are around
140 HTPs affecting mothers and children occurring in almost all ethnic groups of
the country.

Even though the prevalence and the degree may vary these practices, which
have numerous long-term devastating effects, are also performed on all
continents of the world. They also date back to before the birth of Christ.
However, unlike in developing countries like Ethiopia where traditional practices
are performed in more than 80% of the population, some countries in the Middle
East as well as immigrants to Europe and USA have abandoned these practices.
This was the result of the work contributed by religious leaders, government
bodies and the victim people themselves.

The problem in Ethiopia is not only that these traditions continue to be

practiced, but the people who participate in all the practices do not know
about the harmful effects of the acts. Because of this most Harmful

iii
 Traditional Practices are very resistant to change. Therefore appropriate
strategies must be designed and implemented by all community members to
prevent the occurrence of these practices.

To read this module, it is necessary (especially for the sub topics like female
genital mutilation) to be reminded of the basic anatomic structures of the body
related to the subject. The module also includes units for each specific group of
the health center team of Ethiopia (satellite module for public health nurses,
medical laboratory technologist, and environmental health technician and for the
health officer).

In addition later on in the module the specific tasks you are expected to
understand and perform will be summarized based on your category in the health
center team. However, all the team members are asked to thoroughly read the
core-module before dealing with the specific units.

Points to be stressed are highlighted with bullets, tables and brief illustrations.
Pre- and post-tests are also included for you to take.

We hope you will gain from this reading and express your concerns about these
problems.

iv
 About the Authors

Dr. Dawit Assefa: Lecturer and department head of Biomedical and Behavioral
Sciences in Awassa College of Heath Sciences.

Dr. Eshetu Wassie: Lecturer in Awassa College of Heath Science and currently
attending postgraduate studies in public health.

Ato. Masresha Getahun (B.Sc in Nursing): Assistant lecturer in Awassa College

of Heath Sciences.

Ato. Misganaw Berhanesellasie (B.Sc in MLT): Assistant lecturer in Awassa

College of Heath Sciences and currently attending postgraduate studies in
microbiology.

Ato Atsinaf Melaku (B.Sc in EH): Assistant lecturer in Awassa College of Heath
Sciences.

v
 Abbreviations

HTPs - Harmful Traditional Practices

FGM - Female Genital Mutilation
NCTPE - National Committee on Traditional Practices of Ethiopia
WHO - World Health Organization
MOH - Ministry of Health.
TBA - Traditional Birth Attendant
NGO - Non-Governmental Organizations
SNNPR - Southern Nation’s Nationalities and Peoples Region
EC - European Commission
ENI - Ethiopian Nutrition Institute
EHTs - Environmental Health Technicians
TBAs -Traditional Birth Attendants
V/S - Vital signs

vi
 CORE MODULE

Introduction

Tradition represents the sum total of all behaviors that are learned, shared by a group of
people and transmitted from generation to generation. It includes language, religion, types
of food eaten, and methods of their preparation, childrearing practices and all other values
that hold people together and give them a sense of identity and distinguish them from
other groups.
To evaluate a traditional practice as harmful/beneficial we might use the objective
instruments based on the knowledge gained from social and natural sciences. Today we
have ample knowledge about the physical nature of man, his physical anatomy and social
life. It is therefore possible to objectively assess whether a specific traditional practice is
harmful to the physical nature of a human being, his psychology and social needs and
development, and therefore incompatible with scientific theory and practice.
Ethiopia is a country of famous and long-standing history. It is also a country with many
useful and promotional traditions. These major beneficial traditional practices include
breast feeding which is common especially in the rural areas of the country, postnatal
care, social gathering such as “Edir” “Ekub” etc., caring for the aged, the disabled and
others within the family circle.
On the other hand Ethiopia is a country where harmful traditional practices continue to
devastate, especially the health and social condition of mothers and children. These
harmful traditional practices include:

*HTPs that affect mothers

o Female genital mutilation
o Early marriage
o Massaging the abdomen of pregnant woman with butter during difficult labor
o Marriage by abduction
o Shaking women violently to cause placental delivery
o Throat piercing using hot iron rods to remove the placenta
o Giving “Kosso” to pregnant women
1
 o Encouraging excessive fertility
o Plucking finger nails of women prior to weddings
o Application of cow dung etc. to the umbilical cord
.♦ HTPs that affect children
o FGM
o Milk teeth extraction
o Food taboos
o Uvula cutting
1.5 Forbidding food and fluids during diarrhea
1.6 Keeping babies out of the sun
○Feeding fresh butter to new born babies
.♦ HTPs that affect all members of the community
o Eyelid incision o
Rectal ulceration o
Son preference o
Cupping
o Tribal marks
o Cauterization
o Blood letting
o Tattooing
o Emasculation

In this module we will limit our discussion to the most prevalent and severe HTPs in
Ethiopia.

2
 Pre and post-test questions for all members of the
health center team of Ethiopia

Answer both the short answer and the multiple choice questions individually or in groups of
3-5 persons. Put your answers on a separate blank sheet. You will find key answer key on
the end page of the module.

2.3 Short answer questions

What are the most common Harmful Traditional Practices in our country?
List some of the major impacts of HTPs on the victims.
Describe some of the major HTPs in your area of the country where work
and the reasons given for practicing them.
Which population groups are usually most affected by many of the Harmful
Traditional Practices in your working area?
5) What do you think can be done to prevent the occurrence
of Harmful Traditional Practices?

II. Multiple choice questions

For the multiple choice questions, circle the best response
6.3 The most serious immediate complication of Uvulectomy is:
Teeth breakage
Hemorrhage
HIV/AIDS
None of the above.
6.4 Which of the following measures can be taken to eradicate Harmful Traditional
Practices?
Involvement of religious and opinion leaders in educating the community

Empowerment of the most affected population groups, particularly women,

in decision making

3
 C. Use of media like radio messages for propagation of the harmful effects of
the practices
D. All of the above.

1.3 Long term complication of FGM includes:

Painful sexual intercourse
Psychological trauma
Obstructed labor
D. All of the above

1.4 Reasons that are usually given to practice FGM include:

To promote the cleanliness of the female genitalia
To make a woman sexually active before marriage
For treatment of repeated urinary tract infection
All of the above.

1.5In which of the following regions are milk and permanent teeth
extraction commonly seen?
Amhara
Gambella
Somali
Tigray

1.6 Which traditional practice is not harmful to the community?

Early marriage
Female genital mutilation
Prohibition of some food items during pregnancy or child hood
None of the above.

The cut off point for the definition of early marriage according to the current
Ethiopian legislation is:
15 for females and 18 for males
15 for females and 19 for males
15 for females and 20 for males
4
 D) 18 for both sexes
E) 20 for both sexes

- Reason usually given to deprive pregnant mothers of white foods

includes all except:
It makes the child bald
It makes amniotic fluid offensive
It will be plastered on the body of the child
It will make labor difficult.

Pre and post-test questions for Public Health Officers

Give short answer for the following questions on a separate sheet of paper. Key answers
are found at the end of the module.

1. According to the WHO classification, list the four types of FGM.

2. Write at least five complications of female genital mutilation.
3. Write five reasons for and the complications associated with early
marriage.
4. Write the general management principle for a girl raped by her abductor.
5. List major strategies for preventing harmful traditional practices in Ethiopia.

Pre and post-test questions for Public Health Nurses

Give short answers on a separate sheet of paper. Key answers are found at the end of the
module.

1. Write at least four complications of marriage by abduction.

2. Write at least four complications associated with FGM.
3. Write the possible nursing diagnosis for uvulectomy patients.
4. List the nursing interventions for major harmful traditional practices.

5
 Pre and post-test questions for Medical Laboratory Technicians

Choose the best single answer on the following multiple-choice questions and write the
answer on a separate sheet of paper. Answers are found at the end of the module.

1. A spore forming bacteria transmitted by a contaminated blade or when dung is applied

to the cord stump is:

A. Chlamydia

B. Clostridium

C. Streptococcus

D. Staphylococcus

E. None of the above.

2. The specimen of choice for the detection of Gonococcal infection in women is:

A. Vaginal discharge

B. Urine sediment

C. Cervical swab

D. Cerebrospinal fluid

E. None of the above.

3. Which one of the following disease conditions is the consequence of harmful traditional
practices?

A. Anemia

B. Sexual transmitted disease

C. Wound infections

D. Urinary tract infection

E. All of the above.

6
Pre and post-test questions for the Environmental Health Technicians

Short answer questions:

- List the roles of the EHTs in the campaign against HTPs.
- List some of the effects of HTPs.
- How do HTPs conflict with health promotion?
- Why is it important to involve the practitioners of HTPs in the intervention
program?

Multiple Choice Questions (key answers are found at the end of the module):
- Which group should be involved in the fight against HTPs?
Religious leaders
Political organizations
Practitioners of HTPs
Health professionals
All of the above.
- Which of the following health risks is/are associated with HTPs?
HIV/AIDS
Infections
Death
All of the above.

7
Case study 1

The following is a fictitious case and does not refer to any person known or to any
individual.

Abaynesh – A lifetime victim of HTPs.

Abaynesh was born and brought up in remote village called Chico in Sidamo, which is 20
Kilometers south of Yirgalem. She was a very pretty child. At the age of 5 years, her
parents promised to let her marry Alehegn who was then 18 years old. Following the
request of Alehegn’s family a practitioner in the area circumcised her. The circumcision
was done because Alehegn’s first fiancé was uncircumcised and was found to have
committed adultery. During the days following her circumcision Abaynesh became sick;
she developed fever, diarrhea and vomiting. She also used to cry now and then, especially
upon micturition. Her parents thought it was because of the recent milk tooth that had been
pulled out by the woman who performed the mutilation. However Abaynesh become sicker
and was taken to a traditional healer.

The healer cauterized Abaynesh with a hot iron on the abdomen and anus.Soon after she
got well and the marriage with Alehegn was celebrated. It was decided that she should
begin living with her in-laws until she was 12 years old. When Abaynesh became mature
enough she started to live with her husband and got pregnant at the age of 13.

Completing her 9-month pregnancy was the most difficult experience for Abaynesh. She
was still a child and needed parental care and attention but at that early age she was given
the responsibility of motherhood. At the end of her pregnancy Abaynesh was in prolonged
th
labor for 5 days. On the 6 day she was very weak and she was taken to Yirgalem
Hospital where she was operated upon and had a stillbirth. In addition her uterus was ruptured
and surgically removed. As a result of the complications of prolonged labor she also had a
ruptured bladder and could no longer control her urine. The attending physician referred her to
Addis Ababa Fistula Hospital for better management of this condition. At the end of her
treatment and recovery the doctor at the hospital told Abaynesh that she would never be
pregnant again because of the problems that occurred as a result of the difficult labor. When
she completed her treatment at the Hospital she was sent back to her village.
8
Knowing that Abaynesh could no longer have children her husband divorced her and sent
her back to her parents.

