BLOOD TRANSFUSION

Transfusion medicine is comparatively a fairly new

discipline
the history of transfusion evolved from mysticism
and pseudoscience to present-day rational therapy.
benefits of blood have been known for centuries.
lack of scientific understanding prevented the
effective utilization of blood.
combined expansion of knowledge in the areas of:
bioengineering, physiology, immunology,
mechanical engineering, biochemistry and genetics
improved the understanding of blood transfusion
Historical perspective
1613, William Harvey explained the circulatory
system
1628, Giovani suggested transfusion to prolong life
1649, Frances Potter attempted a transfusion
procedure on a pullet
1656, Wren proposed intravenous medication in his
dog
1665, Lower joined the jugular veins of two dogs
1667, Lower and King transfused Arthur Coga
1816, Blundell suggested transfusion as treatment
for postpartum haemorrhage (first to human to
human transfusion)
1900s, Karl Landsteiner MD, discovered the ABO
blood group system.
1

 Studies on the red cell antigens and their

corresponding antibodies constitute
Immunohaematology.
Application of the knowledge of
Immunohaematology constitutes Blood
Transfusion Science or Blood Banking
ABO and Rhesus Blood groups
ABO antigens are by far the most common and
immunogenic of all red cell antigens
Hence, ABO remains the most important blood
group to date
The antigens are found in all body tissues
Antibodies to ABO antigens are naturally
occurring, that is, stimuli for their formation is not
known
ABO incompatibility between donor and recipient is
the foundation on which all other pre-transfusion
tests rest
if ABO red cell and serum typing of recipient are
discrepant no transfusion is done
the Rhesus blood group system is very complex
there are as many as 45 antigens of the Rhesus
blood group system
only the first five ( D, C, E, c and e ) are tested
D is, after A and B, an important antigen in
transfusion practice
D is routinely typed along with A and B
2

 Anti- D is immune type, that is , it is not regularly

found in Rhesus negative individuals.
Its presence is only after immunization through
pregnancy or previous transfusion.
Rhesus positive means presence of D antigen and
Rhesus negative means absence of D antigen
A positive means A+ and D +.
the same for B, AB and O persons
ROUTINE ABO GROUPING
REACTION OF CELLS
WITH:

REACTION of SERUM
WITH:

Anti-A Anti-B Anti-AB

A cells B cells O cells

+
+
0

BLOOD
GROUP

FREQUENCY (%)
W
B
I
O

45

49 79 40

40

27 16 28

11

20 4

27

AB

4 <1

DONOR SCREENING

The safety of blood transfusion:

1. reliable medical history of a health donor

2. proficient laboratory tests for transmissible

infectious agents
3. good and reliable laboratory tests
4. adequate measures to protect the donor
Donor screening is done to protect the donor and
the recipient

QUALIFICATIONS AND MANAGEMENT OF

DONORS
Organisation of donor services
Volunteer donors are primary source of blood for
transfusion
BTS operates all-volunteer blood donor services in
the country. ARC in USA.
BTS has centers in major towns collects blood
from industrial, educational and military institutions.
Standards for blood donor services
Scientific and technical collaboration among
international transfusion organizations needed to
standardize blood collection, qualifications and
management of donors.
Red Cross Directives
AABB Standards for Blood Banks and Transfusion
Services.
AABB Technical Manual
Guidelines for donor qualification and management
established by the ISBT and WHO.
4

A. MEASURES TO PROTECT DONORS

Donors should be fully informed about all aspects of

donation procedure, eg.
Risks
intended disposition of their blood
Predonation information brochures on:
-What you should know about giving blood
-What you must know before donation
-Donor and patient safety
interviewed by qualified or trained member of
donor services.
an ideal donor :
- should be a healthy individual
- should be an informed volunteer
- free of transmissible disease
- capable of donating a unit without adverse
effects.
Interview to be private to encourage:
- truthful responses
- openness in answering questions
- confidentiality

1. Past Medical History

Convulsion, seizure or fainting

- History of seizure or convulsion after the first 2
years of life may be reasons for deferral.
-Accepted if convulsion is controlled by medication.

 Cardiovascular diseases
- History of stroke, myocardial infarction, cardiac
arrthymia, angina
Deferred permanently.

2. Age

- 18 year old may donate with parental permission.

- upper limit is 65 years
- over 65 years may donate after evaluation of
donor's health
- only occurrence of disease may cause deferral.

3. Haemoglobin and Hct (AABB) (ARC)

Hb concentration and Hct should be obtained before

donation not less than 12.5g and 38%,
respectively for females. 13.5g and 41% for males.
Hb Measurements
a) measured by standard spectrometric method
b) Copper sulphate solution (screening test)
S.G of 1.055 for men (13.5g/dL)
S.G of 1.053 for women (12.5g/dL)
-Copper sulphate solution may give false low Hb
estimates, e.g. of 663 unacceptable donors by
copper sulphate 82.5% were acceptable by other
techniques.

4. Intervals between donations

BTS limits donation frequency to not more than

once in 12 weeks from men and 16 weeks from
women.
ARC recommends not more than once in 8 weeks
for men and 12 weeks for women.
Aim of restriction to reduce iron depletion,
prevent iron deficiency anaemia.
Oral iron supplementation.

5. Pulse

- to detect irregularities of heart rate or rhythm such

as bradycardia, tachycardia.
- No specific criteria but must be counted for at least
30 second or 1 minute.
50 to 100 beats / minute.

6. Blood pressure

Measured to avoid phlebetomy related hypotension

Aortic stenosis detected by BP measurements
Systolic BP 90 to 190 mmHg
Diastolic BP 50 to 100 mmHg
May also alert donors to potential risk of high blood
pressure

7. Weight

To reduce risk of acute hypovolaemic reactions.

At least 110 lbs (50Kg) if donating 450mls.
If less than 110 lbs (50Kg) donors may reduce
volume to be donated.
7

 Volume of anticoagulant is proportionally reduced

also.
100 to 110 lbs with previous donation without
reactions is acceptable. Rate of collection may
need to be controlled.
< 100 lbs donors are rejected.

8. Pregnancy

No known causal relationship between pregnancy

and donation.
An issue may be created if there is an adverse
reaction, e.g. abortion, premature labor, birth
defects
6 weeks after normal full-term delivery in an
uncomplicated post partum course.

9. Medications

Indications of a disqualifying illness

Medication of chronic disorders such as
malignancies, infection or autoimmune diseases will
disqualify donor.
Amount of donor drug reaching transfusion
recipient is insignificant.

B. MEASURES TO PROTECT RECIPIENTS

Directed at exclusion of donors likely to transmit

infectious agents by whole blood, blood
components, or plasma derivatives.

1. Prescreening confidential self-exclusion

8

 Donor criteria statement provided to prospective

donors
List past illness which disqualify the donor, e.g.
Hepatitis, HIV,
Describe risk behavior for HIV or other infections
Donor told to have right to terminate blood
donation without specific reason.

2. Voluntary blood donation

Elimination of paid donors.

Paid donors more likely to be implicated in posttransfusion hepatitis B virus, non-A, Non-B virus.
Licensed blood products to be labeled paid donor
or volunteer donor.

3. General appearance

Prospective donors should appear in good health

Asked whether they feel well
Deferment if factors compromising obtaining a
reliable good health history are noticed, eg.
a) acute alcohol intake
b) influence of drugs
c) physical handicap that interferes with
communication and / or comprehension
of donor criteria.
Translation required in non-English speaking
communities.

4. Inspection of phlebotomy site

9

 Visual inspection done to avoid possible infection

that could contaminate donated blood.
Inspection of both arms for signs of skin punctures
or scars.
Tattoos

5. temperature

All donors must have a normal temperature

Not greater than 37.5oC
No lower limit

6. Immunization and vaccination

History of recent immunizations should be obtained

and evaluated for any risk of secondary spread of
infectious immunizing agent, e.g. rabies
vaccination, immune serum globulin and
Hepatitis B immune globulin.
Whether event causing immunization represent
reason for deferment.
Donor immunized with toxoids or killed viral,
bacterial, rickettsial vaccine is acceptable if there
are no symptoms or febrile reaction.
Acceptable vaccines include those for:Hepatitis B
Tetanus
Diphtheria
Pertussis
Typhoid,

10

 2 weeks deferment for attenuated virus; polio,

measles, yellow fever
4 weeks deferment for German measles ( rubella)
1 year deferment if vaccination for rabies is done
after animal bite.

7. Recent dental procedure

recent extractions or other oral surgery should

cause for deferment until the site has healed.
Routine dental care such as polishing teeth, scaling
or filling are no causes for deferment.

8. Malignant diseases

Circulating malignant or oncogenic agents may be

transmissible by transfusion.
History of leukaemia, lymphoma, or any other
malignant disease must be cause for permanent
deferment.
A person with removed local skin cancer e.g. basal
cell carcinoma may be acceptable if evaluated by
doctor.
Person with benign tumours may be acceptable
e.g. uterine leiomyoma, subcutaneous lipoma,
haemangioma, or benign cysts.

11

OBSTETRIC RELATED PROBLEMS

A.

Transfusions

Blood transfusion is a serious and sometimes

predictable event during and after delivery.
The following conditions often require that
transfusion be done:
1. Previous abortion
12

2. Bleeding during pregnancy

3. Operative delivery
4. Caesarian operation
5. Abnormal placentation
6. Multiple pregnancy
Problems sometimes arise:
1) Needless transfusion (single unit
transfusion)
2) Non-bleeding patients with chronic
anaemia
3) Emergency requests for blood
4) Shortage of compatible blood
5) Prevention and management of ABO and
Rhesus diseases.
Reduction of obstetrics related blood
transfusions in the past 20 years.
- improved delivery techniques
- group and retain method

B. ABO and Rhesus diseases

HAEMOLYTIC DISEASE OF NEWBORN

(HDN)
-

A condition in which life span of infant RBCs is

shortened by action of specific antibodies from
the mother by placental transfer. Haemolysis is
13

maximal at the time of birth and thereafter

diminishes as the concentration of maternal
antibodies decreases in foetal circulation.
- Before 1967 Rh HDN was responsible for 800
stillbirths and neonatal deaths each year in UK.
- Anti D was responsible for 94% of Rh HDN.
- Prophylactic anti D (Rhogam) reduced Rh HDN
deaths significantly.
- Other cases were due to anti C and anti E.
- The most frequent causes of HDN are related to
ABO, by anti A,B in O mothers who give birth to
A or B infants.
- ABO HDN is usually mild.

Rhesus HDN
Pathogenesis
- Rh negative (Rh d/d, rr) mother
- Rh positive foetus.
- Rh positive cells cross the placenta into
maternal circulation at parturition
- Mother sensitized (immunized) to form anti D
14

- Mother also sensitized by previous miscarriages

or blood transfusion, amniocentesis or other
trauma to the placenta.
- Anti D crosses the placenta to the foetus
during the next pregnancy with another Rh
positive infant.
- Anti D coats foetal red blood cells and results
in their destruction in the RES.
- Destruction results in severe Anaemia and
Jaundice.
- There is a 50% probability that the foetus will
be Rh positive if father is D/d.
CLINICAL FEATURE
1. Severe HDN characterized by severe
anaemia and intrauterine death
2. Moderate HDN is characterized by anaemia
with: - jaundice
- pallor
- tachycardia
- oedema
- hepatosplenomegaly
- kernicterus
3. Mild HDN s characterized by mild anaemia
without jaundice
LABORATORY FEATURES
1. Cord cells Rh positive
2. Mother cells Rh negative
15

3. Variable anaemia
4. High reticulocyte count
5. Direct Antihuman-globulin Test (DAT)
6. Increased Serum bilirubin
7. Presence of Nucleated red cells on a blood film

Prophylactic anti D (RhoGAM) is given to Rhesus

negative mothers with Rhesus positive infants without

prior immunization.
- Coat foetal red blood cells that enter the
mother, thereby reduce their
immunogenicity.
TREATMENT

A) Exchange transfusion if:

1. Hb < 14.og/dL and positive DAT

2. cord serum bilirubin > 60 mol / L
3. infant serum bilirubin is > 300 mol/ L
4. there is kernicterus
5. infant has moderate disease (one transfusion)
Blood for transfusion should be less than 7 days old
(fresh whole blood),
It should be Rhesus negative and ABO
compatible.
500ml may be sufficient for each transfusion.