She stayed with her parents until she was 20 years old. At 20 years of age she went to
Dilla to look for a job so that she could become economically independent. She got a job
as a housemaid and worked for 3 years. When she started to know the town and the
people in Dilla she decided to work in a hotel as a waitress. Since she was very physically
attractive her friends wrongly advised her to be a prostitute.

Unfortunately Abaynesh contracted the deadly disease HIV/AIDS and could no longer
work in the hotel since she was very weak and sick. Finally she decided to go back to stay
with her parents in the village .She was nursed by her family and died two years after she
left Dilla.
Source:
Modified from NCTPE/EC project fund brochure on early marriageDec.1999, A.A., Ethiopia

Learning activity:
• List the different HTPs that Abaynesh experienced.
• Identify some of the complications that can be attributed to the major HTPs you
have listed above.
• What do you think is your role in preventing the future occurrence of HTPs in
general?

9
 Case Study 2

An African writer called Dahabo Ali Muse wrote the following poem to express the
psychological pain caused by FGM:
Feminine pain
And if I may speak of my wedding night:
I had expected caresses, sweet kiss, hugging and love.
No, never!

Awaiting me was pain, suffering and sadness.

I lay in my wedding bed, groaning like a wounded
Animal, a victim of feminine pain.
At dawn, ridicule awaited me.
My mother announced: Yes she is a virgin.

When fear gets hold of me,

When anger seizes my body,
When hate becomes my companion,
Then I get feminine advice, because it is only feminine pain,
And I am told feminine pain perishes like all feminine things.
The journey continues, or the struggle continue,
As modern historians say.
As the good tie of marriage matures.
As I submit and sorrow subsides.
My belly becomes like a balloon
A glimpse of happiness shows,

A hope, a new baby, a new life!

But a new life endangers my life,

A baby’s birth is death and destruction on me!

It is what my grandmother called the three feminine sorrows.

She said the day of circumcision, the wedding night

10
 And the births of a baby are the triple feminine sorrows.

As the birth bursts, I cry for help, when the battered flesh tears.
No mercy, push! They say.
It is only feminine pain!

And now I appeal:

I appeal for love lost, for dreams broken, For
the right to live as a whole human being.
I appeal to all peace loving people to protect, to support
And give a hand to innocent little girls, who do no harm,
Obedient to their parents and elders, all they know is only smiles.
Initiate them to the world of love,
Not to the world of feminine sorrow!
Source: NCTPE/EC project brochure on FGM, poem by Dahabo Ali Muse (Somalia)
Dec 1999, A.A., Ethiopia

Learning activity

• Discuss the effects of FGM that occurred to the woman in the poem.

Objectives of the Core Module

General objective:

After completion of this core module the leaner is expected to have the knowledge, attitude
and measures used for the management of HTPs that occur in Ethiopia.

Specific objectives:
At the end of the module you are expected to be able to
- Explain female genital mutilation.
- Explain early marriage.
- Discuss marriage by abduction.

11
 3.2 Discuss milk teeth extraction.
3.3 Explain the different food taboos.
3.4 Discuss Uvulectomy.
3.5 List other common HTPs.
3.6 Implement the different measures used for prevention of major HTPs.

Major Harmful Traditional Practices

As described in the introduction there are as many as 140 harmful traditional practices
which exist in Ethiopia. This module addresses the following:

1. Female Genital Mutilation (FGM)

3.3 Description

FGM is a traditional operation that involves cutting away parts of the female external
genitalia or other injuries to the female genitalia for cultural reasons.

1.2. Epidemiology

The historical roots of the practice are not well-known but they appear to date back 2,000
years to ancient Egypt during the time of the Pharaohs. A global review of FGM shows that
the custom of FGM is known to be practiced in one form or another in more than 28
countries in Africa including Ethiopia. According to a survey carried out in 1987 by NCTPE,
more than 80% of women in the country (100% in certain communities) are circumcised. It
also says 60% of Ethiopian woman support the practice. In places where FGM takes place
it is performed during infancy, childhood or adolescence.

12
Table 1: occurrences of FGM (source: NCTPE brochure, HTPs as a risk of HIV Oct. 2001).

Region Occurrence (%)

Tigray 73
Afar 96
Amhara 92
Oromiya 99
Somali 100
Benishangul 63
SNNPR 54
Gambella 2
Addis Ababa 69
Harrari 90

1.3 Types of FGM

FGM is a collective term for the different practices that involve cutting of the female
genitalia. According to WHO (1995) FGM is classified in the following four categories:
1. TYPE I (clitoridectomy): This is partial/total excision of the hood of the clitoris.

Figure 1: Type I FGM.

13
2. TYPE II: This is partial/or total excision of the labia minora (Vaginal inner lip), with
excision of the clitoris. This is also called the Sunna type.

Figure 2: Type II FGM.

3. TYPE III: This is excision of the external genitalia (labia majora) partially or totally
together with the above two procedures. Following the procedure the vaginal opening is
closed/narrowed with stitches (Infibulations/Pharaonic incision) allowing a small opening
for urine and menstrual blood to be excreted.

Figure 3: Type III FGM.

14
The word "infibulation" is derived from the Latin word "fibula," meaning a pin or clasp. The
term has been given to a mulative procedure in which the vagina is partially closed by
approximating the labia majora in the midline. Clitoridectomy may or may not be included,
but the essential part of the operation consists of partial closure of the vulva and the
vaginal orifice. The custom is deeply rooted in the country and has been performed since
remotest time on all social classes in both the interior regions and in the few coastal towns.

TYPE IV: this is any other procedure done together with/other than the above three
Pricking, piercing, or incision of the clitoris and/or labia; stretching of the clitoris and/or
labia; cauterization by burning of the clitoris and surrounding tissues; scraping (anguria
cuts) of the vagina orifice or cutting ( gishiri cuts) of the vagina; introduction of corrosive
substances in to the vagina to cause bleeding, or of herbs in to the vagina with the aim of
tightening or narrowing the vagina; any other procedure that falls under the definition of
female genital mutilation given above.

Such devastating procedures are usually performed by traditional practitioners, often by a

layperson with no or only rudimentary training. These individuals are most commonly aged
with poor visual acuity and well known in the community as experts in the procedure.

1.4 Reasons given for practicing FGM

Though the reasons for practicing FGM vary from society to society, the following are
commonly mentioned as a reason for undergoing FGM:

♦ To maintain the moral behavior of women in the society

Almost all ethnic groups who perform FGM believe that sexual sensitivity and response
are reduced if a female undergoes mutilation. Women who are not circumcised are
believed to have a strong desire for sex and it is believed may indulge in extramarital sex
to satisfy their needs.

♦To preserve virginity

In some areas virginity is highly valued. It is also a reflection of the status of a family and
an integral part of a marriage transaction. Therefore FGM is performed to preserve the
15
virginity of a woman until she is married. Most commonly, infibulation is performed to make
the woman totally handicapped for premarital sex.

♦For hygienic reasons

A woman is considered unclean if she is not circumcised. It is said that FGM decreases
genital secretion and helps a woman to be neat. Moreover uncircumcised women are
believed to produce worms and that their genitalia are foul smelling.

♦To ‘calm’ a girl and make her decent

A girl who is not circumcised is believed to be unreserved (Aynawta), breaks utensils, be
wasteful and becomes absent minded. Many believe circumcision is done to control the
woman’s reaction/emotions, to help her to be decent and reserved.

♦Esthetic reasons
The genitalia of uncircumcised women are believed to be unattractive and uninviting to a
man for sexual intercourse. Therefore FGM is performed so that the genitalia look beautiful
for the man who owns the girl.

♦Religious requirement
Among certain religious groups prayers made by uncircumcised women are believed to be
unacceptable. For some ethnic groups, to be uncircumcised is considered to be an insult
to God. Some even believe that the Holy Book says “that which protrudes from the body is
excessive and should be trimmed”.

♦To avoid difficulty at delivery

Some people believe that FGM helps to shorten the duration of labor and the passage of
the newborn child through the birth canal, despite evidence to the contrary.

♦To increase matrimonial and marriage opportunities

In most ethnic groups marriages are pre-arranged by families. Therefore a woman has to
be circumcised pre-martially to find a husband. A circumcised woman is said to have a
tight perineum that increases the pleasure of a man during intercourse. In some instances,
even in a circumcised woman, if a man is not satisfied with the tightness of the vaginal

16
opening, the woman is made to go back to her parents, saying she is not well and
adequately circumcised.

♦ Myth
There is a myth that says mutilated girls are fertile. Infertility may occur If a woman is not
mutilated. Some also believe that if FGM is not done the clitoris will grow and dangle
between the legs.

1.5 Impact of FGM

FGM is now seen as a health, human rights, women’s reproductive rights and
developmental issue having the following consequences:

1.5.1 Physical consequences

Physical complications often arise as a result of infection due to use of unsterile tools and
other materials. These complications are divided into immediate and delayed
complications.

♦ Immediate complications are those which arise soon (in few seconds) or within a few
days of the procedure:
Hemorrhage
Shock
Severe pain
Damage to the nearby urinary structures
Septicemia
Tetanus
Bone fracture following heavy pressure applied to the
struggling girl
Death

- Late complications are those that occur later on in life:

Heavy scarring (keloid scar)
Dyspareunia (painful intercourse) or apareunia (not being able to have sexual
intercourse)
17
 _ Neuromas from cut ends of the nerves
− Haemotocolpos (accumulation of menstrual blood caused by closure of the vaginal
opening by scar tissue)
− Recurrent urinary tract infection
− HIV/AIDS
− Obstructed labor and other obstetric complications
− Urinary and rectal fistulae usually following delivery

1.5.2 Psychological consequences

It is obvious that most of the psychological impacts are as a result of the trauma and the
physical complications. They are due to the deep-rooted memory of the act of FGM
(especially if performed in older children) and the loss of control of their body. These
psychological consequences include:

♦Lack of sexual desire and/or satisfaction: Absence of the most sexually sensitive part
of her body usually makes a woman sexually unresponsive and fails to achieve
satisfaction during sexual intercourse. Loss of sexual satisfaction may also be because
of painful intercourse (Dysparunia).

18
 ♦Fear of sexual intercourse: Especially if the genital mutilation was done when the child
is old enough to remember painful experiences, she unknowingly may avoid anybody,
such as a physician or even her husband to have a look at or touch her genitalia. This
is mainly because of the deep-seated memory of the pain and suffering she had during
the act of mutilation.

♦Lack of self-confidence (low self esteem): These women may not be confident enough
to participate in certain social gatherings if, for example, they have fistulas and
complications arising from that. In addition they may feel they are not sexually
competent and tend to avoid men.

1.5.3 Social impacts

Female genital mutilation has serious consequences for women’s health both physical and
mental. This in turn may affect their productivity. Ill health, lack of concentration and poor
output, reduce their ability to participate effectively in decision making, in productive
activities and in the care and nature of children who are the future generation and leaders
of the society.

1.6 Measures against FGM:

• Educating the community about the risks and consequences of FGM through
available channels of communication, also its role in the transmission of HIV.
• Involving religious and other influential leaders of the community, TBAs and
circumcisers in educating the public about this issue.
• Strongly encouraging the government to be critically committed to making the laws
more stringent and to develop enforcement strategies.
• Improving the availability, accessibility and quality of modern health services
• Including knowledge about harmful traditional practices in school curricula
appropriate to the grade levels in elementary and secondary schools.
• Integrating active teaching learning strategies about harmful traditional health
practices, especially with favor prevention , in all health sciences curricula.

19
 Education programs that are sensitive to the cultural and religious importance of FGM
seem to be the best hope of eradicating the practice. Education can, however, be a
long process, as evidenced by the UN plan" to bring about a major decline in female
genital mutilation in 10 years and completely eliminate this practice within three
generations." There are some signs, however, that education programs are having an
impact. In Ethiopia, the Ministry of Education has used radio broadcasts to warn about
the dangers of FGM. The broadcasts are sponsored by the National Committee on
Traditional Practices in Ethiopia, a committee that includes UN agencies. These
actions, along with a government ban on FGM, have had "encouraging" results.

2. Uvulectomy

2.1 Description
Uvulectomy is a procedure involving the cutting of the uvula and sometimes the near-by
structures such as the tonsils. The uvula is a small soft tissue that hangs down from the
back of the mouth above the throat and between the two lymphoid tissues (tonsils). It
helps to prevent choking during swallowing and is used in producing certain sounds
necessary for language communication.

Figure 5: Anatomy of the oral cavity

20
Traditional instruments such as a sharp blade, horsetail hair or thread attached to a loop
are used for the cutting of these important structures. Whichever instrument is used the
process starts after an attendant or the mother firmly holds the child. Then the child’s
tongue is pulled out and a flat piece of wood is placed in the mouth to prevent injury. The
practitioner inserts the thread or the horsetail hair through a hollow stick in the mouth to
trap the uvula, which is then cut by pulling the strings or by the use of a sharp blade.
Sometimes following this procedure the tonsils are made to bleed after being scratched by
a sharp object.

2.2 Epidemiology

Uvulectomy is commonly practiced in the northern part of Ethiopia but rarely seen in
Gambella region. According to the national survey of 1997G.C. about 84.3% of the general
population practice uvulectomy despite some 48.6% of them being aware of the harmful
effects of the act indicating the enormous need for education about the damaging results.

Table 2: Occurrences of Uvulectomy (source: NCTPE brochure, HTPs as a risk of HIV

Oct.2001).

Region Occurrence (%)

Tigray 98
Afar 99
Amhara 89
Oromiya 89
Somali 100
Benishangul 73
SNNPR 54
Gambella 39
Addis Ababa 72
Harrari 90

21
2.3 Reason given for this practice
The swelling of the uvula and the tonsils has been considered a serious health problem
and some feel it to be a life threatening illness. Other reasons given for the practice among
communities in the country include:
• Prevention of headaches
• Ensuring a clear voice
• Prevention of blindness and change of eye color
• Avoiding the swelling of the body and depression of the anterior fontanel.

Figure 6: The process of Uvulectomy.

2.4 Impact of Uvulectomy

The procedure of Uvulectomy may easily introduce harmful micro-organisms which could
lead to serious complications. These complications could be either local or disseminated
(systemic).

The local complications are the most prevalent ones and include:
• Speech problems
• Injury to the tongue (e.g. through being pulled)
• Broken teeth as a result of a struggle

22
 • Local infection
• possibility of no teeth or deformity of teeth
• Excessive bleeding
• Long term dental problems

And some of the systemic complications include:

• Sepsis
• Tetanus
• HIV/AIDS

2.5 Measures against Uvulectomy

• Educating people about the normal functions of uvula and tonsils
• Educating people about the complications of uvulectomy, with special emphasis on
blood borne infections like HIV.
• Using active teaching learning strategies to prepare all health care providers in
effective and appropriate preventive techniques.

3. Milk Teeth Extraction

3.1 Description:
Milk teeth extraction is the procedure of pulling out the early teeth of children. The growth
of milk teeth begins when the child is 5-7 months old and starts from the bottom front and
is later followed by the upper front teeth. Teething does not make the child ill but it is often
painful and makes the child restless. The mother or other care giver usually rubs the gum
to relieve pain, which can help the introduction of microorganism causing diarrhea and
other infections in the child. With the introduction of diarrhea and other infections the
mother will take her child to a traditional healer to remove the child’s teeth.

3.2 Epidemiology

As mentioned in the introduction, milk teeth extraction is another serious HTPs occurring in
almost all regions of Ethiopia. According to the National Baseline Survey on HTPs carried
out in 1997, milk teeth extraction is practiced in more than 80.2% of the general

23
population. In Gambella region there is very high prevalence of this practice that is used as
a marker for ethnic identity in the region. The practice here is not only the extraction of milk
teeth but also extraction of permanent front lower teeth. This should increase our concern
about this practice.

Table 3: Occurrences of milk teeth extraction (source: NCTPE brochure, HTPs as a risk of
HIV Oct.2001).

Region Occurrence (%)

Tigray 85
Afar 99
Amhara 93
Oromiya 89
Somali 99
Benishangul 72
SNNPR 94
Gambella 98
Addis Ababa 42
Harrari 66

st
Milk teeth extraction is carried out in early childhood, as early as the 21 day after birth
and is typically done by creating an incision with a razor blade, knife or sharp iron. These
materials are usually not hygienic. After the incision the “root” is then extracted using a
pointed iron (“wosfe”) or pincers (“worento”) or the practitioner’s fingernails which are
especially grown for this purpose. The wound is finally cauterized using heated mustard or
garlic, or rubbed with salt to stop further bleeding.

3.3 Myths given for practicing milk teeth extraction

According to traditional beliefs diarrhea and fever at the time of milk tooth eruption may be
due to a worm in the child’s gum. This problem is believed to be relieved by having the
teeth extracted or the gum being drilled and the primary teeth or the new permanent teeth

24
extracted or carved out. The practice of milk tooth extraction is also listed as a treatment of
poorly growing older children.

Summary of myths given for milk teeth extraction

● To avoid/as a treatment for diarrhea and vomiting

• As a tribal marker
• For a remedy to poor body growth of children

3.4 consequences of milk teeth extraction

Like Uvulectomy milk tooth extraction provides easy access for micro-organisms to cause
local infections or sepsis. Other complications include:

• Possibility of no teeth growth

• Injury to the tongue
• Excessive bleeding
• Tetanus
• HIV/AIDS and other blood borne infections

3.5 Measures against the practice of Milk Teeth Extraction

Educating people about the complications of milk teeth extraction, especially emphasizing
the danger of blood borne infections like HIV, helps prevention of the practice.

4. Early Marriage/Arranged Marriage

To be free from early and arranged marriage is a reproductive right.

4.1 Description
According to Ethiopian law early marriage occurs before the age of eighteen. There are
three marriage arrangements for early marriage:

25
 • Promissory marriage: Oral promises made by two families to give each other their
children in marriage right after or even before the birth of their children.
• Child marriage: Marriage of children less than ten years of age. Girls then live with
their in-laws (Madego) or live with their parents until the age of twelve with the
frequent visits of their in-laws (Mililis).
• Elderly marriage: In some cultures very young girls are forced to marry an old man
for matrimonial or other benefits.

Figure 7: Early (elderly) marriage.

4.2 Epidemiology
In most parts of Africa early marriage is considered a norm. In Ethiopia, 34.1 percent of
girls have their marriage contracted before the age of 15 and 75% get married at the age
of 17. They may stay with their husband’s family until they are considered old enough to
establish their own family. A survey conducted in Northern Ethiopia revealed the mean
preferred age at first marriage to be 14.2 years for girls.

According to the baseline survey carried out in 1997 early marriage occurs in the regions
of the country as follows:

26
Table 4: Occurrences of early marriage (source: NCTPE brochure, HTPs as a risk of HIV
Oct.2001).

Region Occurrence (%)

Amhara 82
Tigray 79
Benishangul 64
Somali 26
Harrari 29
Gambella 64
SNNPR 51
Oromiya 49
Addis Ababa 51
Afar 46

Early marriage is not only a practice of rural and remote areas but also occurs in
significantly high numbers in urban centers like Addis Ababa. The problem is entrenched
in the minds of the people and it is useful to ask why the practice of early marriage is so
prevalent.

4.3 Reasons given for practicing early marriage

Parents are motivated to give their female children for early marriage for several reasons:
• To ensure virginity:
• This is a very common reason given for early marriage. A girl who is not a virgin
at her first marriage is considered unfit for family life and is a disgrace to the
family. The groom will severely beat the bride, take away the entire dowry given
to her and chase her out. The marriage is immediately dissolved. Therefore girls
are forced to get married early so that the above mentioned consequences will
not happen.
• To increase fertility:
If a girl marries early she will produce many children which is considered as an
asset for the family.
• To conform to societal norms:

27
 Early marriage avoids stigmatization by the community. If a girl does not marry
early in her life people call her “Komakerech” which literally means she is no
longer wanted for marriage.
• To get early access to material benefits:
If a girl marries early especially with her virginity intact her family gets early
access to material benefits.
• To secure the future:
It is desirable for children to marry when the two parents are still young so that
the children’s future is secured before the parents get old and die.

4.4 Consequences of early marriage

Early marriage has many consequences. The most common are

Health related consequences:
8. Early pregnancy
9. Unwanted pregnancy
10. Early child bearing
11. Risks to the babies born from them, e.g. low birth weight, prematurity, etc.
12. Obstetric complications like prolonged labor, obstructed labor, fistulae, etc.
13. Sexually transmitted diseases and infection from HIV/AIDS early in life
14. Premature death mainly as a result of obstetric complications
15. Physical trauma (genital)
16. Vaginismus (being tense during sexual intercourse) which interferes with sexual
pleasure

Social consequences:
Early marriage, adolescent pregnancy and child bearing completely disrupt the life of the
victims. They are denied the opportunity for education. The lack of education restricts the
girls’ capacity to make their own choices in life. Without proper education the girls’
chances of being gainfully employed are highly limited. An illiterate mother is very likely to
raise illiterate children. In addition, the following adverse consequences may also follow
early marriage:

28
 9. Little chance of re-marriage if the marriage does not last long
10. Social stigmas which may follow obstetric complications, like fistula
11. Too many births which may have an adverse effect not only on the family’s
economic status but also on the needs of the community as a result of
overpopulation
6. Maternal death has serious impact in disrupting the family hence migration,
street kids or malnutrition can be the end result
• Urban migration and the possibility for being involved in risky behaviors such
as commercial sex work.