B) Phototherapy (UV light) in order to degrade

bilirubin for excretion.
16

Handling a Rhesus Negative Pregnant

Woman
1. ABO and Rhesus typing
2. Serum screened for anti D.
3. A confirmed Rhesus negative woman:
- Serum rechecked for antibodies every
trimester.
- Antibodies identified and quantified if
present.
4. If first newborn is Rhesus Positive
- Give Rhogam within 72 hrs after birth to
avoid immunization
- No prior immunization no HDN.
- No HDN affecting first Rhesus positive infant
- No HDN if the mother is also Rhesus positive
- Antibody should be of the IgG type
- Antibody should be reactive at 370C

ABO - HDN
- In 20% of births, ABO is incompatible between
mother and baby.
- Occurs when O mothers gives birth to A or B
infants.
- An IgG anti AB in mother is responsible for
the HDN.
17

Clinical features
- mild anaemia without jaundice
- Mild due to:
1. fairly weak A or B antigens in infants
2. neutralization of maternal IgG antibodies
by A and B antigens on other cells, in
plasma or tissue fluids.
- Exchange transfusion has been done only once
in 3000 cases.
- First pregnancies affected.
- May or may not affect subsequent
pregnancies.
Laboratory features
- DAT on A or B infant cells may be negative or
positive (weakly)
- Agglutination of rbc on blood film
- Spherocytosis
- Polychromasia
- Erythroblastosis
COMARISONS OF ABO AND RHESUS HDN

Antibodies
Antibody type
Placenta

Rhesus HDN
Anti-D
IgG
Crosses
18

ABO HDN
Anti-AB
IgG
Crosses

Prior
immunization
Mother
Children
Severity
Treatment

Yes

No

Rhesus negative
Rhesus positive
Very
Prophylactic anti-D
exchange transfusion

Infants
affected

Subsequent D+
pregnancies

Frequency

Now very rare

O
A or B
Mild
Exchange
transfusion
in severe
forms
All A or B
infants.
Does not
involve AB
infant.
Most
common

Preparation of blood components

Aphaeresis
- is the removal of blood from the donor, retention
of a portion and return of the remainder
- leukopheresis is the removal and retention white
blood cells
19

- plateletpheresis is there removal and retention of

platelets
- plasmapheresis is the removal and retention of
plasma
- cytapheresis removal and retention of cells in
general
Plasmapheresis
is done for fractionation into plasma components
such as antihaemophilia factors, albumin,
cryoprecipitate, fresh frozen plasma (FFPs)
and gamma globulins
pre-identification of units is important
Plateletpheresis
Good to harvest as many platelets as possible
from a single donor
Donor should have at least a platelet count of
150 000 plts/ l.
No aspirin should have been taken during the
previous 48 hours.
6 to 8 units can be harvested from a single
donation.
Leukopheresis
Is done for an aggressive use of many
granulocytes
20

 Good treatment for bacterial infections

As many as 3 x 1010 must be harvested.
Plasma exchange
Removal of plasma in order to:
1) decrease the concentration of toxic
substances such as excess antibodies,
immune complexes and toxic
metabolites.
2) carter for adjustive therapy.

COMPONENTS
A.Packed Red Cells
Involves separation of plasma and red cells into
satellite bags using a plasma expression or
centrifugation
250 ml per unit of red cells are harvested

21

 Haematocrit (PCV) is about 70%

Leukocyte-poor red cells (Buffy coat removed)
Have a PCV of 90%
These are used to avoid febrile transfusion
reactions
Washed red cells:
Have few or no micro-aggregates
Trace amounts of plasma
Reduced febrile reactions
No metabolites and plasma proteins
RED CELLS ARE USED FOR THE TREATMENT
OF SEVERE ANAEMIA

B.Platelets
They are prepared before refrigeration to avoid
clumping
Platelet rich plasma (PRP).
Prepared by light spin (150g) from red cells
Platelet concentrate:

22

 Is prepared by a heavy spin of PRP at 22oC

30 to 50 ml should be the volume per unit if to
be stored at room temperature (24oC).
20 to 30 ml should be the volume per unit when
stored at 1oC to 6oC.
Continuous agitation is required during
preparation and storage to prevent aggregation
Each unit must contain a minimum of 5.5 x 1011
platelets
PLATELETS ARE USED FOR THE

TREATMENT OF HAEMORRHAGES AND PLT

DISORDERS

C. Fresh Frozen Plasma

Prepared by a single heavy spin of red cells
225 ml of plasma per unit
Must be frozen using dry ice-alcohol bath at 30oC within 6 hours after collection of blood
May be stored for 12 months at -18oC
Is thawed at 37oC before use
23

 FFPs ARE USED FOR THE TREATMENT OF

HYPOVOLAEMIA, MULTIPLE COAGULATION
FACTOR DEFICIENCIES AND EXCHANGE
TRANSFUSION.
D.

Cryoprecipitate

Contains factors VIII, V and I

Prepared by separating plasma from red cells
within 4 hours of blood collection and freezing at
-30oC within 2 hours
The plasma is thawed at 1oC to 6oC overnight
The AHF-poor plasma is separated from
cryoprecipitate (the remaining solid mass) after a
light spin.
10 ml of cryoprecipitate is left
It is refrozen at within 4 hours t -18oC
A unit of cryoprecipitate contains 80 to 100 units
of AHF and can be stored for 12 months at -18oC.
Is thawed at 37oC before use
CRYOPRECIPITATE IS USED TO TREAT

FACTOR VIII, V, VON WILLEBRAND

DISEASE AND I DEFICIENCIES.

24

E. Other components
Factor VIII concentrate
5% albumin
Plasma proteins
Factor IX complex
Immune serum globulins
Anti-D (Rhogam)

25

MASSIVE TRANSFUSION SYNDROMES

A) Definition:
Transfusion of approximately or exceeding
patient's total blood volume (5-6 litres in
adults) or a minimum of 10 units in an
adult within a 24-hour interval.
Often done in an emergent situation.
Exchange transfusion in infants is also
massive transfusion.
In many trauma patients the volume
replacement is done in very short space of
time.
Blood losses between 30 to 50% represent
massive haemorrhage.
Trauma patients are predominantly young
men around mid-thirties.
Indications:
Massive haemorrhage is usually associated
with gastrointestinal bleeding, ruptured
aneurysms and surgical procedures
Blunt and penetrating traumas represent
the leading indications for massive
transfusion!
Blunt trauma requiring massive
transfusion:
-Pelvic
-Abdominal and thoracic
-Liver
26

-Kidney
-Near amputations
Penetrating injuries requiring massive
transfusion:
-Vessels of the neck
-Thoracic outlet
-Aorta
-Great vessels of the chest and
abdomen
-Hepatic vascular injury
B) Mortality:
The survival rate is about 40 to 60% and
correlates with the number blood
transfusions.
Survival ranges from 38% in patients with
liver failure to 100% in obstetric patients.
The rate increases to 70% with proper
mobilization of medical personnel and
other logistics in those patients requiring
more than 25 units.
In blunt trauma the survival rate is 52%
Duration and magnitude of shock are
critical factors affecting the mortality rates
in massively transfused patients.
Mortality rates increases with age, severe
head injury, underlying medical conditions,
non-traumatic surgical emergencies in
relationship to blood transfusion.
27

C) Dilutional Coagulopathy
With exchange transfusion the remaining original
blood elements drop in concentration (15% to 5%)
respectively after two to three volume exchanges.
Trauma patients on massive transfusion tend to
have abnormal coagulation.
Dilution of coagulation factors to levels below 30%
activity may cause abnormal coagulation tests.
Dilutional thrombocytopaenia is predictable after
large volumes of blood replacements.
This results in microvascular bleeding characterized
by oozing from mucosa, raw wounds and puncture
sites.
Factor replacement in trauma patients
1. First define coagulation status using
appropriate laboratory tests
2. Clinical guidelines
- Extent and location of trauma
- Duration of shock
- Response to initial fluid
Replacements
- Risk of complications, eg.
intracranial bleeding
3. Replacement of components to
correct specific abnormalities.
4. Guidelines for specific components:
- platelets if count < 80-100 x
28

109/L
- FFPs if PT /APTT > 1.5 x
normal value
- Cryoprecipitate if fibrinogen
level < 10 g/L
D) Disseminated intravascular coagulation
(DIC)
The location and extent of trauma and duration of
shock are important factors in development of DIC.
Severe trauma may cause release of tissue
thromboplastin which activates coagulation factors
Present in 5 to 30% of trauma patients on massive
transfusion due to induced hypothermia
Associated with high mortality of nearly 70%
E) Laboratory features in massive transfusion
Laboratory testing and interpretation of results in the
diagnosis dilutional coagulopathy and DIC in
massively transfused patients seem to be
contradictory, eg.:
a) Prolonged PT/APTT in the absence of haemorrhage
b) Moderate thrombocytopaenia in acute DIC
dilutional coagulopathy
c) Normal coagulation tests in transfusion induced
hypothermia

29

d) No single laboratory test can be used to diagnose

DIC and dilutional coagulopathy.
e) No clinical observation of coagulopathy is confirmed
by PT/APTT tests.
But a combination of the following tests may be
helpful in the diagnosis of DIC and dilutional
coagulopathy:
1. Low plt count
2. Low fibrinogen levels
3. Elevated D-dimer concentration
4. Presence of soluble fibrin monomers
E) Complications of Massive Transfusion
Despite its anticoagulant-preservative, blood
undergoes aging during storage
The aging process results in:
- low blood pH
- loss of potassium from the red blood cells
- loss of ionized calcium due to chelation by
citrate
- deterioration of labile coagulation factors
- loss of 2,3-DPG
Massive transfusion may result in physical, chemical
and physiological effects on a patient.
Blood transfusion has immediate and delayed
adverse complications in as many as 10% of
recipients.
30

 Massive transfusion may cause several metabolic

problems such as acute haemolytic reactions,
citrate toxicity, hyperkalaemia, acidosis, and a shift
of O2 dissociation curve to the left.
hyperbolic

Sigmoid

haemoglobin

10

20

30

40

Emergency Issue

Immediate blood needs

Medical or surgical problems created by

hypovolemia and hypotension pose greater risks
than those caused by blood transfusion
Transfusion services must respond quickly but
without sacrificing patient's safety.
If immediate transfusion is required blood must be
given without completing the necessary laboratory
tests.
A written request by the attending clinician must be
made.
31

 Preferably, group O Rhesus negative red blood

cells must be given immediately in case of unknown
patient blood group.
If there is time to determine patient's ABO and
Rhesus status uncrossmatched group-specific whole
blood or red blood cells can be issued.
When volume replacement is critical Whole blood (if
available) is the preferred component.
Group O negative blood must be made available
for females of childbearing age who are Rhesus
negative.
Only one or two units of uncrossmatched O
negative blood are recommended to allow time for
completion of appropriate laboratory tests.

Effects on recipient blood type

Large volume blood transfusions often alter blood

composition of the recipient such that pretransfusion specimen ceases to represent patient's
current blood status.
Investigation of possible transfusion reactions
becomes difficult.

Conservation of scarce blood

Transfusion services should establish guidelines for

switching to different blood groups during massive
transfusions

32

 It is usually preferable to switch ABO groups before

Rhesus. Eg., A Rh- to O Rh- group than to A Rh+
The transfusion medical director must be consulted
when switching to different blood groups.
Immune haemolysis
ABO- incompatible haemolytic reactions are the
most common and are mainly due to human error.
There is increased risk to transfusion reactions with
the number of transfused units.
Clinical findings include haemoglobinuria,
hypotension, fever and microvascular haemorrhage.
Prevention of clerical errors requires utmost care in
patient identification.
Immunosuppression
Infection is common in massively transfused
patients and is the leading cause of death.
The mechanism is not clearly understood.
But it is thought the severity of injury, amount of
bacterial contamination, pre-existing immunological
condition of the patient, and the systematic effects
of the haemorrhagic shock can impair the immune
response to infectious agent.
Many plasma constituents such as opsonins
contribute towards immune system of the patient.