8. Measures to prevent early marriage

Educating and informing the public on the harmful effects on early marriage
using different media channels including radio, TV, discussions, workshops,
public debates, newspapers etc.
Educating parents and the community on the importance of gender equality,
education for girls, empowerment of women and the negative effects of early
marriage. Here ” idir” and ”mahber” can be used.
Strengthen the educational opportunities for girls to attend secondary, college
and postgraduate levels of education.
Youth education (both in-school and out-of-school youth) on the harmful effects
of early marriage.
Educating and informing community, religious and political leaders or decision
makers on the effects of early marriage.
Undertaking legislative measures to revise laws including the legal age of
marriage for girls and boys and the legal measures to be taken if early marriage
is practiced.
Establishing pressure groups in communities, schools, etc that would educate
the public and fight against early marriage.

29
5. Marriage by abduction

5.1 Description

Abduction is the illegal carrying away of a woman for marriage or rape. Marriage becomes
a holy act if it occurs with the consent of both the bride and the bridegroom. The act
becomes more meaningful if the families or relatives of the couples are aware of the
process.

In Ethiopia and other African countries unlawful marriage (marriage by abduction) is a

common practice. This occurs by kidnapping a girl without the knowledge of the girl or
family. It is sometimes known to the girl that abduction will happen. This usually occurs if
the girl is in love with the kidnapper and the kidnapper is not the one to be selected by her
family. In this case marriage by abduction occurs and her family is informed. When the girl
is willing and ready the process of abduction and its consequences are usually less
complicated.

In most cases rape follows the forced marriage. Rape is a guarantee that the abductor will
most likely succeed in keeping the girl after some negotiation through local elders or by
paying some compensation to the girl’s family. It is believed that once this has happened
the girl is less likely to get another husband.

Figure 8: The process of abduction.

30
5.2 Epidemiology

Like the other harmful traditional practices discussed earlier marriage by abduction is
prevalent in Ethiopia. According to the base line survey for more than 50% of young girls in
the community marriage occurs by abduction. Its prevalence is also high in regions like
Oromia and SNNPR.

Table 5: Occurrences of marriage by abduction (source: NCTPE brochure, HTPs as a risk

of HIV Oct.2001).

Region Occurrence (%)

Tigray 36
Afar 66
Amhara 33
Oromiya 80
Somali 33
Benishangul 69
SNNPR 92
Gambella 41
Addis Ababa 18
Harrari 43

5.3 Reasons given for practicing marriage by abduction

The following are the most common reasons given for the practice:
• Inability to pay (excessive) dowry
• Difference in ethnic status
• To avoid the excessive expenses of the conventional wedding ceremony
• Refusal or anticipated refusal by the girl or her parents
• If there are prior secret commitments or promises made by the couples or they have
fallen in love.

31
5.4 Impact of marriage by abduction

Some of the harmful effects of marriage by abduction include:

• Rape, which is sexual violence occurring to the abducted woman without her
consent
• The girl may be physically injured, occasionally to the extent of disability and/or she
may be killed by her abductors
• Conflict between families or ethnic groups
• Tremendous expense for conflict resolution
• Unstable marriage
• Psychological stress of the woman which may lead to suicide
• Sexually transmitted infections such as HIV/AIDS and unwanted pregnancies.

5.6 Measures to prevent marriage by abduction

• Education of the public about the harmful effects of abduction through media, etc.
• Involving the influential leaders, NGOs and other institutions in educating and
mobilizing the community
• Empowering women to play an active role in education, health, management and
planning
• Taking legal action at all levels against those who practice abduction

6. Food taboos
6.1 Description
Food taboos are a common practice of prohibiting certain food items for pregnant and/or
lactating women or girls in general. Children, especially girls, are most vulnerable to this
practice.

Foods that are good sources of energy and protein are not allowed to be consumed by
pregnant women for reasons such as difficult and prolonged labor due to fears of a large
baby. Similarly sources of vitamins and minerals are restricted during pregnancy mainly
due to the fear of offensive discharges during delivery and skin diseases on the body.

32
6.2 Epidemiology
Studies conducted among 25 ethnic groups in central, eastern and southern parts of
Ethiopia (ENI 1995) have reported that food items that are white in color (e.g. milk, fatty
meat, porridge, potato, banana, etc.), clean vegetables, colostrum and fruits are prohibited
to be consumed by pregnant/lactating women and children. Hence, the diet of a pregnant
woman is limited to food items such as ‘teff,’ bread made of sorghum, corn, etc. Only a few
studies have been done on the different food taboos in Ethiopia.

Table 6: Occurrences of food taboos (source: NCTPE brochure, HTPs as a risk of HIV
Oct.2001).

Region Occurrence (%)

Tigray 53
Afar 39
Amhara 42
Oromiya 52
Somali 38
Benishangul 37
SNNPR 56
Gambella 59
Addis Ababa 13
Harrari 29

6.3 Reasons given for food taboos

It is believed that certain food items which are white in color will be plastered on the body
of the baby and cause labor difficulties and will also produce offensive uterine fluid
(amniotic fluid) during delivery. In addition green vegetables, like green peppers, are
believed to causes a bad odor in both the mother and the baby and make the newborn
baby bald. In some communities girls are forced to consume enormous quantities of food
items like milk and meat, causing obesity. This is due to the strong desire of families to
have their girls marry as early as possible.
Most mothers discard the colostrum produced in the first few days postpartum. They think
colostrum causes abdominal pain and diarrhea for the new born baby. Sources of vitamins
33
such as mango, orange and banana are also restricted since they are believed to cause
worms, malaria and diarrhea to the growing child.

6.4 Impact of food taboos

Restricting the food of pregnant and lactating women and children may cause the following
health effects:
Under-nutrition of the pregnant mother leading to increased risks in pregnancy and labor,
such as
− Anemia and other micro-nutrient deficiency illnesses
− Low resistance to infection
Restricting children from important dietary items leads to
− Increased risk of infection
− Protein-energy malnutrition like kwashiorkor and Marasmus
− Poor physical and mental growth

6.6 Measures against food taboos

Educating woman and the community about the harmful effects of depriving mothers and
children of important food items can help to change attitudes.

7. Other HTPs
As was stated earlier, there are nearly 140 HTPs known to be practiced in Ethiopia. The
major ones have already been discussed. The following are also commonly practiced in
some regions:

2 Incision of eyelid: This is a common practice in the northern part of the country as a
treatment of eye disease (commonly eye infections). It is performed with a razor
blade and often results in secondary skin infections and excessive bleeding.
3 Bloodletting through vein resection on the scalp and on the arms: These practices
are called “wagemt” and “mognbagegn” in local terms. They are commonly
practiced in the highlands of the Amhara and Tigray regions. The rationale behind
blood letting is that the body is believed to be decaying internally causing tissue

34
 swelling and deteriorating health. It is prescribed for treatment of encephalitis,
rheumatism, high fever and headache (usually seen during epidemics of
meningitis). The most serious complication is the puncture of an artery and the
patient bleeds profusely.
8. Cauterization (“Tattate” in local terms): Ailments such as conjunctivitis, headache,
ear infections, tuberculosis, and bone fractures are sometimes treated with
cauterization (burning with hot material). It is most common in the East (Dire Dawa
and Somali region). The belief is that intense heat destroys the pathogenic
substances inside the body. Although the modalities of the procedure differ it is
usually carried out with hot charcoals, a hot iron or a burning stick.
9. Uterine Massage: Kneading and squeezing a woman’s abdomen with the intention
of inducing labor. This act may cause excessive bleeding, uterine rupture and
incomplete placental separation.
10. Rectal Ulceration: A procedure occasionally seen in the eastern part of Ethiopia as
a treatment for diarrhea and rectal prolepses. Little is known about this practice.
11. Son preference: The expectation of most parents in developing countries like
Ethiopia about the overall contribution to a family’s income from girls is very small
once she marries and leaves home. Because of this parents with limited resources
invest more in their sons than their daughters. In addition girls are made to work at
an earlier age than their brothers and work harder and longer at home while the
boys are allowed recreation after school hours. Because of son preference women
are often exposed to repeated pregnancies in a bid to have sons to the detriment of
the mother’s health and well being. Sons are sent to school more than girls. One in
five girls is sent to school compared to 50% of boys. Girls are made to discontinue
school earlier than boys. The reasons include the danger of girls losing their
virginity, the need to help their mothers at home, or that they will be exposed to
unplanned pregnancies. There is also an attitude of “what better can a learned
female does except serving her husband”.
Wife inheritance: In this case a woman, if her husband dies, is made to marry his
brother or close friend. Similarly, if the wife dies, the husband will immediately
marry her younger sister. This practice is common in many parts of Ethiopia
particularly among followers of Islam. This has an effect in the transmission of HIV.
35
 Learning activity

We hope you have read the core module thoroughly. Answer the following questions
based on case study 1.

1. What are the HTPs you have seen in relation to the victim?
2. Which complications are related to the HTPs?
3. What measures can be used to save the lives of other victims in the future?

In addition try again to answer the post-test questions presented at the beginning of the
module. We hope you will do well and continue with the satellite module for your
respective group in the Health Center.

36
 Conclusion
There are a variety of both beneficial and harmful traditional practices in Ethiopia. The
harmful traditional practices are done for various reasons but can cause a spectrum of
health and social problems.
In addition to the deep-rooted beliefs, customs and rational attitudes, lack of knowledge
and being unaware of the effects of these practices help maintain these problems.

Especially with the advent of the recent pandemic, HIV/AIDS, these HTP’s may impose
even greater health and economic consequences. Therefore, encouraging beneficial
practices and the eradication of the harmful ones is the responsibility of the whole
community members and all health professionals.

Health professionals especially, those close to the community, play a major role in
designing and implementing strategies to eradicate HTP’s in Ethiopia.

37
 UNIT ONE

SATELLITE MODULE FOR PUBLIC HEALTH OFFICERS

Introduction
This satellite module contains four units, one for each group of the Health Center team of
Ethiopia i.e. Satellite modules for public heath officers, public health nurses, medical
laboratory technicians and environmental health technicians.

Before going to the satellite module for your specific group make sure that you have read
and thoroughly understood the body of the core module and attained the objectives stated
at the beginning of the core module. It is also our expectation that you will have taken the
pre/post-test questions, the case studies questions and the learning activities mentioned
earlier in the module.

In the later pages of this module the details of the satellite modules are summarized giving
more emphasis on your duties, roles and tasks to enable you to handle the existing
problems of HTPs in Ethiopia.

We hope that you will work hard to attain the objectives for the satellite module of your
specific group.

38
SATELLITE MODULE ON MAJOR HARMFUL TRADITIONAL PRACTICES
IN ETHIOPIA FOR PUBLIC HEALTH OFFICERS

Introduction

This satellite module is prepared for Public Health Officer Students to give an emphasis on
specific issues that were not covered by the Core Module.

Instruction for using the satellite module:

• You must carefully study the core module before reading the satellite module
• Do the pre-test and post-test of the satellite module before and after studying the
module and try to appreciate the difference in the results
• Refer to the Core Module where indicated
• Carefully analyze the tasks expected to be accomplished by you

Learning objectives

After reading this satellite module, the student should be able to:

• Identify the different types of HTPs in Ethiopia

• Diagnose and manage major complications resulting from HTPs for victims and their
families at Health Center level
• Play a leadership role in designing appropriate strategies and measures against
HTPs in your working area

1.1 Female Genital Mutilation (FGM)

Refer to the core module for the types of FGM and reasons given for the practice. The lists
on the complication and consequences of FGM are also addressed as follows:

39
1.1.1 Complications and consequences of FGM (Also see the core module)

Health consequences
Immediate complications:
• Hemorrhage, shock, even death from severing of blood vessels
• Infection of the incision and of the urinary tract, tetanus from using
non-sterile equipment
• Damage to the urethra and surrounding tissue
• Acute urinary retention from very severe pain

Intermediate complications:
• Adhesion of vaginal lips that can prevent normal urine and menstrual blood
outflow
• Delayed wound healing
• Bartholin's cyst and abscess formation
• Keloid scar formation
• Dyspareunia
• Hepatitis

Late complications:

• Hematocolpos from adhesion of vaginal lips, scarring and narrowing of vaginal

hymen
• Infertility from infection of the uterus, fallopian tubes, and ovaries
• Obstructed labor and its consequences like fistula formation
• Recurrent urinary tract infections
• Inability to deliver vaginally without tearing
• Urinary calculus (from urine stasis, bacterial infection)
• HIV/AIDS from use of unsterilized materials

40
 Psychological consequences

These can be life long and have a grave impact on the quality of life (See the core
module).

Socio-economic implications (Also refer to the core module).

1.1.2. Management of complications of FGM

Table 7: Management of FGM (N.B: General management is similar with any trauma
patient).

Complications
Hemorrhage
Shock (hemorrhage)
Infection
Wound infection
Urinary tract infection
Tetanus
Acute urinary retention
Injury to the surrounding tissue
Adhesions of vaginal lips
Delayed wound healing
Cyst and/or abscess formation
in the Bartholin's duct
Keloid scar
Hepatitis
Other Consequences
Neuromas
Measures
Arrest bleeding, take vital signs
Administer IV fluid based on body weight amount of blood
lost, and stability of vital sign
Follow standard surgical wound management, give broad
spectrum antibiotics
Give Amoxicillin or Cotrimoxazole as first choice
Wound management + immediate referral to hospital
- Give procaine penicillin, sedate the patient with diazepam,
chlorpromazine, and refer urgently
Analgesics + catheterization to evacuate urine
Follow the principles of management of trauma patient
Try to separate the two lips (defibulations). If difficult refer to
hospital
Identify the cause and treat accordingly
Incise and drain the abscess using sterile procedures
Give broad spectrum antibiotics against mixed infections
Refer to hospital
Treat according to the cause and condition of the patient
Needs inter-sectoral collaborations
Refer to hospital after giving analgesia

41
1.2 Early Marriage
Refer the core module for the definition, epidemiology, reasons and consequences of early
marriage.

1.2.1. Management of complications of early marriage

Before dealing with the measures, ask yourself the questions listed on the left hand
column of the following table:

Table 8: Management of early marriage.

Does the victim have Measure

Forced sexual assault? See the management of sexual assault (rape)
Obstetric complications (teenage Follow the usual antenatal care procedures
pregnancy and its effects, obstructed Close antenatal follow up
labor)? Institutional delivery
Treatment of complications if any
Refer the mother if beyond your scope
Unwanted pregnancy and its Provide the usual management of abortion and
complications like unsafe abortion? give post natal care
Other complications? Treat individually
Psychological and emotional trauma Ideally, locate a support system for the victim.
Refer for psychological assistance if available.

1.3 Marriage by abduction

For definition, epidemiology and consequences refer to the core module.

1.3.1 Management
Victim is very unlikely to seek medical help. As almost all abductions are followed by rape,
see management of rape victim below in case they visit you.

1.3.1.1 Carefully diagnose the incident

• History of sexual penetration using force against the person’s will

42
 • Evidence of signs of force such as physical injury (abrasions, scratch marks,
bleeding on the face, eyes, extremities, genitalia). Torn clothes including underwear
etc.
• Evidence of sexual contact like wetting of the genital areas, the perineum, and
thighs or underwear like pants
• Freshly torn hymen in a previous virgin rape victim, bleeding around perineum,
blood-soaked underclothing, etc.
• Psychological distress
• Other signs of consequences listed in the core module
N.B Do not do digital or speculum vaginal examination

1.3.1.2. Management principles for abduction followed by rape at health center level

• All cases must be reported immediately to the appropriate law enforcement agency
like a police station
• General management is similar to any trauma patient, but you should take some
special considerations. Follow these steps to assist the rape victim:
• If possible, you must always refer the patient to where there is a doctor or hospital
after doing all of the following
o Listen to the victim carefully
o Advise the victim not to do anything that might destroy evidence
o Don’t clean wounds
o Don’t change or discard clothes
o Don’t bath
o Handle clothing that was removed as little as possible. Bag each item
separately and transport with the patient to the hospital or next higher level
as evidence
o Reassure the victim of safety
o Believe the victim’s story
o Avoid unnecessary touching of the victim
o Ask for permission before proceeding to treat the victim

o Let the victim know what the plan of action is

43
 o Do not examine the genital area unless the wounds are severe enough to
require some inspection before treatment
o Stay with the victim unless the victim specifically requests a private time
o Talk to the victim about the victim’s fear and pain
o Allow the victim to cry or otherwise express emotions
o Protect the victim's privacy
o Do not offer false hopes to the victim
o Document all evidence encountered or observed on the patient’s card and
referral sheet
o Provide accurate information to the victim
o Treat victim with respect, kindness, and gentleness
o Immediately refer victim to a hospital with a relative
N.B.
• Don’t question the victim about the details of the situation; this type of questioning is
best left to the investigation team from the police department
• Don’t ask if penetration occurred
• Don ‘t inquire about the patient’s sexual history or practices and don’t ask questions
that may lead to feelings of guilt such ask “why were you out so late alone?”
If the victim is unlikely to or refused to go to a hospital or next higher level where there is a
doctor, document all the evidence carefully and do all the following:
• Increase psychological support with subsequent follow-up
• Follow the usual management of trauma patients including careful wound care,
prophylactic antibiotics and tetanus prophylaxis
• Prophylaxis for sexually transmitted diseases (Gonorrhea and Chlamydia):
- Doxycycline (100 mg PO Bid for 7-10 days) + Spectinomycin (2 gm IM stat)
according to patient's condition and drug availability
- Alternatives: TTC (for age above 15) or erythromycin for Chlamydia 500 mg
PO QID for 7-10 days + Cotrimoxazole or other drugs effective against
gonorrhea
• Give prophylaxis for unwanted pregnancy by administering emergency
contraception pills (post-coital pills) if she arrives within 72 hrs of the incident. It
could be:
44
 Neogynon
First dose: 2 tabs within 72 hours of unprotected sex, repeat 2 tabs 12 hours later
Microgynon
First dose: 4 tabs within 72 hours after unprotected sex
Repeat: 4 tabs after 12 hours of giving the first dose
Follow for pregnancy and STD in case your prophylaxis fails
8. Always help the patient (victim) any time she wants

Table 9: Other common harmful traditional practices in Ethiopia.

Traditional practice Reasons Complications

A Affecting the health of women
• During labor and delivery
- Violent shaking of the - To shorten labor - Trauma both to the mother and
laboring mother - To rapidly expel fetus
- Throat piercing with hot iron placenta - Postpartum hemorrhage
rod - To remove - Pain, bleeding, infection, etc
placenta
• During pregnancy
a) vigorous abdominal a) To avoid drying of a-i) Pre-term labor and delivery,
massaging with butter the abdomen trauma to the placenta, etc.
a-ii) Harmful effects both on the
fetus and mother e.g. Affects the
labor if excess
b) Giving ‘kosso’ (herbal b) To clean the b) Liver damage of both the mother
medicine) abdomen and fetus
c) Prohibiting foods c-I) Avoid bad smell c) Under nutrition of the mother
commonly carbohydrates during labor, avoid and baby e.g. Anemia,
(potato, gruel, linseed, difficult labor from hemorrhage, low birth weight,
porridge, banana, sugarcanes large baby infection, vitamin A deficiency.
etc.) Protein (meat, milk c-ii) Avoid baldness
products, chicken, egg, etc), in the new born.

45
 fatty meat, vegetables.
• Food discrimination for girls - Avoid fast growth - Malnutrition
e.g. eggs, chicken and green so that avoid early
vegetables. sexual activity
B Affecting infants and children To prevent - Immediate complications
• Milk – teeth extraction and - Diarrhea, vomiting, - Severe pain, bleeding, shock,
Uvulectomy fever death
- Use unsterile material - Itching and rest less - Swelling of mouth
ness - Difficulty in eating and breast
- Growing of worms feeding
in the root of milk - Infection (wound infection,
teeth, etc. tetanus etc)
- Late complications
- HIV/AIDS

1.4 General management approach of milk teeth extraction and Uvulectomy

Table10: General management approach of milk teeth extraction and Uvulectomy.

Complications Principles of management

Early complications
• Severe pain Analgesics
• Bleeding Arrest bleeding, fluid replacement
• Swelling and wound Wound care, mouth care etc
• Infections (wound) Antibiotics, wound care
• Shock Treatment of pain, bleeding, infection

• Difficulty in eating and breast Use of soft and fluid diets, IV feeding if
feeding necessary

Late complications
• Absence of teeth then after Counseling, reassurance and education
• HIV/AIDS on the problems and how to cope with
them

46
1.5 Strategies and measures to eradicate harmful traditional practices (HTPS) in
Ethiopia (See the conventions and policies against HTP in Ref 1and4).

Strategies
• Education of the public through:
Organizing seminars, workshops and training courses on harmful effects and the
role of each individual on eradication efforts:
Employees of different community groups, organizations and
institutions
Leaders (of the community, religion, politics organizations)
Creating general public awareness using different communication
channels such as radio, posters, films, etc
• Community participation
• Strong government (political)
commitment (Law enforcement, police)
• Multi-sectoral collaboration
• Mobilizing civil organizations
Human right’s organizations, professional associations youth associations and women's
groups
• Empowerment of women
To play an active role as educators, trainers, planners, managers, evaluators, and
decision-makers.