33

 Opsonins are stable in CPD blood stored under

refrigeration for 28 days.
They diminish in patients undergoing massive
transfusion with RBCs, crystalloid or albumin
solutions.
Citrate toxicity
To prevent blood from clotting in the unit citrate is
used as an anticoagulant.
Blood collected in CPDA-1 and Adsol contain about
5 and 2 mg of citrate respectively.
Plasma from Adsol alone contains about 30 mg of
citrate.
During massive transfusion plasma containingproducts such as FFPs and platelets are the major
source of citrate.
Citrate is normally metabolized to bicarbonate in
the liver.
Increased citrate levels may cause transient
decrease in ionized calcium.
Patients with less ability to metabolize citrate such
as those with hypothermia, hypotension and
hepatic injury, citrate toxicity may cause muscle
tremors and decreased cardiac output.
Calcium salts should, therefore, be reserved for
patients undergoing massive transfusion with
clinical manifestations of citrate toxicity.

34

Hyperkalaemia
This may occur during rapid blood transfusion in
patients with severe shock or renal failure and
those with extensive muscle necrosis.
As both hyperkalaemia and hypokalaemia are
associated with cardiac dysfunction, close
monitoring of potassium in massively transfused
patients is important.
2,3-Diphosphoglycerate (2,3DPG)
This compound allows for the release of O2 from
the haemoglobin to the tissues.
Normal levels are maintained up to 7-10 days of
blood storage.
Loss of 2,3 DPG during storage causes the O2
dissociation to move to the left preventing the
haemoglobin to release O2 resulting in tissue
hypoxia.
3 to 8 hours are required for restoration of 2,3-DPG
to half its original level.
24 hours for full restoration.
Massively transfused patients must be given fresh
red cells (if available).
Hypothermia
Rapid transfusion of blood right out of the
refrigerator (1 to 60C) can cause hypothermia
Cardiac arrhythmia will result
35

 DIC is usually promoted under cold conditions

Warming of blood to close to 370C is recommended
if rapid transfusion is contemplated.

ADVERSE EFFECTS OF TRANSFUSION

Transfusion is usually a safe and effective way of
controlling haematological defects:
1. Anaemia Rbc
Platlets
2. Haemorrhages
Factors
FFPs
3. Leukopaemia (infections)
4. Hypovolemia
- Untold results may occur
- Many are called transfusion reaction.
- Some reactions are immediate others are delayed
- Some can be prevented; others cannot be prevented.
- Health care providers should know the risks of blood
transfusion. Should evaluate them against potential
transfusion benefits.
- Short time between suspicion of T.R and investigation and
the appropriate therapy is recommended.
- Responsibility for recognising reaction lies with the person
transfusing nurse / physician
- If transfusion reaction is suspected:
1. Stop the transfusion immediately. Notify the responsible
person.
36

2. Keep the intravenous line open with infusion of normal

saline.
3. Check all labels, forms and patients identification on to
confirm if the patient received the right component.
4. Report the suspected reaction to Blood Bank personnel
soon.
5. Send other blood samples for evaluation by Blood Bank as
soon as possible, together with discontinued bag and
other intra-venous sets of equipment, solutions and
related forms and labels.
6. Send other samples for the evaluation of acute haemolysis
Outline for Laboratory investigation
1. Check identification of patient and donor blood.
-Notify patients physician of any discrepancies
-Check records to see if any donor or patients
blood has been wrongly identified.
2. Compare patients pre-transfusion and post transfusion
specimen for colour of serum or plasma.
-Pink or red colour in post transfusion sample
means destruction of transfused RBCs.
3. Perform DAT:
-Positive with transfused RBCs.

37

IMMEDIATE AND DELAYED ADVERSE

EFFECTS OF TRANSFUSION

IMMEDIATE EFFECTS
Immunological effects
Usual Aetiology
Haemolysis with symptoms
Red cell incompatible
Febrile non-haemolytic reaction
Donors granules
Anaphylaxis
Antibody to IgA
Urticaria
Antibody to plasma proteins
Non-cardiac pulmonary oedema Antibodies to leukocyte and
Complement activation
Non-immunological effects
Marked fever with shock
Congestive heart failure
Haemolysis with symptoms

Bacterial contamination
Volume overload
Physical destruction of blood
Eg. Freezing and overheating
Mixing non isotonic solutions with
red cells.

DELAYED EFFECTS
Immunological effects
Usual Aetiology
Haemolysis
Anamnestic anti body to red
antigen
Graft-versus-host disease
Engraftment of transfused
Functional lymphocytes
Post-transfusion purpura
Development of anti-platelet
Antibodies eg, anti-PIA1
Immunisation to RBCs and PLT
Exposure to antigens of donor
antigens
origin
Non-immunological effects
Iron overload
Hepatitis
Acquired immune deficiency
syndrome
Protozoal infections

Multiple transfusions (100+)

NANB, occasionally B (rarely A)
HIV-1, HIV 2
Malaria parasites, babesia

38

ANAEMIA
A term used to describe red cell deficiencies
that result in hypoxia
It is manifested by decreased red cell count,
haematocrit and decreased OR dysfunctional
haemoglobin or both.
Usually a manifestation of an underlying
condition
General clinical signs and symptoms of
anaemia are:
1) Weakness and loss of breath, especially
after physical exercise
2) Dizziness or fainting
3) Gastrointestinal disturbances, loss of
appetite and indigestion
4) Confusion, insomnia or hallucinations
5) Headaches
6) Lethargy
7) Cardiomegaly
8) Pallor

39

 Specific clinical signs and symptoms of

anaemia are:
1) Koilonychia (spoon shaped nails)
2) Jaundice
3) Leg ulcers
4) Bone deformities
5) Mental retardation
The severity of the clinical signs varies from
none to life-threatening depending on:
a) The severity of red cell deficiency
b) Rate of onset of anaemia
c) Age of patient
d) The general health of the patient
It is important to note that anaemia is always
not a primary disease, but a secondary
manifestation of another disorder.
The underlying disorder must be investigated
clinically and in the laboratory.
The ideal way to resolve anaemia is by
identification and correction of the underlying
condition.
The appearance of red cells on a blood film
sometimes gives evidence of the cause of
anaemia.
40

Classification of Anaemia

Anaemias are classified on the basis of

morphological characteristics, aetiology and
pathological manifestations.
The first two are the mostly widely used
methods.
1. Morphological classification
Based on the descriptive and quantitative
criteria to classify red cells by maturation
stage, shape, size and haemoglobin content.
Size is derived from determination of mean
corpuscular volume (MCV); haemoglobin
content is derived from mean corpuscular
haemoglobin concentration (MCHC) and mean
corpuscular haemoglobin (MCH). These
calculations are referred to as red cell
indices.
Normal reference ranges for the above are:
a) MCV
= 80 - 97 Fl
b) MCHC = 31% - 36%
c) MCH
= 27 - 31 pg
An MCV between 80 and 97 fL is normocytic,
above 97 fl is macrocytic and below 80 fl is
microcytic.

41

 An MCHC between 31 and 34% is

normochromic, above 36% is
hyperchromic and below 31%, it is
hypochromic.
Anaemia can be classified morphologically as
normocytic normochromic, microcytic
hypochromic, normocytic hypochromic,
or macrocytic normochromic depending on
the values of the red cell indices of interest
2. Etiological Classification of Anaemias
Based on the mechanisms by which
haemoglobin or red cell count is decreased.
Three possibilities of causes of anaemia are:
a) Impairment of marrow production
which results in fewer or
defective cells
b) Normal production but circulating
cells are being destroyed or lost
c) Red cell survival is normal, but
plasma volume dilutes the red cell
count and haemoglobin
concentration.
Identification of cause of anaemia is done
after investigation of morphological patterns
of the red cell.
42

 Eg, iron deficiency anaemia is always

suspected if the red cells show a microcytic
hypochromic picture.
Laboratory findings in anaemia
Initial findings in anaemia are:
1. Decreased haemoglobin levels; <12.5 g/dl in
adult females, < 13.5 g/dl in male and 8.5
g/dl in children:
2. Decreased haematocrit value; < 36% in
females, < 40% in males and <30% in
children.
3. Decreased or normal red cell count.
Treatment
1. In severe forms of anaemia there is need to
quickly restore the body's capacity to
transport O2 to the tissues by restoring Hb
through red cell transfusion.
2. If known, treatment of underlying disease
is important.

43

RED CELL MEMBRANE DISORDERS

These are usually intrinsic red cell defects.
Intrinsic red cell defects are almost always
hereditary.
Extrinsic forms are almost always acquired.
Extrinsic red cell disorders are due to external
forces destroying the cells, eg. Malaria infections,
isoimmunization and burns.
Intrinsic defects are due the abnormalities of the
red cells themselves, eg. membrane disorders
Normal red cell
Should be biconcave in shape
Should be flexible enough to pass easily through
the micro-circulation
Should maintain Hb in a reduced state
Should maintain osmotic equilibrium
Should be able to deliver O2 to the tissues with
easy

44

Common membrane disorders

1. Hereditary spherocytosis
2. Hereditary elliptocytosis / ovalocytosis
3. Hereditary stomatocytosis
4. Hereditary acanthocytosis

A.Hereditary spherocytosis

Characterized by presence of spherocytic cells in

circulation.
inherited as autosomal dominant.
commonest inherited haemolytic disorder affecting
about 1 in 5000 in USA.
pathogenesis is associated with defective spectrin
- reduction in spectrin content
- unstable spectrin molecule
- defective spectrin binding to ankyrin
-these may lead to release of parts of lipid bilayer
in circulation.
-surface area to volume ratio is decrease.
-cells become mostly spherical in shape.
-removed from circulation leading to anaemia
clinical features:
- anaemia presents at any age
- jaundice is present
- splenomegaly is found in most cases
- aplastic crisis may cause a sudden
increase in the severity of the disease
laboratory features:
45

-severe anaemia.
-reticulocyte count is increased (5-20%).
-microspherocytes found on a blood film.
-polychromasia and occasional nucleated red
cells on blood film.
-Osmotic fragility test is increased.
-direct antiglobulin test (DAT) is negative.
-51Cr studies show a shortened red cell
survival.

Treatment:
-Splenectomy (avoided in early childhood).
-folate administration in severe cased.
46

-pneumococcal and haemophilus vaccination

before splenectomy.

B.Hereditary Elliptocytosis

Characterized by cells that are elliptical or

oval
-inherited as autosomal dominant
less common than HS and affect about 2 to 5
persons in 10 000
pathogenesis is associated with:
-defective spectrin molecules ( OR
mutants).
-abnormal interaction of spectrin and other
cytoskeletal proteins.
-defects in protein band 4.1 or glycoprotein

-double heterozygous forms present with severe

anaemia with microspherocytes, poikilocytosis
and splenomegaly.
-This variant is known as hereditary
Pyropoikilocytosis.
clinical and laboratory features:
- are the same as those found in HS.
- 25% to 90% of red blood cells on blood
film are elliptocytes / ovalocytes.
- newborn infants may have jaundice severe
enough to require transfusion.

47

C. Hereditary stomatocytosis (Hydrocytosis)

48

 Characterized by presence of red blood cells with

slit-shaped central pallor (mouth-shaped).
Some stomatocytes (xerocytes) may be present as
a normal transient stage in response to
dehydration rehydration conditions.
Inherited as autosomal dominant.
Pathogenesis is associated with:
- increased levels of phosphatidylcholine
(type I ).
- increased surface area.
- increased ion passage across the cell
membrane.
- potassium and sodium content of the cell
is increased.
- lack of certain red cell Rhesus antigens
(Rhesus null phenotype) (type II).
- Moderate cell lysis results from increased
H2O entry.
- It may be found in association with acute
alcoholism and liver disease.
clinical features:
- same as those found in HS and HE

laboratory features:
- mild to severe anaemia.
49

- reticulocyte count is normal to moderately

elevated (10-20%).
- red cells are 10-50% stomatocytes on a
blood film
- OF is increased
- 51Cr studies show a shortened red cell
survival

treatment:
- splenectomy may or may not help in
the treatment of hereditary stomatocytosis

D. Hereditary Acanthocytosis(Abeta
lipoproteinaemia)
50

 Characterized by presence of acanthocytic red

blood cells on blood film.
These are cells with bulbous extensions or
spicules.
Pathogenesis is associated with:
- absence of beta-lipoproteins.
- these are major red cell membrane
components.
- usually found in individuals with no Kell
blood group antigens (K null phenotype)
clinical features:
- the disorder manifest itself during the first
few months of life.
- growth failure
- abdominal distention
- steatorrhea
- retinitis pigmentosa
- neurological damage
laboratory features:
- mild anaemia
- normal or increased reticulocyte count.
- increased acanthocytes on a blood film.
- decreased cholesterol levels in serum.
- absence of beta lipoproteins and triglycerides in
serum.
- K null phenotype identification.
- 51Cr studies show a decreased red cell survival

51

treatment:

no definitive mode of treatment

52

MICROANGIOPATHIC HAEMOLYTIC ANAEMIA

is a term designated for a haemolytic anaemia that
results from the red cell fragmentation usually in
association with small vessel diseases or conditions.
it is an acquired (extrinsic) haemolytic disorder.