Measures
There is a need for a concerted effort from each and every part of the society. In general:
• Giving the public adequate information about harmful traditional practices
• Understanding the community and how it lives/acts and which traditional practices
are more prevalent
• Building alliances and partnerships in efforts to eradicate HTPs with both
governmental, non-governmental organizations and private sectors
• Follow-up and refresher training courses to maintain and ensure the continuity of
the intervention programs

47
• Mass media, using both locally available electronic and print media to reach all
segment of the population.
• Arranging with the concerned bodies annual campaigns with key thematic
messages both at local and national levels
• Training sensitization workshops for influential members of the community,
teachers, health workers, students, etc so that the trainee will be able to conduct
education and training to others
• Educating practitioners of harmful traditional practices and helping them to be
aware to the complications of their practices. They can in turn teach the community
• Using the existing traditional structures like ‘edirs’, ‘Ikub’, 'mahiber', etc for teaching
the community. Leaders of these traditional structures can organize and help
teaching the community
• Strengthen Information, Education and Communication (IEC) activities such as
Production and distribution of IEC materials such as posters, booklets, leaflets,
pamphlets, etc.
Organizing drama and song contests, exhibitions, etc to disseminate information
regarding the nature and effects of harmful traditional practices
• Creating awareness on policies related to the issues of HTPs for individuals and
groups e.g. Information on women’s health and human rights policies, legislations on
rape, abduction, and early marriage.
• Creating alternative employment opportunity for the practitioners
• Formal education for girls (future mothers) is one key long term strategy. So, we
should be able to under score the benefits of girls education not only to minimize or
abolish HTPs, but for overall development of the society.

48
 UNIT TWO
SATELLITE MODULE ON MAJOR HARMFULTRADITIONAL
PRACTICES IN ETHIOPIA FOR PUBLIC HEALTH NURSES

The detailed description of harmful traditional practices is presented in the core module.
Review the core module while using this satellite module.

Learning objectives
At the end of this session the student will able to:
• Define the major types of harmful traditional practices in Ethiopia
• List the major types of harmful traditional practices in Ethiopia
• Identify the health related effects of harmful traditional practices in Ethiopia
• Determine the preventive measures against harmful traditional practices in Ethiopia
• State the possible nursing diagnosis for major harmful traditional practices
• Describe the nursing intervention for complications of major harmful traditional
practices.

The nursing management for major harmful traditional practices is as follows:

2.1 Nursing management

2.1.1 Marriage by abduction

Victims of abduction are very unlikely to seek medical help but if you happen to encounter
a case of abduction you should follow the following nursing processes:
Nursing processes
Assessment
• Take their history
- Ask the reasons for seeking health care (C/C)
- Carry out a physical examination
• Take and record V/S
• Assess ABC (Air way, Breathing, Circulation)
• Examine injury site for size, depth and fracture

49
 - Examine for STD (sexual transmitted disease)

Possible nursing diagnosis

- High risk of infection related to exposure
- Anxiety related to fear of family and other community members
- Ineffective family coping: compromised related to
poor communication between client and abductor
- High risk for fluid volume deficit related to bleeding during rape

Nursing plan
8. Prevent infection
9. Promote effective coping
10. Relieve anxiety
11. Maintain hydration
12. Provide support

Intervention
Follow usual management of rape patient including
6.2.5Careful wound care
6.2.6 Prophylactic antibiotics and tetanus prophylaxis

Prophylaxis for sexually transmitted diseases (Gonorrhea and Chlamydia)

• Doxycycline (100 mg PO Bid for 7-10 days) + Spectinomycin (2 gm IM
stat) according to patient's condition and drug availability
- Alternatives: TTC or erythromycin for Chlamydia 500 mg PO QID for 7-
10 days
- Co-trimoxazole or other drugs effective against gonorrhea

50
Give prophylaxis for unwanted pregnancy by administrating an emergency contraception
pill. It could be:
Neogynon First dose: 2 tabs within 72 hours of unprotected sex, repeat 2 tabs 12
hours later.
Microgynon First dose: 4 tabs within 72 hours after unprotected sex. Repeat: 4 tabs
after 12 hours after first dose.
Then follow for pregnancy or STD in case your prophylaxis fails and decide accordingly.
Always help the patient (victim) any time she wants.

N.B. Rape is a legal term and the examining health worker is encouraged to use
terminology such as “alleged rape” or “alleged sex".
- Create psychological support with subsequent follow-up (address
both social and cultural factors)
- Demonstrating a caring attitude is essential in providing emotional
support for patients
- Empower the patient by increasing their sense of control. Patients
also may be taught activities to decrease anxiety and to gain a
sense of control over their reactions. The most common
approaches are relaxation exercises, music therapy and guided
imagery.
- Establish good relations with members of the family to
deal with problems
Advise the patient how to cope with this problem in the future
Assess V/S frequently and give fluid if necessary.

6.2.6 Female Genital Mutilation

Nursing Processes
FGM is mostly carried out by traditional practitioners and often by laypersons. The practice
has many grave consequences. It is the nurse’s responsibility to manage both immediate
and later complications.

51
Assessment
Physical examination:
Early cases
• Assess vital signs
• Assess for other signs and symptoms of shock
• Look and assess for signs and symptoms of infection
• Look and assess for pain
• Assess for anxiety, guilt feelings or poor self-esteem
Later Cases
• Urinary problems
• Recurrent infection of kidney (UTI) problems
• Decreased sexual enjoyment

Nursing Diagnosis:
• High risk for infection related to the use of contaminated instruments
• Alteration in comfort related to severe pain of injury site
• Fluid volume deficit related to bleeding
• Anxiety related to difficulty of becoming pregnant and injuries during
birth
• Body image disturbance related to excessive growth of tissue on the
scar

Nursing Plan:
• Prevention of infection
• Relief of pain
• Maintenance of body fluid
• Relieve anxiety
• Refer to hospital if necessary
• education for prevention of this practice

52
Intervention:
- Infection can be prevented by
• Appropriate wound care
• Providing prophylaxis (see abduction management)
• Regular follow up for recurrent infection

- Relieving pain:
Effective pain management can occur only when systemic and regular assessments
take place. Assess the nature of acute pain including the location, intensity, quality,
timing (on set, duration, frequency, cause) and provoking and palliative factors.
• Use of analgesics
• Assess and record the effectiveness of analgesics
• Give analgesics to prevent or minimize pain

- Distraction and relaxation techniques:

Patients can be taught to modify their sensory input to control pain by activities that
promote distraction or relaxation.
11. Maintain hydration
12. Assess vital signs frequently
13. Measure input and output
14. Give appropriate IV fluid if necessary
15. Relieve anxiety
16. Give psychological support
17. Advise the reason for each problem
18. Arrange follow-up program
19. Body image disturbance
20. Provide gentle persuasion to explore altered body part
21. Help significant others to support and assist the patient
22. Offer opportunities for social contact with persons who have
had similar experience
23. Introduce patient to support groups
24. Assist the patient to express anger, frustration, and

53
 disappointment
Actively listen
Assist the patient to engage in problem solving activities
Identify behaviors that will help to restore self-esteem
Assist the patient to develop strategies to help cope with
loss, stress or environmental factors that can cause low
self-esteem.

5. Early Marriage
This is the most common problem in Ethiopia and has various impacts.
Nursing process
Assessment:
• Determine age at marriage
• Assess reason for early marriage
• Assess for possible complications like trauma to genital organs,
pregnancy, infection
• Assess the response of the family and client regarding this
marriage
• Assess social and economic problems of the family
(income, occupation, community attitude, and schooling).

Possible nursing diagnosis:

• Altered elimination of urine related to trauma of injury site
• Anxiety related to fear of pregnancy and delivery
• Knowledge deficit related to pregnancy and possible
complications
• High risk for infection related to injury site
• High risk for altered growth and development related to
potential disruption of peer relationships and interruption
of secondary schooling to unplanned pregnancy.

54
 Nursing Plan:
• To establish normal urinary elimination pattern
• To relieve anxiety
• To give information on possible complications of pregnancy
• To prevent infection.
Nursing intervention:
• Catheterization if there is an alteration to the elimination of
urine
• Psychological support and give appropriate advice about
family planning
• Ask what kinds of social and financial support she needs
• Give information on possible complications of pregnancy
• Advice on the importance of Antenatal Care (ANC)
• Advise on the need for referral
• Ask if the girl is planning to continue with school.
• Counsel on ways to deal with potential barriers for continued
education

If she is pregnant, help her to see that the months of pregnancy will go faster if she is
busy. Remaining in school and doing well is a way of keeping busy as well as preparing for
the future. A high school education is necessary to obtain marketable skills. Girls will have
little chance of supporting themselves and their babies later if they are not allowed to
continue with their education .Once they delivered the baby, returning to school will be
difficult because they may have baby-sitter problems and because they may feel that they
are more mature than the other girls.

2.1.4 Uvulectomy and milk teeth extraction

Nursing process
Assessment
• Take history
• Physical examination (Ask about time and place
of procedure conducted)

55
 • Take V/S
• Assess for difficulty in swallowing and eating
• Assess for vomiting
• Assess for fever, headache or other pain
• Assess for swelling in the mouth, throat, nose
• Assess for respiratory problems (difficulty in breathing,
shortness of breathing)
• Assess for bleeding.
• Assess for broken teeth

Possible nursing diagnosis:

• High risk of ineffective airway clearance related to
edema and constriction of airway
• Pain related to the procedure conducted
• Knowledge deficit related to harmful effect of uvuloectomy
and tooth extraction
• Fluid volume deficit related to bleeding and inability to take
liquids or foods orally
• High risk for infection related to use of contaminated
instruments /objects during the procedure.

Nursing plan:
• Maintain effective airway clearance
• Relieve pain
• Provide information
• Maintain hydration
• Prevent infection.
Intervention:
• Take and record respiratory and pulse rate every 15 minutes
• Encourage fluids if able to swallow
• Clear the airway
• Give analgesics to relieve pain

56
 • Give appropriate management
• Monitor V/S, secure Iv fluid if necessary
• Give appropriate prophylaxis and treatment.

2.1.5 Other HTPs (see Public Health Officer Module and

Core module)

57
 UNIT THREE

SATELLITE MODULE ON HARMFUL TRADITONAL PRACTICES

IN ETHIOPIA FOR THE MEDICAL LABORATORY TECHNICIAN

Introduction

This satellite module assists medical laboratory technicians to participate in the team work
of the fight against harmful traditional practices, with specific emphasis on laboratory
investigation of diseases and ill-health conditions that result from the complications of this
practice.

Direction for using the satellite module

For a better understanding of this module the laboratory technicians are advised to follow
the succeeding directions:

• Do the pretest

• Read the core module thoroughly before going to the

satellite module

• Read the satellite module

• Consult listed references and suggested reading materials

• Evaluate yourself by doing the post test and referring to

the keys given.

Learning Objective

After the completion of this module students will be able to:

• Describe the laboratory methods that are used to diagnose disease conditions
related to harmful traditional practices (HTPs)

58
 • Relate specific consequences of harmful traditional practices with the laboratory
tests to diagnose them

• Describe how to collect, handle, process and refer different specimens

• Perform various laboratory techniques that are used to diagnose disease

conditions resulting from HTPs

• Describe the principles of tests that cannot be done in a health center laboratory.

N.B Refer to the Core Module for the detailed reading of HTPs in Ethiopia.

3.1 Laboratory diagnosis

The laboratory diagnosis for HTPs is based on investigating the causes of infections
(wound infections, STDs, UTIs, HIV and Hepatitis) and conditions such as anemia and
pregnancy that occur as a consequence of the harmful practices.