Pathogenesis

red cell fragmented by;1) fibrin mesh within the

small vessels.
Red cells are also destroyed by; 2) toxic chemical
substances from vascular pathologic lesions
Red cells are destroyed by;3) physical trauma
caused by restricted vessels as a result of
intravascular tumours.
Presence of; 4) Artificial heart valves provide an
abnormal surface which may not tolerate normal
red cell survival.
Red blood cells are destroyed by; 5) excessive
force applied to limps, eg. marching and playing
bongos
The destruction of red cells takes place in the
vessels (intravascular haemolysis).
Conditions that are characterized by deposition of
fibrin in small vessels or abnormal vascular
endothelium are:
a) prosthetic heart valves,
b) vasculitis
c) malignant hypertension
d) eclampsia
e) renal graft rejection
53

f) giant haemangioma
g) thrombotic thrombocytopaenic purpura (TTP)
h) haemolytic uraemic syndrome (HUS)
i) disseminated intravascular coagulation (DIC)
j) march haemoglobinuria

Clinical features

severe chronic anaemia

jaundice
high blood pressure
haemorrhage
other features related to the underlying cause

laboratory features

very characteristic blood changes ( fragmented red

cells, helmet cells, triangular red cells,
microspherocytes, pincer cells
a classic feature is the presence of schistocytes
nucleated red blood cells may present on a blood
smear
(fragmented red cells) on a blood smear.
Prolonged APTT and PT
abnormal liver function test
abnormal renal function tests
thrombocytopaenia
haemoglobinuria and haemosiderinuria in severe
cases

54

treatment

treat the underlying condition

transfusion in the most severely anaemic and
bleeding cases is recommended

HAEMOGLOBINOPATHIES
Haemoglobinopathies are structural abnormalities
involving one of the four types of polypeptide chains
causing Hb to be functionally abnormal or altered
physical and chemical properties.

Thalassaemia normal structure but the rate of

synthesis is abnormal.

Homozygous Haemoglobinopathies - HbSS,

HbCC.
Heterozygous Haemoglobinopathies - HbAC,
HbAS
Haemoglobin variants
Over 300 human Hb variants detected.
Majority discovered incidentally during Hb surveys
in populations.
These do not cause clinical syndromes
(haemoglobinopathies).

55

Genetics:
90% are single amino acid substitutions in , , ,
and chains.
Single base substitution in the DNA synthesis.
Inherited as autosomal co-dominant traits.

Molecular bases of Human Haemoglobin Variants

Basic mechanism

Chain

Example

A. Amino acid substitution

1. One substitution

I, Memphis
S,C,D, E, Seattle
F Texas I, F Hull
A2 Sphakia

2. two substitution

J Singapore
C Harlem
Gunhill (5)
Lyons (2)
Tochigi (4)
Leiden (1)
Coventry (1)
The Lepores
P. Congo
Kenya
Constant Spring

Seal Rock

B. Amino acid deletion

C. Fusion Haemoglobins

D. Elongated sub units

56

1.) SICKLE CELL SYNDROMES

Discovered in 1910 by Herrick on a black student

with haemolytic anaemia and peculiar and
elongated cells, sickled cells.
Most detailed chapters of molecular biology
HbS
2 2 6Glu
valine.
A) SICKLE CELL TRAIT:
Red cells contain Hb S and Hb A.
8% of black Americans are heterozygous for S i.e.
they have genotype AS.
30% of gene in parts of central Africa where
malaria is common.
Heterozygotes are slightly protected against P.
falciparum malaria species.
Clinical features:
Life expectancy is normal
SA red cell require far lower oxygen tension for
sickling than SS.
SA heterozygotes rarely develop sickling unless
exposed to hypoxia e.g. during a flight.
Renal medulla is unusually susceptible to sickling
Impaired urine concentration
Recurrent painless haematuria due to medullary
infarction.
Diagnosis:
Sickle cells are looked for after deoxygenating Hb
with metabisulfite.
Solubility test (dithionite test)
57

 Hb electrophoresis 35% Hb S , 60% Hb A.

Genetic defects in major abnormal Hbs
Hb

Chain

Position

AA

HbS

Beta

Glu val SCA

HbC

Beta

Glu lys

HbC di'se

HbD

Beta

various

Various

Mild HA

HbE

Beta

26

Glu lys

Mild HA

121

Glu lys

Mild HA

HbO Arab Beta

SICKLE CELL ANAEMIA

Disorder

A hereditary defect in beta chain synthesis resulting

in intra-cellular crystals (tactoids) distorting the
cells and culminating in their destruction by RES
macrophages.
The genetic defect is substitution of valine for
glutamic acid at position 6 of beta chain: 6 glu
val.
This results in less soluble Haemoglobin especially
under conditions of deoxygenation
Upon deoxygenation a red cell with HbS acquires
an elongated sickle shaped structure.
Bundles of fibre, 180A of precipitated Hb form
tactoids.
58

 The sickling phenomenon is affected greatly by

other non-Hb S haemoglobins, e.g. Hb C
participates in the co-polymerisation of Hb S
while Hb F tends to inhibit polymerisation of Hb
S.
Clinical severity of SCA is in the order of: SS > SC
> SF > SA.

Varying clinical severity of Sickle cell

Syndromes
Genotype

%HbS

% Non-HbS

Severity

SA

30-40

60-70(A)

SF

70

30(F)

SS

80-90

5-15(F)

++/++++

S-Thal

80

20(A+F)

++/+++

SC

50

50(C)

++/+++

30-40

60-70

++/++++

SO, SD

At cellular level sickled red cells become very rigid

and may obstruct capillaries leading to tissue
hypoxia resulting in further deoxygenation.
Cells are destroyed by the spleen causing
extravascular HA.
There is also intravascular HA.

59

Complications of SCA

1. Hand foot syndrome (painful crisis)

Painful swelling of dorsa of hands and feet.

Sickling and capillary stasis.
Lasts for 2 weeks.

2. Sequestration crisis

sudden pooling of blood in spleen.

Rapid splenomegally
Hypovolaemic shock.
Common in early childhood.

3. Functional asplenia

inadequate antibody response

RES unable to clear bacteria due to blockage.
Increased Salmonela and pneumonia infections.

4. Autosplenectomy

Vasoocclusive episodes, progressive infection,

fibrosis and contraction.

5.

Vaso-occlusive crisis

Chest, abdominal and bone or joint pain.

Fever
Plugging of small vessels
Bone necrosis
Many organ system damage

6. Aplastic crisis

Failure of blood production due to:

- infection
- drug exposure
- folate deficiency

60

7. Priapism
Involuntary painful erection of the penis.
Caused by vascular damage to the penile skin and
accumulation of blood in the organ.
Priapism may result in damaged vascular erectile
system leading to impotence.
8. Enuresis
Nocturnal enuresis is bed wetting at least 2 nights
per week
Caused by increased urinary volume and lower
functional bladder capacities due to renal vascular
damage.
Laboratory
Decreased Hb.
Increased reticulocyte count
Positive solubility test (screening)
Confirmation of sickle cells on blood film and also
target cells.
Hb S identification on electrophoretic strip (85100%HbS) and increased Hb F.
Decreased ESR
Increased MCV due to reticulocytosis and folate
deficiency.

61

Treatment
has progressed during the past few decades
largely supportive.
prevention of infection major concern
prophylactic antibiotics
Close monitoring and rapid intervention to minimize
problems.
Periodic transfusion to forestall haemolytic crisis.
Future
Hydroxyurea administration
Insertion of normal DNA sequences

2.) THALASSAEMIAS

The are hypochromic microcytic anaemias that

result from the hereditary defect in the synthesis of
globin chains.
Derived from Greek word thalassa (sea).
First cases described in patients from the
Mediterranean region.
Skeletal changes representing prehistoric examples
of thalassaemia have been found (50 000 year
ago).
Excavated from submerged sites in Italy and
Greece.
Forms of thalassaemia are found in Africa and Asia.
Severe and mild forms have been designated :
THALASSEMIA MAJOR OR THALASSEMIA MINOR

62

 Designation based on degree and system

complication.
Forms of defect could either be:
1. alpha chain defect
OR
2. Beta chain defect

A.) BETA THALASSEMIA MAJOR (Cooleys

anaemia)

Results from homogenous or double heterozygous

abnormal gene.
Beta chain production can vary from zero (o o) to
moderately decreased (+ +)
Gamma chain production compensates for loss.
Hb F is increased (20 to 90%).
Hb A2 may be normal or increased.
Symptoms
Clinical symptoms more severe in homozygous
thalassaemia than Hb H disease.
Most symptoms are associated with:
-Anaemia
-skeletal abnormalities
-iron over load.
Death due to
1. recurrent infection
2. iron overload cardiac failure
3. severe anaemia
63

-chain
(in F cells)

Exc. -synthesis

Blocked

Excess precipitate
Hb F formation
Increased Hb F
Increased O2
Affinity

Blood
RES trapping

Bone marrow
ineffective
erythropoiesis

Membrane
damage
Increased Iron
storage
iron
overload

Increased EP
production

Haemolysis
Anaemia
transfusion

endocrine,
liver, heart
damages

Increased erythroid
cell production
Splenomegaly

El'ted uric acids

Hypermetabolism
Skeletal changes
Folate deficiency

Pathophysiology of Beta Thalassemia

64

DOUBLE HETEROZYGOUS FOR -thal and Haemoglobinopathies

1) S/Bo Thal more severe than S/B+
Clinical findings vary from slight to more severe
and is similar to sickle cell disease.

Laboratory feature

microcytic hypochromic blood picture

Sickle cells uncommon
Target cells present
MCV and MCH lower
Electrophoresis
Solubility test positive
HbA absent
HbS > 50%
HbF 5 to 20%
HbA2 > 4.5%
(2) S/B+-Thal less severe

Laboratory features

HbA 15 to 30%
HbS - >50%
HbF 1 to 20%
HbA2 over 4.5%
MCV and MCH are lower.

65

3.) HbC/-Thal disease found mainly blacks

causing slight disability.

Laboratory features

Blood MCV and MCH reduce

Hypochromasia
Target cell and fragmented rbc
Microspherocytosis
Electrophoresis:
-HbC 60 to 95%
-HbF variable
-HbA 20%
-HbA2 not known

4.) HbE-thal SEA disorder.

Clinical picture similar to Thal major.
Hb E is 15 to 95%
HbF-5 to 85%
Hb A is absent.

Laboratory features
1.
2.
3.
4.
5.
6.
7.
8.
9.

Anaemia (Hb <8g/dL)

Anisopoikilocytosis
Microcytic hypochromic blood picture
Target cells, tear drops and basophilic stipplings.
Bone marrow and serum iron increased.
Nucleated rbc and polychromasia.
Positive Kleihauer-Bekte test.
Increased HbF and HbA2.
Decreased chain in rbc.
66

B.) BETA THALASSEMIA MINOR

Caused by inheriting a single abnormal gene.
No clinical disorders
Target cells, poikiloanisocytosis present.
Normal Hb levels
Decreased OF test.
Normal HbF and increased HbA2 OR
Normal HbA2 and increased HbF
Slightly increased iron levels
Tissue iron is normal.
C.) ALPHA- THALASSEMIAS
Caused by deletion of genetic material of the
gene.
Clinical severity based on the amount of deleted
material as follows:
Clinical state
% alpha chain
Amount of
production
functional alpha
genes
1. Normal
100%
4
75%

3. Trait (-thal-1)

50%

4, Hb H disease

25%

2. Silent carrier (thal-2)

5. Hydrops fetalis
with Hb Bart

67

 Inheritance patterns:
i.
ii

iii.

I)

Silent carrier x normal = 50% silent carriers

Silent carrier x trait:
-25% silent carriers
-25% normal
-25% Hb H
-25% trait
Trait x trait:
-25% normal
-25% hydrops fetalis
-50% trait

HOMOZYGOUS ALPHA THALASSEMIA

(Hydrops foetalic with Hb Bart (4)

Incompatible with life
Infants are stillborn with:
-severe edema
-marked anaemia
-marked hepatosplenomegaly
Blood - marked anisopoikilocytosis
- marked microcytosis and erythroblastosis.
Electrophoresis
No HbA
No Hb F
Increased Hb Bart (4)
Some HbH (4)

68

II.) HbH Disease

Caused by deletion of 3 of alpha genes.

Chronic anaemia
Clinical picture similar to thalassaemia intermedia.
Found in almost all racial groups

Laboratory features

Blood MCV and MCH decreased

hypochromic microcytic cells
anisopoikilocytosis, target cells
reticulocytes 4 to 5%
Heinz bodies present after splenectomy.
Electrophoresis Hb H, 4 to 30%
-Traces of Hb Bart- 25% at birth

III.)
Heterozygous thalassemia (alpha-thal1 trait)

Deletion of 2 alpha genes

Mild anaemia
Microcytic hypochromic cells
Electrophoresis 5 to 60% Hb Bart in cord blood
/ synthesis ratio is 0.6

IV.) Silent carrier of -thal (-thal-2)

Deletion of one of four - genes
No haematological abnormalities
Hb Bart 1 to 2% in infants.

69

V.) Hb Constant Spring

- chain variant
Found in 40% cases of Hb H disease in South Africa
and East Asia.
31 extra amino acids
synthesis is very slow
Homozygous mild anaemia
microcytic hypochromic
5 to 6% Hbs C/S
Normal Hb A2
The rest is Hb A

VI) Hereditary persistence of fetal Hb (HPFH)

HbF production persists beyond infancy
No significant abnormalities
In 1% black Americans also Greek variants
Caused by deletion of gene complex.
In homozygotes
100% Hb F
- no HbA or HbA2
in heterozygotes
- HbF is 20 to 30%
- HbA2 1 to 2%
- Remainder HbA (-70%)
- Positive alkaline denaturation test.

70

Morphological Classification of anaemia

1. Hypochromic microcytic
MCV less than 80 fl
MCHC less than 31 %
Causes include:
Iron deficiency
Thalassaemia trait
Anaemia of chronic disease
Lead poisoning
Sideroblastic anaemia
Hb E disease
Anaemia of chronic blood loss
2. Normochromic normocytic
MCV 80-97 fl
MCHC 31-36 %
Causes include:
Haemolytic anaemias
Anaemia of chronic disease
Anaemia of acute blood loss
Mixed deficiencies (iron and B12 or folate)
Bone marrow failure (Aplastic anaemia)
Renal disease
3. Normochromic macrocytic
MCV great than 97 fl
MCHC 31-36 %
Causes include:
Megaloblastic anaemia (B12 or folate
deficiency)
71

 Non-megaloblastic causes (alcohol, liver

disease)
Some haemolytic anaemia
Intrinsic factor deficiency
malabsorption

72

MACROCYTIC NORMOCHROMIC
ANAEMIAS
Defined as anaemias with MCV of more than 97 fLs.
Can be divided into megaloblastic and nonmegaloblastic.
A) Megaloblastic refers to characteristic
abnormalities of erythroblasts in the BM in which the
maturation of the nucleus is delayed relative to the
cytoplasm.
Megaloblastic anaemias account for the most
common forms of macrocytic anaemias.
Vitamin B12 or folate deficiencies account for the
vast majority of megaloblastic anaemias
Vitamin B12 deficiency is caused by:
Pernicious anaemia caused by
autoimmune gastritis which results in
decreased IF levels (80%)
Decreased dietary intake in a strict
vegan diet
Gastrectomy or gastric bypass surgery
Blind lope syndrome
Ileal resection
Malabsorption
Crohn's disease
HIV infection

73

 Vitamin B12 deficiency takes four years to develop

if supplies are totally cut off by malabsorption.
PA occurs in all races, but most common in
northern Europeans (127:100000)
PA is found mostly at 60 years and above
A positive family history is found in 30% of the
patients with PA.
Folate deficiency may occur because of the
following:
Inadequate intake (body stores
sufficient for only four months)
Malabsorption, eg. in Crohn's disease
Increased demand, eg. during
pregnancy, infancy, rapid cell turnover
in haemolysis and leukaemia.
Increased urinary excretion, eg.
during heart failure, acute hepatitis,
dialysis.
Drug-induced, eg. alcohol,
antiepileptics, methotrexate,
sulfasalazine.
During pregnancy demand for folate is high.
30% of pregnant women in the third trimester
have reduced levels if they are not on folate
supplements.
A minority may develop megaloblastic
anaemia.
74

Clinical symptoms

Fatigue
Breathlessness
Anorexia
Indigestion and episodic diarrhoea or both
Reversible sterility
neuropathy:
sub-acute combined degeneration of
the spinal cord
visual disturbances
psychiatric disturbances- mild
neurosis, depression, dementia

Clinical signs

pallor
mild jaundice
atrophic glossitis and angular cheilosis
hepatomegaly
mild pyrexia
optic atrophy
tachycardia, murmurs, cardiomegaly

Complications

heart failure
angina
dementia
gastric carcinoma (4%)
neural tube defects in fetus of B12 def. mothers
sterility
75

Prognosis

macrocytic anaemias due to folate and B12

deficiency respond very well to replacement
therapy
neuropathy due to folate/B12 depletion is
usually irreversible.

Laboratory manifestations

low Hb and increased MCV

pancytopaenia in severe cases
low reticulocyte count
dimorphic blood picture if iron deficiency is also
present (MCV is normal)
macrocytes on blood film
hypersegmented neutrophils
low serum and red cell folate
low serum vitamin B12
50% of patients show positive anti- IF
autoantibody screening in PA.
Antibodies against gastric parietal cells present
in 85% of patients with PA.
Schilling test determines causes of B12
deficiency:
- excretion of > 10% 58Co = dietary
deficiency
- corrected by giving IF (57Co) = PA
- no correction = malabsorption

76

 response to B12 injection:

- patient feels better in 1-2 days.
- reticulocyte count increases in 2-3 days
and peaks in 5-8 days.
- red cell count increases within 1 week and
normalizes in 4-8 weeks.
- MCV increases in 3-4 days, then decreases
and normalizes in 25-78 days.
- Hb increases by 2.3g/dl every 2 weeks.
- White blood cells and platelets normalize
in 7-10 days.
Bone Marrow aspiration shows megaloblasts
and giant metamyelocytes.
B). Non-megaloblastic anaemia
1. Excess alcohol intake:- is the most common
cause of macrocytosis due to poor diet in
spirit drinkers (beer has a high folate
content).

2.

3.
4.
5.

6.

Liver disease
Severe hypothroidism
Reticulocytosis
changes in plasma proteins
drugs that affect DNA synthesis, eg.
hydroxyurea, azathiopine

Treatment

Replacement of B12 intramuscularly.

Vegans should be given B12 orally.
77

Intramuscular injection for those who have

undergone gastrectomy or illeal resection.
For folate it is important to assess dietary
intake and consider alcohol excess.
Oral folic acid is given, 5mg daily and
treatment continues for four months.
First exclude B12 deficiency before folate
treatment.
Patients with chronic haemolytic anaemia
should be given folate supplements.
400 micrograms daily of folate is
recommended for pregnant women, starting
from prior conception continuing up to 12
weeks of pregnancy.

MICROCYTIC HYPOCHROMIC
ANAEMIAS
Iron deficiency anaemia
sideroblastic anaemia
anaemia of chronic blood loss
78

vitamin B6 deficiency
thalassaemias
anaemia of chronic disease
Hb E disease
porphyrias
1) IRON DEFICIENCY ANAEMIA
-

most common cause of anaemia world wide

Caused by iron depletion
No longer sufficient iron for normal Hb
synthesis.
Iron utilization exceeding iron intake.
An average Western diet has 15 mg of iron
daily only 5-10% of which is absorbed.
Absorption is in ferrous form
4000 - 5000 mg iron in the body and 70%
is in Hb in the circulating blood
1 ml of RBC contains about 1mg of iron.
40% of iron is stored as ferritin and
haemosiderin in the liver and RES.
each day 20-25ml of RBC are lost through
normal aging and 1mg is lost. 19 to 24 mg
is re-utilized.
Amount of iron lost is equal to amount
absorbed.
Deficiency results from increased utilization
in:
79

Pregnancy and childhood

OR
Blood loss (excessive)
-

Commonest cause of iron deficiency is blood

loss due to:
1.
2.

Gastrointestinal bleeding
Excessive menstrual blood loss

antacids and tetracyclines can block iron

absorption.

iron deficiency staging:

a)

First step - iron depletion due to increased

utilization (pregnancy bleeding).
-Dietary intake is not adequate
iron stores are utilized.
- normocytic normochromic blood
picture

b) Second step - iron deficiency erythropoiesis

taking place.
- iron stores become
exhausted but anaemia may
not be present.
- microcytic blood picture.

80

c) Third step - iron deficiency anaemia now

present.
- intake is unable to meet
demand.
- stores have now been
depleted.
- microcytic hypochromic
blood picture.

clinical features

No clinical abnormalities at the beginning.

Commonest symptoms are lethargy and dyspnoea
Later specific symptoms and signs of anaemia start
to show.
angular stomatitis
spoon nails (koilonychia)
unusual dietary cravings (pica)
reduced gastric secretions

laboratory features

Diagnosis is usually straight forward

Reduced MCV
MCHC
MCH
Microcytic hypochromic blood picture
Poikilocytosis
Low reticulocyte counts
Dimorphic blood picture due to:
- presence of folate and vitamin B12
deficiency
- present transfusion
81

- iron therapy
platelet count decreased
No iron stores with Perls stain in the BM.
No siderotic granules in developing in the BM
Erythroblasts are small with ragged cytoplasm
decreased iron and increased TIBC
decreased ferritin
increased free erythrocyte protoporphyrins (FEP)

82

83

Treatment
identification and treatment of underlying cause
iron replacement therapy orally with ferrous salts
(200 mg per day)
side effects; epigastric pain, constipation and
diarrhoea
blood transfusion

84

2) SIDEROBLASTIC ANAEMIA

A group of disorders characterized by iron

loading and its accumulation in the mitochondria of
the erythroid precursors.
Can be inherited OR acquired.

a.

Hereditary Sideroblastic anaemia

Sex-linked recessive trait that occurs primarily in

males.
Anaemia is generally severe with Hct of 20%
Blood pictures is:
dimorphic
normocytic normochromic
microcytic hypochromic
moderate anisopoikilocytosis
target cells and basophilic stipplings
increased iron stores
erythroid hyperplasia
10 to 40% ringed sideroblasts

85

Primary idiopathic sideroblastic anaemia

c.

Secondary sideroblastic anaemia

more common than hereditary

acquired and found in adults over 50 years.
Hct is about 25-30%.
Macrocytic normochromic picture mostly small
proportion of microcytic hypochromic found.
Erythroid hyperplasia - large number of ringed
sideroblasts.
10% of patients develop acute leukaemia

may be caused by certain agents: toxins or drugs

that interfere with haeme synthesis.

86

 Found in alcoholism, lead poisoning, TB therapy

and large doses of chloramphenicol.
Reversible condition
some patients respond to pyridoxine therapy.
3. HAEMOCHROMATOSIS
accumulation of iron in the parenchymal cells and
causes tissue damage.
If it accumulates in the macrophages without
causing a lot of damage to parenchymal cells it is
known as haemosiderosis.