3.1.1 Wound infections

Wound infections associated with harmful practices, such as FGM, incision and
uvulectomy; mostly occur due to the use of unsterile techniques to perform the traditional
practices. The infected site may form an abscess containing pus; hence the swab material
from wounds and abscesses is required in the laboratory to diagnose the causative
organisms.

Collection of specimens from wounds and abscesses:

Specimens of pus should be collected by an officer or an experienced nurse. Pus from

abscesses is best collected at the time the abscess is incised and drained or after it has
ruptured naturally. In a Health Center laboratory take from a gram stain of the infected site
an evenly spread smear of the pus which is put on a clean slide and allowed to air-dry in a
safe place.

If the pus specimen is to be dispatched to a referral laboratory for culture, it is preferable to

collect the pus using a needle and syringe to aspirate the drainage. This avoids the

59
collection of normal flora and also enhances the recovery of anaerobes, which are often
associated with wound infections and abscesses. Transfer the collected pus (about 5 ml)
to a leak-proof sterile container. If pus is not being discharged, collect a sample with a
sterile cotton wool swab and insert it in a container of Amies transport medium, breaking of
the swab stick to below the bottle top which is to be replaced tightly. In all cases label the
specimen containers with the necessary patient identification and accompany with a
request form to reach the microbiology laboratory as soon as possible. Swab samples
from infected uvular areas are collected using a sterile tongue depressor to hold the
tongue down and a sterile swab to collect the specimens. Take the specimen by directly
swabbing of the infected site, being careful not to touch the other parts when inserting or
removing the swab. Smears for Gram stain can directly be made from the swab sample.
To refer the specimen for culturing, the swab containing the specimen can be placed in
Amies transport medium.

Gram staining technique: To perform Gram stain a thin smear from the wound material
is made as shown in the following figure

Figure 9: Preparing a bacterial smear using a swab.

The air-dried and heat fixed smear is placed on a staining rack and the primary stain,
crystal violet, is poured onto the slide for one minute. After one minute, the slide is rinsed
by gently pouring tap water on it. Gram’s iodine (a mordant) is then poured onto the slide.
After one minute the slide is again rinsed with tap water. A decolorizer, such as alcohol or
an alcohol-acetone mixture is added to the slide. After the decolorizing procedure is
finished the slide is again rinsed with water to remove all decolorizer. The slide is then

60
flooded with the counter stain, safranine, for one minute and placed to air-dry. After the
slides are completely dry they are ready to be viewed microscopically. Report the
abundance, Gram reaction and morphology of the bacteria including the presence of pus
cells, and whether the organisms are intracellular.

Common possible pathogens in gram-stained smear of wound and abscesses:

• Gram positive large rods with straight ends that could be Clostridium
perfrings

• Gram positive cocci that could be Staphylococcus aureus

• Gram positive streptococci that could be Streptococci pyogens

• Gram negative rods that could be proteus species E.coli,

Pseudomonas aeroginosa, or other coliforms.

NB: Also look for spermatozoa in vaginal discharge at the posterior fornix after rape/sexual
assault.

3.1.2 Sexually transmitted infections

Since marriage by abduction is often followed by rape, victims may acquire STIs. For the
laboratory diagnosis of STIs, the specimen can be taken from the urethra, vagina and the
cervix. Laboratory techniques; Gram stain, KOH and wet mount preparations are carried
out in the laboratory. Uro-genital specimens should be collected by an officer, an
experienced nurse or laboratory personnel. Urethral pus from a urethra to detect N.
gonorrhea can be collected on a sterile cotton wool swab by gently massaging the urethra
down wards from above. If pus is not appearing the swab must be inserted approximately
2 cm in to the urethra and rotated gently before withdrawing. Make a smear of the
discharge on a slide for staining by the Gram technique. For culturing, insert the swab in a
container of Amies transport medium breaking off the swab stick to allow the bottle top to
be replaced tightly.

N.B.: If possible the patient should not have passed urine, preferably for 2 hours before
the specimen is collected.

61
Vaginal swabs from vaginal secretions are collected with the swab inserted well into the
vaginal canal. The outer lips of the labia must be held apart so that the swab does not
touch them. Samples are commonly required to detect yeasts, fungi T. vaginalis and clue
cells. An endo-cervical swab is necessary for suspected gonorrheal infections and requires
the use of a speculum.

Gram stain:

Perform gram stain as described above.

N.B.: Fix by alcohol to preserve the intracellularity of N.gonorrhoea (Gram-negative

intracellular diplococcic are a presumptive diagnosis for gonococci infection).

Saline wet preparation:

The saline wet preparation is prepared by mixing a drop of vaginal specimen with a drop of
0.85% saline on a microscope slide. Examine this preparation to detect Trichomonas
vaginalis, a parasitic protozoa and clue cells which are vaginal epithelial cells covered with
the bacteria Gardenella vaginals.

Figure10: Saline wet preparation showing A. Trichomonas vaginalis B. Clue cells.

The KOH preparation:

A drop of vaginal material is mixed with one or two drops of 10% KOH solution on a
microscope slide .Examine the preparation under microscope to look for fungi, such as
Candida albicans. The KOH destroys structure such as epithelial cell and WBCs. Many
fungi present will appear as tangled masses resembling hairs or threads.

62
 Figure 11: KOH preparation showing presence of fungal elements.

3.1.3 Laboratory Diagnosis of UTIs and pregnancy

UTIs may occur in patients who have undergone FGM, as the bacteria may ascend and
infect the urinary tract. Urine microscopy, Gram stain and culture help to diagnose UTIs in
the laboratory. In early marriage and marriage by abduction pregnancy can occur and a
urine specimen is needed to rule out pregnancy.

Specimen collection:

Urine samples must be collected in clean, dry and disposable plastic containers with
screw-top lids. For urinalysis, including urinary sediment constituents, and HCG tests, a
freshly voided, randomly collected specimen of urine is usually satisfactory. However, an
early-morning urine specimen is the most satisfactory one to use because the urine is
usually more concentrated and contains the highest HCG concentration, which also
increases the chance of finding certain components.

A gram stained smear of the urine is prepared by transferring a drop of the urine sediment
to a slide and spreading it to make a thin smear and allowing it to air-dry. If significant
bacteria are indicated on properly collected urine, through examination of the gram stained
smear, the urine specimen must be cultured.

63
Quality assurance is employed in the blood bank to support error-free performance to
insure the highest quality patient care. Important factors in a routine quality assurance
program include evaluation of reagents, equipment, and personnel qualifications.

Urine microscopy:

To obtain sediment; pour 10 ml of urine into a centrifuge tube and centrifuge the tube at
1500-2000 rpm for 5 minutes. Decant the supernatant sediment leaving approximately 0.5 ml
in the tube. Re-suspend in the 0.5 ml of urine by gently tapping the tip of the tube. Place a
drop of the mixture on a microscope slide, cover it with cover slip and examine it under a
microscope. Report the examined WBCs, RBCs, epithelial cells, bacteria and yeasts.

Gram stain:

Follow the procedure described earlier

Possible pathogens E.coli, pseudomonas and proteus species.

Pregnancy (HCG) test:

Pregnancy tests are based on the detection of human chorionic gonadotropin (HCG) by
immunological methods. Many types of urine pregnancy tests are available and are based
on the interaction of HCG in the specimen with anti-HCG, an antibody specific for the
hormone. The rapid latex slide tests for the direct and indirect (inhibition) type are the most
commonly available.

Direct Method:

In the direct slide test, the latex reagent consists of particles coated with anti- HCG
antibodies. This reagent is mixed directly with the urine. If HCG is present in the urine it
will combine with the antibodies and cause agglutination of the latex particles. If no HCG is
present in the urine, there will be no agglutination of the latex particles. In the direct slide
test, therefore, agglutination of the particles indicates a positive test and no agglutination
indicates a negative test.

64
Indirect (Inhibition) Method:

In the inhibition test, urine is first mixed with the antiserum and the latex reagent is added.
If HCG is present in the urine it will combine with the anti-HCG antibody. This will leave no
antibody free to combine with the latex HCG and therefore there will be no agglutination of
the latex particles. If there is no HCG in the urine, the antibody will be free to combine with
the latex HCG and cause agglutination of the latex particles. In the inhibition slide test,
therefore, no agglutination indicates a positive test and agglutination indicates a negative
test.

Test for HIV and hepatitis infections:

HIV and hepatitis infections can be diagnosed by detecting the antibodies produced
against the viral antigens. In the laboratory antibodies to HIV or HBs Ag are detected by a
serological procedure such as ELISA. Summarized steps to detect HIV antibodies are as
follows:

Patient’s serum and control serum are added to a test well of a micro titration plate to
0
which HIV antigen is fixed by the manufacturer and the test is incubated at 37 C. HIV
antibody in the patient’s serum will attach to the HIV antigen in the well forming an antigen-
antibody complex. After washing, an enzyme conjugate reagent (usually an anti-human
IgG to which an enzyme peroxidase has joined) is added which in turn binds to any
specific antibody already bound to the antigen on the well and the test is re-incubated.
After washing to detect any conjugate bound to the well a substrate chromogen reagent is
added.

This is acted on by the enzyme resulting in the formation of a colored product. Samples
not containing specific antibody will not cause the conjugate to bind to the well.

After incubation, ‘a stop’ reagent is added to stop the reaction and the color produced in
the patient’s and control well is measured by spectrophotometer as it is directly related to
the amount of conjugate and hence the HIV antibody level.
Students are advised to read the Core Module for the theoretical
aspect of HTPs.

65
 UNIT FOUR
SATELLITE MODULE ON HARMFUL TRADITIONAL
PRACTICES IN ETHIOPIA FOR THE ENVIRONMENTAL HEALTH
TECHNICIAN

Introduction
The problem of HTPs is not only a health issue but also developmental, human rights,
women, gender, child survival and development, education and social service issues.

The environmental health technicians are in a position to improve the behavior and
practice of the community by health education and creating awareness. Different
approaches and strategies to address the issues of HTPs make it easier to bring the
expected change of attitude. In this regard EHTs play an important role.
[For the detail please refer to the core module]

This module can be used in the training of EHTs that are in actual training or those already
in service for management and prevention of HTPs.

Directions for using the module

• Do the pre-test pertaining to your profession in the core module
• When using this satellite module it would be profitable if the EHTs
follow the knowledge gained from the core module with his/her
knowledge of the community he/she is working in
• Read the reference materials listed to supplement your understanding
• Do the post-test and evaluate yourself by referring to the module.

4.1 Effects of HTPs

The most common effects of HTPs are:

66
I) Infection
These practices may lead to infections through repeated use of the same instruments and
contamination from normal bodily functions. Physical complications often arise as a result
of infection due to use of unsterile tools, unsanitary conditions and substances used to
stop the bleeding and heal the wound.

ii) Psychosocial stress

Leading to psychosocial stress, the girls/women will have poor practice in the household
such as poor sanitary practice, poor care for the child, and infrequent visits to the health
institutions.
[Also refer to the core module]

- Prevention
• Awareness creation to influential target groups on the
effects of HTPs and on management of these practices
• Their role in changing the behavior of the community
• Health and hygiene education.