Hereditary haemochromatosis:

is a rare disease
inherited as an autosomal recessive disorder.
found in middle-aged mean due to disorders in
iron absorption.
excess iron is stored in tissues of the pancreas,
liver
and spleen.
Patients generally show signs of:
(i) hepatosplenomegaly
(ii) bronze-colored skin pigmentation
(iii) Rheumatoid Arthritis type of symptoms
(v) Diabetes mellitus symptoms
often referred to as 'bronze diabetes'
Weakness and weight loss
Cardiac abnormalities, cirrhosis of liver and loss of
hair.

87

laboratory features
decreased transferrin
increased serum iron
increased transferrin saturation
increased transferrin saturation
normal Hb, Hct, blood smear
Iron overloaded parenchymal cells of the liver
(Biopsy).
treatment
1. Phlebotomy procedure (removal of 500ml) of
blood at regular intervals.
2. Chelation of iron using deferoxamine in patients
with Hb of less than 10 g/dl.
Iron overload may also be caused by:
1. Increased number of blood transfusions resulting in
iron being stored in macrophages.
2. Increased dietary intake (over 100mg per day).
3. Contributory factors such as abuse of alcohol and
liver disease.

88

BONE MARROW EXAMINATION

It is one of the largest organs representing 3.54.5% of total body weight (about 3.5 kg in adults)
It is found in the skeletal shaft.
Haematopoietic BM is organized around the
vasculature of the bone cavity.
Its main function is to produce and supply mature
blood cells to the circulation in a steady state.
It also responds to increased physiological and
pathological demands.
Consists of red or active marrow (1.5 kg/ 70 kg
man) and yellow or inactive marrow.
Major marrow-containing bones are: Skull,
Clavicles, Scapulae, Sternum, Ribs,
Vertebrae, Pelvis, Proximal ends of long
bones.
AGE DISTRIBUTION:
1. At Birth- marrow spaces filled with red marrow.
2. At 20 years- shafts of long bones filled by
fatty (yellow) marrow. Red marrow at proximal
ends
3. At advanced age 2/3 fat and red
- gradual red marrow
replacement from:
- Periphery
Central
- birth
advanced age

89

 Distribution of red marrow (functioning )

by weight (40 years)
Site

Wt (gm)

% of total
BM

Cranium and mandible

136.6

13.1

Humeri, scapulae and

86.7

8.3

Sternum

23.4

2.3

Ribs

82.6

7.9

Vertebrae

297.8

28.4

Pelvis

418.6

40.0

clavicle

BM can be sampled easily by a needle aspirate and/

or needle biopsy.
These are usually performed concurrently.
BM studies are done to aid in the diagnosis,
staging, and monitoring of several diseases.
Diseases such as the following are the most
common indications of BM examinations:
-Iron deficiency anaemia
-aplastic anaemia
-megaloblastic anaemia
-haemolytic anaemias
90

-thrombocytopaenia and thrombocytosis

-leukaemias
-multiple myeloma
-Guachers disease
-Niemann-Pick
Diagnostic cells in BM
- Sea blue Histiocytosis
-metastatic tumors
Systemic diseases may affect the BM secondarily
and may require bone marrow examination.
These include solid tumors such as; lymphoma
sarcoma and carcinoma which may metastasize to
the BM.
Sites for BM sample collection
Sternum in adults
Iliac crest (posterior and anterior)
Spinous process of the lumber
Tibia in children
Types of Bone Marrow samples

1.

Needle aspiration biopsy:

-involves the aspiration of the BM mass

-it is simple, safe and relatively painless
-but must not be done on cases of known blood
coagulation disorders.
-arrangement of marrow architecture is distorted
-blood may be aspirated also causing
Haemodilution.
-provides useful information

91

Micro trephine biopsy

4.

Surgical biopsy

-involves the removal of BM core

-more complicated to use
-now widely used than surgical
-relatively painless
-provides perfect view of large pieces of marrow
-good for the assessment of BM cellularity
-more superior to the aspiration method
-both aspirate and trephine give complementary
information.
-marrow is obtained by surgical means
-provides a large sample
-there are several disadvantages:
a) done in operation theatre
b) not suitable for patients with
bleeding disorders
c) can not be repeated
d) may leave an ugly scar at the site.

Equipment

1.

Needle:
a)

University of Illinois sternal needle consists

of a heavy duty short external needle with a
stylet.
b) Jamshidi
c) Islam

92

2.

Zenker's fluid:

3.
4.

1% Procaine
Slides, syringes, glass tubes, gloves, band aids
and alcohol
Decalcifier

5.

- 5% glacial acetic acid

- Potassium dichromate
- acts as a fixative

EXAMINATION
Peripheral blood
- full blood count results
- peripheral blood film
Cellularity
-based on the ratio of haematopoietic cells
to marrow space
-vary with age and site
-increased ratio is hypercellularity or
hyperplastic marrow
-decreased ratio is hypocellularity or
hypoplastic marrow
-best judged by the trephine biopsy sample.
Distribution
distribution of various cell types
ME ratio:
-based on total granulocyte to total
normoblast count
- normal range is 2:1 to 4:1
93

- increased ME (>6:1) found in CML,

infection, and erythroid hypoplasia
- decreased ME (<2:1) found in
leukopaenia and erythroid
hyperplasia
Normal number of megakaryocytes should be 1 to 3
per field at 100x.
Maturation
- balanced nuclear / cytoplasm development
- impaired cytoplasm maturation in Hb synthesis
- impaired nuclear maturation in megaloblastic
Anaemia
- Bizarre maturation in drug poisoning
Presence of Abnormal or rare cells
- tissue mast cells are found in aplastic anaemia
and refractory anaemia
- Osteoblasts are mistaken for myeloma cells
- Osteoblasts are mistaken for megakaryocytes
- both are very rare in the BM
-metastatic tumor cells resemble myeloblast, but
appear in clusters.

94

 Evaluation of trephine biopsy :

- gives better estimation of marrow
cellularity and the number of
megakaryocytes in the bone marrow.
- good for the diagnosis of lymphomas and
other types of neoplasm.
"Dry tap"
- marrow can not be aspirated easily
- occurs in myelofibrosis with myeloid metaplasia
(MMM) or granulomatous disease.
- Trephine biopsy should be done.
Appearance
- state marrow particles
- state whether there is haemodilution
- state the staining quality
Iron stain
-Use Perl's Prussian blue stain
-evaluation of iron should be done on every BM
sample.
Comment
- Diagnosis
- Other

95

BONE MARROW FAILURE

APLASTIC ANAEMIA

Basic defect is failure of the bone marrow to

produce cells - wbc, rbc, platelets.
Pancytopaenia - decrease in all formed elements
of the blood due to:
depletion in cell precursors
damage to haematopoietic stem cells
aplastic anaemia may be acquired :
1. Chemical exposure
2. Drugs (chloramphenicol)
3. Radiation
4. Infection
viral hepatitis
TB
5. Autoimmune disorders
it may also be due to unknown causes (idiopathic).
It may be a congenital disorder

1. Acquired aplastic anaemia

Physical and chemical agents

Clinical features
- Clinical course is characterized by rapid onset and
progression to death.
- slow onset and chronic course.

96

Laboratory features
- pancytopaenia
Wbc
Rbc
plt
-

normocytic normochromic blood picture

rarely macrocytic
varying degrees of anisopoikilocytosis
Basophilic stippling, polychromatophilia and
nucleated RBCs are absent
decreased reticulocyte count
BM is hypoplastic with increased fat
Bleeding time and clot retraction tests are abnormal
Serum iron is increased and TIBC is decreased

treatment
1. Removal of causative agent
2. Red cell transfusion
3. Platelet transfusion, if necessary
4. Antibiotic administration
5. Bone marrow transplant, (< 50 years)
6. Immunosuppressive therapy

2. idiopathic aplastic anaemia

-

An acquired condition of an unknown cause

Accounts for 50% of cases of aplastic anaemia
Laboratory tests and symptoms are similar to
those of the acquired aplastic anaemias.

97

3. Fanconi's anaemia (familial Aplastic

anaemia)
-

Congenital form of pancytopaenia found in

children.
Chromosomal defect identified
Autosomal recessive
Mental and physical developmental defects
present.
Deposition of melanin showing patches of brown
pigmentation of the skin.

Laboratory features
Normochromic normocytic blood picture
target cells present
Immature rbc and wbc
Bone marrow is normocellular to hypocellular as
the disease progresses
increased Hb F

4. Pure red cell aplasia

Anaemia associated with RBC diminished

production.
Other cell lines are not affected.
RBCs might also not be normal
Three forms are recognized:
a) Congenital hypoplastic anaemia (Diamond-Blackfan Syndrome)

98

Moderate to severe anaemia

associated with retarded sexual maturation,
osteoporosis and portal hypertension.
Females predominate, but it is autosomal
dominant disorder
First seen in infants with Hb <3g/dl
Marrow shows dearth of erythroid precursors
Plasma erythropoietin levels are increased
may result from:
a, innate deficiency in erythroid stem cells
b, selective defect of DNA synthesis
c, presence of haeme synthesis inhibitor
d, T-lymph mechanism that inhibits
erythropoiesis
progressive disease

Treatment:
1. Steroid administration
2. BM transplantation (limited by need donor
HLA compatibility)
3. Occasional transfusion (iron accumulation
problems)

b). Acute acquired erythroblastopaenia

-

Transient form of PRCA

found mostly in children after treatment with
antibiotic chloramphenicol
Occasionally found in association with
malnutrition
99

It is a reversible condition

c). Chronic acquired erythroblastopaenia

-

50% of cases found patients with

thymoma.
These patients have autoantibodies that
inhibit erythroid stem cells.
Also found in SLE and lymphoma patients
Anaemia is generally normocytic
normochromic
Normal WBCs and platelets
bone marrow has marked decrease in
maturing RBCs
decreased reticulocyte count or absent
increased serum iron and decreased TIBC

Treatment:
a, corticosteroids
b, immunosuppressive drugs

d). Congenital dyserythropoietic anaemia

(CDA)

Characterized by abnormal erythroblast with

multinuclearity, karyorrhexis and bizarre
malformations as a result of dyserythropoiesis.
Anaemias are divided into three groups:
Type I, II (HEMPAS), III

100

a). Type 1 (CDA I)

-

Rare condition
inherited as autosomal recessive trait
Mildly macrocytosis anaemia
a, anisocytosis
b, poikilocytosis
c, Cabot rings
d, basophilic stipplings
e, bone marrow megaloblastic erythropoiesis
f, binucleation and karyorrhexis

b). Type II (HEMPAS)

-

Hereditary erythroblast multinuclearity with

positive acid test serum test.
Most common form of CDA
inherited as autosomal recessive trait
Hepatosplenomegaly present
Usually a normocytic normochromic anaemia
Anisopoikilocytosis
Basophilic stipplings
Bone marrow shows Megaloblastic features
Multinuclearity of normoblast
RBC display haemolysis in acid serum test, but
none in sugar
water test.
RBCs contain blood group i antigen (HEMPAS
antigen)
Benign course

101

c). Type III (CDA III)

-

Very rare condition

inherited as autosomal dominant trait
Anaemia is slightly macrocytic
30% multinuclearity in bone marrow

102

COMMON WHITE CELL DISORDERS

Granulocyte kinetic changes in Disease

3 factors influencing granulocyte concentration:

Rate of input from storage pool
Proportion of circulating cells to marginal granulocyte
pool (MGP).
Rate of leaving circulation.
1. Neutrophilia
can be due to: pathological causes and non pathological causes such as vigorous exercise and
epinephrine administration which decreases MGP cells.
No change in BM output.
But anaesthesia increases cell margination.
Bacterial Causes of neutrophilia

Organism

Example

Cocci

Staph., Strepto.,
Pneumo., Gono.
E. Coli, Pseudomo.,
Corynebacterium
Actomyces
Rabies, herpes zoster
Typhus

Bacilli
Fungi
Viruses
Rickettsia

103

 Other major cause of neutrophilia

Cause

Example

Inflammation

surgery, burns, myocardial

infarction, rheumatic fever,
rheumatoid arthritis.

Intoxication

uraemia, drug and chemical

poisoning
liver, gastrointestinal, bone marrow

Haemorrhage
Acute haemolysis
Neoplasms
Physiological
Myeloproliferation

exercise, emotional problems, new

born, pregnancy.
CML, PV

2. Neutropaenia
can also be due to pathological causes and nonpathological
defective BM and acute infection may cause
inadequate input to CGP
during inflammation more bands than mature
neutrophils appear in circulation
the BM normally contains more bands than
neutrophils

104

 Principal causes of Neutropaenia

Cause

Example

Gram-negative bacteria
Viral
Protozoal
Physical agents
Rickettsial
Haematologic

typhoid, paratyphoid
flue, measles, Hepatitis B
malaria, leishmaniasis
radiation, chemical
Rocky Mountain Fever, typhus
Pernicious anaemia, aplastic
anaemia, leukaemia, Hodgkins
Disease, myeloma.
Lupus erythematosus, rheumatoid
arthritis, infectious mononucleosis
chronic granulocytopaenia of
childhood, hereditary Neutropaenia
cyclic Neutropaenia

Immunoneutropaenic
Pediatric
Miscellaneous

Laboratory findings in infection

Bands, other immature cells.
Toxic granulation - infection
Often confused with Chediak-Higashi and
Alder's anomaly
Dohle bodies may be found as round, oval or
spindle shaped structure staining grey-blue
in septicaemia, scarlet fever burns, pneumonia,
measles.
NBT test is positive in bacterial infection,
meningitis, candidiasis and Septicaemia.
AP is increased in leukocytosis due to bacterial
infection

105

3. Eosinophilia:
An increase in the number of eosinophils (>2%)
Principal causes of Eosinophilia

cause

Example

Allergy

Asthma, hayfever, drug

sensitivity.
Pemphigus, dermatitis,
tissue parasites, intestinal
parasites
CML, lymphoma, HD,
E-leukaemia
Acute TB, Brucellosis

Dermatoses
Parasites
Haematologic
Infection

4. Basophilia:
An increase in the number of basophils (>1%)
Principal causes of Basophilia

Cause

Inflammatory
Hypothyroidism
Immunological
Haematological

Examples

infection, varicella, variola

Myxedema, antithyroid therapy.
foreign protein injection, nephrosis.
chronic haemolysis, HD, CML,
erythaemia, MMM

106

5. Monocytosis:
An increase in the number of monocytes
Principal causes of Monocytosis

Cause

Examples

Bacteria + other microorganisms

TB, streptococcal infection

T. Pallidum, Plasmodium,
Trypanosome, Rickettsia.
Myeloproliferative disorders,
myeloma, lymphoma,
Histiocytosis
Ovary, stomach, breast.
LE, arthritis
Ulcerative colitis, regional
enteritis

Haematological disease
malignancy
Collagen vascular diseases.
Chronic inflammation

6. Lymphocytosis
An increase in the number of lymphocytes
Principal causes of Lymphocytosis

Cause

Examples

Acute viral infection

IM, mumps, chicken pox, Hepatitis

B.
Brucellosis, syphilis, tuberculosis
CLL, ALL, Lympho-sarcoma, NHL.

Chronic infection
Haematological

107

7. Lymphopaenia
A decrease in lymphocyte count
Principal causes of Lymphopaenia

Cause

Examples

Physiological

Stress, exercise,
haemorrhage.
Corticosteroids, cytotoxic
agents, irradiation.
Immune deficiencies, acute
infection, liver disease.

Pharmacological
Pathological

In lymphocytosis the count is:

>11 x 109/L in
adults

>8.8 x 109/L in children

108

LEUKAEMIA
unregulated malignant proliferation of
haematopoietic tissue that progressively displaces
normal cells.
Classified by: - predominant cell
- degree of maturity
- manifestations
Identification:- morphological criteria
- cytochemical tests
Etiology: - still not clearly understands
- factors associated with leukaemia:
1. Genetic (fourfold in siblings with
Down syndrome)
2. Exposure to chemical and physical
agents.
3. Viral infection.

1.

Hereditary

2.

Radiation
- causes acquired leukaemia
- Nodal irradiation for Hodgkins disease
developed AML.

- the most important aetiology of leukaemia.

- Down-trisomy 21 results in unstable cells
causing
further leukogenic changes
- Fanconi's anaemia.

109

- Nuclear explosions, e.g. Hiroshima and

Chenybryl.
3.

Chronic exposure to chemicals and toxins.

benzene and benzyl derivatives - acute
leukaemia.
Alkylating drugs - busulphan, chlorambucil,
and cyclophosphamides cause AML.

4.

Human leukaemia viruses were discovered

that also cause autoimmune deficiency
syndromes (AIDS).
Lower primates - RNA C-type virus
Epstein-Bar virus in Burkitt type leukaemia

Incidences of deaths from leukaemia

6.9 in 100 000
60% of deaths due to acute leukaemias
20% of deaths due to CML and CLL
Male to female ratio is 1.3:1
ALL usually affects 3 to 4 years age group
AML usually affects 15 to 20 years age group
CML usually affects 35 to 50 years age group
CLL usually affects those above 50 years.
Acute monocytic leukaemia favours middle aged
and rarely affects those below 30 years.

110

ACUTE LEUKAEMIA
Traditional classification:
morphological appearance
-subjective
-lack of cytochemical marker
-difficult separation of ALL on AMLs.
FAB (Bennett, et al. 1976).

can separate ALL from AML

subdivides each type

uses Romanowsky stained blood and BM cytochemical stains.

ALL classification modified (1981)
Reclassification of AML (M3) proposed in 1980, by
Golomb, Benette, et al.
Type 1: blasts lack cytoplasmic granules. Auer rod
increased.
Type 2: blasts possess a small number granule.
M5a - mainly undifferentiated cells - monoblast
predominate. Rare Auer rods.
M5b - cells mainly differentiated. Monoblasts
promonocytes and monocytes predominate.
WHO (2001)
WHO (2008)

111

1. Acute Myeloid Leukaemia (AML)

- appears suddenly with fever, body pains, often soar
throat, haemorrhage and severe anaemia.
Laboratory:
Anaemia
Moderate leukocytosis
Thrombocytopaenia
Invasion of the BM and PB.
Myeloblastic cells
Sub classification of AMLs
M1- immature blasts - non granular
3% Peroxidase positive.
Auer rods
M2- maturation beyond promyelocyte
30% of cells are blasts.
Frequent nucleolation
chromosomal translocation t(8;21)(Catovsky, 1981)
M3 - predominant cell is the promyelocyte
- heavy cytoplasmic granulation
- variant with minimal cytoplasmic granules with
multiple Auer rods
- DIC
- Chromosomal translocation t (15q+;17q), (Testa
1978).
- CML may transform into M3.
- Ph' aberration (9q+;22q-)
- No t(15q+17q)
112

 M4- both granulocytic and monocytic

differentiation.
- 20% of cells are monocytic and promonocytes.
- rare Auer rods.
M5 - differentiation based on Romanowsky and
cytochemical methods

- Two variants recognized.

- Rare Auer rods.
M6 - abnormal proliferation of both erythroid and
myeloid precursors.
- 50% of nucleated cells are erythroid cells.
- Bizarre morphological features
-multilobulation
- Multinuclei
- Megaloblastic changes
- Giant forms
- Mitotic figure often seen
M7- Megakaryocytic leukaemia
M8- Undifferentiated stem cell leukaemia

113

114

Classification of Acute Leukaemias based on

Cytochemistry
Peroxidase OR SB
Pos

Neg

AML

ALL

CLE
NSE

AUL

CLE-Pos
NSE-Neg

CLE-Pos
NSE-Pos

M1 M2 M3
M6

M4

CLE-Neg
NSE-Pos

Acid Phos.

M5a
M5b
Pos.
focal
T- ALL

115

Pos.
diffuse
B-ALL
or
AUL

Chromosomal abnormalities in Acute

Leukaemias
Banding techniques - (Rowley 1973).
60% AML and a smaller % ALL show non random
chromosomal abnormality
most common abnormalities are trisomy 8,
monosomy 7, t (8q-;21q+), and t(15q+; 17q-).
(1) t(15q+; 17q-) = M3
(2) t(9q+; 22q-) Ph' chromosome CML in blastic
transformation.
(3) terminal deletion of long arm of chromosome No.
11= M5
(4) terminal deletion of long arm of chromosome No.
11= M4
(5) In ALL frequency of abnormal chromosome is
50%:
Ph' 25% (ALL) in adult (L2)
9/22 translocation
(6) t(8q-; 14q+) in B-ALL (L3)
(7) t(8q-; 21q) in M2.

116

Acute lymphoblastic Leukaemia

Characterized by increased number of immature
lymphocytes.
Infiltration of the bone marrow.
The most prevalent malignancy in children peaking
at 307 years.
Rapid onset of - fatigability
- Infection
- bleeding problems
- Anaemia
FAB classification (L1, L2, L3)
L1 - small cells predominate, characterized by scanty
cytoplasm.
- Vacuoles uncommon
- Some cells have clefted or indented nuclei
- The most common type of ALL with 85% of
cases in children.
- Prognosis is favourable.
- Immunologic studies show L1 also includes
undifferentiated T-cell and null cell ALL.
- T-cell ALL has poor prognosis.
L2: - Large and small cells predominate
- moderately abundant cytoplasm
- vacuoles are uncommon.
- Irregular nuclei with clefts and indentations.
- Like L1, cells carry CALLA, T-cell, and null cell
117

markers.
- 14% of children with ALL have L2 type.
- No B-cell markers
L3: - Rarest form (<1% of childhood cases)
- most distinct morphologically and
immunologically.
- Cells are homogenous and large.
- Cytoplasm has large prominent vacuoles
- Cytoplasm is heavily basophilic
- Marker show these are 100% of B-cell type
- commonly known as a Burkitts lymphoma ALL.
- it has the worst prognosis
- clinically characterized by a swollen jaw.
Haematological and clinical remissions of ALL were
maintained for 2 - 10 years.
-White children fared better than black
children (due to greater dissemination
of disease at first presentation).
Wbc <10 000 (l = better response)
wbc >50 000 (l = poor response)
poor prognosis included CNS involvement
(Leukaemic cells in spinal cord).
Only 10% of Acute Leukaemia in adults is of
lymphoid origin and 80% of AL in children is ALLs.

118

119

CHRONIC LEUKAEMIAS
1) Chronic Myeloid Leukaemia
Clonal stem cell disorder of unknown aetiology
Associated with acquired chromosomal disorders
(90%).
May develop after radiation exposure.
Clinical manifestations
usually insidious
Malaise, night sweats, and weight loss are the main
features.
fever
bone pain
haemorrhage - depending on degree of
thrombocytopaenia
anaemia and hepatosplenomegaly may be present
Rarely diagnosed before 20 years of age.
Most common after 30years of age.
Clinical course
Phase 1: asymptomatic granulocyte proliferation
Phase 2: persistently elevated leukocyte count.
Phase 3: acceleration of cellular proliferation.
No extramedullary involvement:
- Malignant cells confined to haematopoietic
tissue:
- Bone marrow
120

- Spleen
- Liver
Chronic phase is:
Unstable
Transformation into aggressive form
Blastic phase
Resistant to chemotherapy
Blastic phase characterized by maturation
arrest at progranulocyte stage.
blastic crisis can be myeloid or lymphoid.
Lymphoid blastic crisis is found in 25% of
CML
Myeloid blastic crisis
- Myeloid cells
- Myeloid antigens and cytoenzymes
- Heterogeneous cells
Laboratory Findings:
Extreme leukocytosis (>750 x 109/L)
Differential count show increased neutrophils,
bands, metamyelocytes and myelocytes.
Predominant cell is NEUTROPHIL
eosinophils and basophils also increased
chronic normocytic normochromic anaemia
P.B. with varying degree of anisocytosis
poikilocytosis, polychromasia, basophilic stippling,
reticulocytosis and nucleated rbcs.
Thrombocytosis also present early.
Bone marrow - hypercellular
121

- increased myeloid cells

- Chromosomal abnormality in 90%
deletion of long arm of G22 to G9 t(9q+; 22q-)
Ph' chromosome present
also found in rbc precursors and megakaryocyte
precursors.
Not found in lymphoid cells
< 10% are Ph' -negative
Plt function is abnormal due to lack of secondwave epinephrine induced aggregation and
impaired collagen induced aggregation? Storage
pool deficiency.
Uric acid production increased due to increased
catabolism of granulocytes
decreased production of leukocyte - alkaline
Phosphatase (LAP)
Increased levels of serum B12.