Health and hygiene education programs should be planned to help community members
understand the importance of hygienic and safe practices in the prevention of infections
and general health promotion. The strategy should be supported by:
5. Simple health and hygiene education
6. Convincing programs and demonstrative programs
7. Different ways of communication to transmit the messages
8. Sensitization activities like posters, leaflets, video films, dramas, T-
shirts and other teaching materials
9. By selecting targets and the right person for this task
10. Language, culture and training on hygiene education
methods, principles and the prevention of HTPs
11. Educating practitioners of HTPs on their negative effects, on
the health of the population and involving them in the
intervention programs.

67
8.5 Teaching the local community using traditional associations
like Idir, Mahiber, Ikub, etc.
8.6 Political religious, social, educational and other leaders can
help by publicly acknowledging the negative effects of the
practices on the health of women and children and the
need to work against the HTPs
8.7 School Curricula
Any possibility of working with teachers
Any possibility of proposing changes to diversity of education to

include some age appropriate information in school curricula

68
 TASK ANALYSIS
1. TASK ANALYSIS ON HTP FOR PUBLIC HEALTH OFFICERS

KNOWLEDGE – OBJECTIVES AND ACTIVITIES

Learning objectives Learning activities

To describe the major HTPS in Define traditional practices
Ethiopia Identify HTPs
Discuss major HTPs and their Epidemiology in
Ethiopia
Study how HTPs and factors associated with
them affect human health
To describe the customs behind Identify and enumerate major reasons why
major HTPs people practice HTPS
To describe the major impacts of Study the complications and consequences of
HTPs major HTPs on the victim, on the family and on
the society at large
To describe the management State the management principles of the
approach of the complications and complications and consequences of the major
consequence of the major HTPS HTPS
To describe strategies and Set appropriate strategies and measures in
measures in the prevention and controlling and preventing HTPs and thereby their
control of major HTPs in Ethiopia complications and consequences

69
 ATTITUDE- OBJECTIVES AND ACTIVITIES

Learning objectives Learning activities

To accept HTPs as major Give emphasis on description and prevention

problems of public health Stress on prevention and control
importance Give emphasis on use of health education
To consider reasons, impacts, Give emphasis to rationale, complications and
and complications of HTPs as a impacts of HTPs in educating the public about
key step in behavioral change of them
the population
To appreciate that HTPs are Stress on health education, inter-sectoral
eradicable and their impacts are collaboration, community involvement and
highly preventable through appropriate media technology
different approaches Give emphasis to multi-sectoral and
interdisciplinary approaches

PRACTICE – OBJECTIVES AND ACTIVITIES

Learning objectives Learning activities

To participate and encourage Identify major HTPs in the community,

other health center team members reasons impacts and complications
participate in the prevention and Prevent and treat complications of HTPs
control major HTPs in Ethiopia Promote health education activities
Involve the community in the prevention and
control of major HTPs
Asses the effects of different prevention and
control strategies and act accordingly

70
2. TASK ANALYSIS ON HTP FOR PUBLIC HEALTH NURSES

Learning objective (expected Task analysis

outcome)
Define the major types of HTPS in Define the major types of HTPS
Ethiopia in Ethiopia
Identify the presumed reasons for Identify the presumed reasons
practicing HTPS for practicing HTPS
Identify the health related effects of Identify the health related effects
Knowledge HTPS of HTPS.
Identify preventive measures against Identify preventive measures
HTPS against HTPS
Advocate the effect of HTPS Educate the community about
Encourage knowledge of human right the consequences of HTPS on
Attitude issues health.
Accept that some of the traditional Design appropriate preventive
practices are harmful for health measures to intervene HTPS
Able to introduce appropriate nursing Early detection and treatment of
Skill interventions for victims of HTPS complications using the nursing
process.

71
7. TASK ANALYSIS FOR LABORATORY TECHNICIAN

Learning objective (expected Task analysis

outcome)

Define HTPs Define HTPs

List the common HTPs in Ethiopia List the common HTPs in

Ethiopia

Enumerate the basic complications of the Enumerate the basic

above listed HTPs. complications of the above listed
Knowledge HTPs.

Describe the different laboratory methods Describe the different laboratory

to investigate the consequence of the methods to investigate the
HTPs. consequence of the HTPs.

Educate the CHW about the harmful Educate the CHW about the
consequences of these practices. harmful consequences of these
Attitude
practices.

Advise patients and clients to raise their Advise patients and clients to
awareness on the bad health effect of the raise their awareness on the bad
HTPs. health effect of the HTPs.

Advocate the CHW on how to handle Educate the CHW on how to

laboratory specimens collected to handle laboratory specimens
diagnose the complications of HTPs. collected to diagnose the
complications of HTPs.

Perform the different laboratory Do laboratory investigation for

Practice
investigation for HTPs HTPs

72
- TASK ANALYSIS FOR ENVIRONMENTAL HEALTH TECHNICIANS

KNOWLEDGE OBJECTIVE AND ESSENTIAL TASKS OF THE EHTs

Learning objective Environmental health activities

Define and describe the most Define and describe the most
important HTPs in the country prevalent HTPs in the country

List common effects of HTPs List the common effects of HTPs

Describe the magnitude of HTPs and Describe the magnitude of HTPs

KNOWLEDGE its effect on health promotion and its effect on health promotion

ATTITUDES OBJECTIVE AND ESSENTIAL TASKS OF THE EHTs

Learning objective Environmental health activities

Believe in the harmful effects of HTPs Instruct practitioners, responsible

bodies about the harmful effect of
HTPs

ATTITUDE Believe in promoting proper Advocate hygienic /safe health

hygienic/safe health practice practices

Believe in utilization of health services Advocate utilization of health services

with special emphasis on children
and women

73
PRACTICES OBJECTIVE AND ESSENTIAL TASKS OF THE EHTs

Learning objective Environmental health activities

Demonstrate the effects of HTPs in Demonstrate the effects of HTPs in

health promotion health promotion

Organize exhibitions of posters,

PRACTICES photographs, and artwork on HTPs-
related subjects, and invite the
community.

Demonstrate proper communication to Display effective communication skills

community members, practitioners of with community members,
HTPs, and responsible bodies practitioners of HTps , and with
different officials and non- officials

74
 References
Cheesbrough Monica, District Laboratory Practices in Tropical countries 2000 Cambridge
University Press, UK.
Dnnessa Maria Delost. Introduction to Diagnostic Microbiology, A text and work book
Mosby Year book, Inc. St. Louis, Missouri, USA, 1997.
Estridge Barbara, Reynolds Anna P. , and Walthers Norman J.. Basic Medical Laboratory
Techniques Fourth edition, Delmar Thomson Learning, USA, 2000.
Joergenson Jean Linne Munson Karen Ringsrud. Clinical laboratory science. The basics
and routine techniques. Fourth edition, Mosby, Inc, st. Louis Missouri- USA, 1999.
Marke R. Dambro. ‘Rape crisis syndrome’ in Griffith’s 5-minute clinical consult. Lippincott;
USA 1999
Shirley A. Jones et.al (editors). ‘Sexual Assault’ in Advanced emergency care for
paramedic practice. Lippincott, Philadelphia, USA 1992.
NCTPE/EC. Major harmful traditional practices in Ethiopia: Resource material for higher
training institutes, National committee on traditional practices of Ethiopia, (NCTPE) Addis
Ababa, Ethiopia, Dec. 1999
NCTPE/EC. Children’s teeth and their care. National committee on traditional practices of
Ethiopia, Addis Ababa, Ethiopia. Dec. 1999
NCTPE/EC. Uvulectomy. National committee on traditional practices of Ethiopia, Addis
Ababa. Ethiopia Dec. 1999.
NCTPE/EC. Resource material on harmful traditional practices for policy makers, National
committee on traditional practices of Ethiopia, Addis Ababa Ethiopia Dec. 1999.
NCTPE/EC. Female Genital mutilation. National committee on traditional practices of
Ethiopia, Addis Ababa Ethiopia, Dec. 1999.
NCTPE/EC. Early marriage. National committee on traditional practices of Ethiopia, Addis
Ababa Ethiopia Dec. 1999.
NCTPE/EC. Marriage by Abduction. National committee on traditional practices of
Ethiopia, Addis Ababa Ethiopia Dec. 1999
NCTEP. Baseline survey on harmful traditional practices in Ethiopia, NCPTE, Addis Ababa
Ethiopia Sept. 1998.

75
R.J. Cook et. al. International Journal of Gynecology and Obstetrics. Ethical and legal
issues in reproductive health: Female genital cutting (mutilation/circumcision): ethical and
legal dimensions, 79(2002) 281-287, Canada

76
KEY ANSWERS

Answers for pre-test and post-test questions for all groups of Health Center teams of
Ethiopia.

Answers for the short answer questions:

9.2 a. Female genital mutilation
Early marriage
Uvulectomy
Marriage by abduction
Milk teeth extraction
Food taboos.
9.3 a. Health impact
Psychological impact
Socio-economic impact.
Cultural impact

9.4 Depends on the place where you work.

9.5 Women and Children

9.6 a. Education
Women empowerment
Involvement of key persons in the community
Involvement of high government officials etc.

Answers for MCQs

1 .B
4. D
5. D
6. D
7. B
8. D
9. D
10. A

77
Answers for pre-test and post-test questions for HO
- Type I Clitoridectomy
Type II Excision of labia minora and clitoris
Type III Excision of labia major (partial or total) including the clitoris referred to as
infibulation
Type IV any other act than the above.

4. Obstructed labor
Wound infection
Dysparunia
Psychological trauma
Repeated UTI etc (for the detail see module).

5. Refer to the Core module.

6. Refer to the Core module.
7. Refer to the Core module.

Answers for pre-test and post-test questions for PHN

8. Early marriage.
Infection
Psychological problems (fear, hopelessness)
Unstable, loveless marriage with high likely divorce
injury /trauma.

9. Promote cleanliness
Avoid sexyness
Preserve virginity by avoiding pre-marital sex
Conformity to tradition
To make it attractive for touch and sight.

78
 3. Infection Hemorrhage (shock)
Damage to urethra and surrounding tissue
Excessive out growth of tissue on the scar Difficulty during birth
Pain
Adhesion of vaginal lips.

4. High risk for ineffective air way clearance Pain related to injury site
Knowledge deficit Fluid volume deficit High risk for
infection.

5. Wound care
Providing prophylaxis (e.g. normogym) Psychological support
Monitoring vital signs
Providing analgesics and relaxation technique
Provide appropriate ordered IV fluid

Answers for pre-test and post-test questions for MLT

1. C
2. B
3. D
4. E

Answers for pre-test and post-test questions for EHT

Short answer questions (refer to the module). MCQ
1. E
2. E

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