122

123

2. Chronic lymphocytic leukaemia

-

is an abnormal, uncontrolled proliferation or

accumulation of lymphocytes.

TYPES
1. COMMON CLL (B-CLL)
Most common CLL in the west, uncommon in
oriental.
Rare bellow 40 years
Male: Female ratio 2:1
onset is insidious
presenting symptoms
- Anaemia
- Infection
- X-lymph
routine blood examination
Blood picture
Moderate anaemia - autoimmune
- Bone marrow replacement
wbc 15 x 109/L
absolute lymphocytosis - 98% lymphocytes (small)smudge cells.
Cells are monotonously similar
Positive Coomb's test

124

Bone marrow picture

No involvement earlier
moderate lymphocytosis
Bone marrow crowding later
2. B-PROLYMPHOCYTIC LEUKAEMIA (B-PLL)
Clinically distinguishable
- massive splenomegaly
- no lymph node involvement
- found in men over 60 years.
Blood picture
- wbc 100 x 109/L
- Prolymphocytes predominate
- Distinct nucleoli
125

3. HAIRY CELL LEUKAEMIA

- CLL type
- Common in male 40-60 years
- Pancytopaenia
- Splenomegaly
Morphology
- Hairy cells - medium size with projections and
Irregular nucleus
- Bone marrow aspirate difficulty
- Weak phagocytic activity.
Techniques:- full blood count
- Differential count
- Cytochemistry
- Monoclonal Ab testing

PRELEUKAEMIAS
Definition - haematological syndromes of different
severity with outcomes linked to a common risk of
developing into leukaemia disease in retrospect after
the diagnosis of acute leukaemia.

Five Etiological groups

(1) Diseases with high incidence of leukaemia

- Polycythaemia Vera
- Idiopathic myelofibrosis
126

(2)
(3)

(4)
(5)

- PNH
patient with chromosomal abnormalities but
have no haematological disorder
- Down Syndrome.
Patients with non-haematological disorders but
have risk to develop leukaemia if treated with
cytotoxic drugs.
- Carcinoma of breast
- Carcinoma of ovary
- Renal transplant patients on
Immune-suppression drugs
Individuals exposed to known leukaemia
agents.
Those with miscellaneous disorders such as:
-Idiopathic acquired sideroblastic
Anaemia
-chronic erythroid myelosis

127

PARAPROTEINAEMIA
Definition- a group of blood disorders that are
characterized by presence of increased levels of abnormal
plasma proteins
1. Multiple myeloma
- Neoplastic proliferation of plasma cells mainly in the
bone marrow and occasionally in the lymph nodes and
spleen.
Clinical findings:
- Most common in elderly patients
- Characterized by - bone pain
- Anaemia
- Uremia
- Infection
- Kidney involvement associated with renal tubular
damage due to Bence-Jones protein precipitation.
- Infection caused by impaired antibody synthesis
- Haemorrhage sometimes present due to decreased
Platelets or impaired function.
- Blood viscosity is increased by abnormal proteins
causing circulation impairment and heart failure

Laboratory features
PB
normocytic normochromic anaemia, but sometime
hypochromic features seen
there is Rouleaux formation on blood film
ESR is elevated.
Plasma cells in the blood indicated an advanced stage
There is tissue infiltration. ?Leukaemic phase
128

BM
hypercellular
plasma blasts and proplasma cells present
plasma cells are the same as normal, but:
may lack pronuclear halo
less tense staining
more open chromatin material
large nucleoli
Russell bodies (cytoplasmic protein inclusions that stain
pink with Romanowsky) present
Mott cells (plasma cells with grey-blue inclusions) present

129

Principle laboratory feature

- Serum myeloma proteins
- Urine monoclonal light chains ( )
- M type protein on electrophoretic strip
Protein Abnormalities in MM
Serum M-type Urine Bence-Jones Protein (light
chain)
IgG
k,
IgA
k,
IgD mixed
Variable
None
l,

130

incidence
52%
21%
2%
25%

2.Waldenstrom's Macroglobulinaemia
- Principally found in the elderly patients
- characterized by a lymphocytic and plasma cell
proliferation and the presence of at least Ig /dL of monoclonal
IgM protein.
Clinical findings
- Those related to presence of abnormal proteins
and hyper viscosity of blood.
- Lymphadenopathy and hepatosplenomegaly
- Haemorrhage
Laboratory findings
PB: - Normocytic normochromic anaemia
- Due to haemolysis
- Blood loss
- Thrombocytopaenia
- Hyperuricaemia
- Relative lymphocytosis
- Rouleaux formation
- Occasionally positive Coomb's test
BM: - difficult to obtain
- Numerous lymphocytes with positive PAS
Chemistry:- Serum globulin elevated
- Immunoelectrophoresis show u-heavy chains.
- Positive SIA water test
3. Heavy Chain disease
- Very rare disease
- resembles malignant lymphoma
- Patients present with:
- Hepatosplenomegaly
- Lymphadenopathy
- Fever
- Infection
Laboratory findings
Normocytic normochromic anaemia

131

MYELODYSPLASTIC SYNDROMES
(MYELODYSPLASIA)
Definition:
A large group of acquired neoplastic diseases of the BM,
most common in the elderly population and characterized
by increased bone marrow failure with quantitative and
qualitative abnormalities of all myeloid cell lines
(erythrocyte, granulocyte/ monocytes and platelets) due
to a defective stem cell.
Most patients are usually over 50 years, but younger ones
are being diagnosed with MDS, especially after prolonged
treatment with cytotoxic drugs or radiation.
There is marrow hyperplasia and peripheral cytopaenia.
They include severe forms of refractory anaemias and a
type of chronic leukaemia.
Major characteristic is ineffective haematopoiesis with
cytopaenia accompanying a marrow of normal or
hypercellularity.
The disease tends to progress to acute leukaemia (AML or
ALL).
MDS are, therefore, preleukaemias.
Causes:
Cytogenetic abnormalities are more frequent in secondary
than in primary MDS.
Thought to be caused by partial or total loss of
chromosomes 5, 7 or Y or trisomy 8.
RAS oncogene mutations are found in 20% of MDS and
FMS mutations in about 15% of the cases.

132

Morphological and functional Abnormalities.

Abnormal clone coexists with normal cells.
Erythroid line:
There is a dimorphic blood picture- macrocytic and
normocytic cells.
Normocellularity varies with age.
Erythroid precursor population varies from 5% to 60%
(normal ME ratio = 3:1)
Exhibition of distinct immaturity.
Presence of ringed sideroblasts
Megaloblastoid cells
Multinuclear normoblasts with coarse clumped chromatin
Defective haemoglobinization is evident
Low reticulocyte count is evident of dyserythropoiesis.

Myeloid line

Dysgranulopoiesis is characterized by neutropaenia

Many neutrophils are without granules
Some may be bizarre and large in size.
Immature granulocytes are increased (should be less than
30% to exclude acute leukaemia)

Platelet line

Ineffective platelet production is common resulting in

peripheral thrombocytopaenia, although normal or high
counts can be found.
Megakaryocytes may be small or large, with single or
hypersegmented nuclei.
Megakaryocytes may exhibit hypogranulation or no
granulation.
Platelets are usually dysfunctional resulting in bleeding.

133

Step1
PRESENCE OF MYELODYSPLASIA? (BMhyper/dys; PBPancyto.)

Step 2
Are BM Blasts> 20%?
YES

NO

AML

Step 3
Are Monocytes > 1 x 109/L?
CMML

Yes

10 - 19%
RAEB2

No
Step 4
% Blasts?
<5%
Step 5
Any deleted (5q) in cyto.?

5-9%
RAEB1

Yes

No

MDS (del. 5q) Step 6

>15%
Multilineage dys.?
( 2 or more cell lines)
(>10% dys.)

% Ring sideroblasts?
<15%
Multilineage dys.?
(2 or more cell
lines;>10% dys.)

Yes

No

Refractory Cytopaenia
with multilineage dysplasia
(RCMD)

Refractory Anaemia
with ringed sideroblasts
(RARS)

Yes

Refractory Anaemia
with multilineage dysplasia (RAMD)

134

No
Refractory Anaemia (RA)

Current WHO Classification

1. Refractory Anaemia (RA)
-

dyserythropoiesis is the main finding

anaemia is the main outcome
there is occasional bicytopaenia and
pancytopaenia

2. Refractory Anaemia with Excess Blasts (RAEB 1 & 2)

RAEB I (5 - 9% blasts and granular)

RAEB II (10 - 19% blasts and agranular)

3. 5q Syndrome
-

Bone marrow is normocellular

Normal or increased platelet count
Decreased and dysplastic erythroid cells
Dysplastic granulocytes
Good prognosis

4. Refractory Cytopaenia with Multilineage Dysplasia

(RCMD)
-

bicytopaenia or pancytopaenia
neutrophils are occasionally hypogranular and
dysfunctional
Prognosis is worse with frequent leukaemic
changes

135

5. Refractory Cytopaenia with Multilineage Dysplasia

and Ringed Sideroblasts (RCMD-RS)

-Extensive erythroid hyperplasia with variable

dyserythropoiesis.
-Poor haemoglobinization.
-Iron stain reveals marked iron deposits in nuclear
margins.
-10% of patients show neutrophil dysfunction.

6. Chronic Myelomonocytic Leukaemia (CMML)

-presents of many monocytic precursors in the BM

(5-20% blasts) and blood (<5%).
-Other cells are abnormal and cytopaenia is
present.
-Hepatosplenomegaly is common
-BM is dominated by myelocytic elements
containing Auer rods in some blasts.

Clinical Features:

50% of patients are > 70 years and 25% are less

than 50 years.
Males are more commonly affected than females.
Disease may be found by chance.
Symptoms are those related to anaemia, infection
or bleeding.
Splenomegaly is usually found in CMML only.
Laboratory Features:
Pancytopaenia is the most frequent finding.
Blood picture varies from macrocytic, dimorphic,
and occasionally hypochromic.

136

Reticulocyte count is low.

Agranular granulocytopaenia is present.
In CMML there is monocytosis
Platelets are unusually very large.
In poor prognosis, myeloblasts may appear in
circulation.
BM cellularity is increased
Ringed sideroblasts may occur in all types of MDS.
Multinuclear normoblasts are common in the BM.
BM biopsy shows fibrosis in 10% of the patients.

Treatment:
Often very difficult as there is no therapy to revert
to normal haematopoiesis.
Those patients with Low-grade MDS (<5% blasts)
rely on red cell and platelet transfusion or
antibiotics.
High-risk MDS (5% or more blasts) patients
-General supportive care only (elderly patients
with other medical problems).
-Low-dose cytotoxic therapy to reduce blasts
and promote cell differentiation.
-Administration of growth factors such as
CSF-GM.
-Single agent oral chemotherapy for CMML
or RAEB.
-Intensive chemotherapy for younger bad-risk
patients (< 60 years).
-BM transplantation for younger patients (50-55
years) with HLA-matching siblings or HLAmatching unrelated donors.

137

MYELOPROLIFERATIVE DISEASES
Definition:
A group disorders characterized by clonal
proliferation of one or more haematopoietic
components in the bone marrow, spleen or liver.
The diseases are closely related to each other in
that: 1) transitional forms are present; 2) they tend
to evolve from one entity to another during the
course of the disease.

BM STEM CELL
Acquired abnormality

CP Granulocyte
Precursor

Disease CML
Entity
70%

Red cell
Precursor

PV

Mega'cytes----Reactive fibrosis

10%

ET
30%
10%

AML

MMM

AML= Acute Myeloblastic Leukaemia, CP= Cellular Proliferation, CML= Chronic

myeloid leukaemia, PV= Polycythaemia Vera, ET= Essential Thrombocythaemia,
MMM= Myelofibrosis with Myeloid Metaplasia

138

Current WHO Classification:

Myeloproliferative diseases are classified as Polycythaemia
Vera (PV), Essential Thrombocythaemia (ET),
Myelofibrosis with Myeloid Metaplasia (MMM) and Chronic
Myelogenous Leukaemia (CML).
PV, ET and MMM are collectively known as non-leukaemic
myeloproliferative disorders.

Polycythaemia (Erythrocytosis) is a pattern of blood

changes that is characterized by red cell count >6 x 1012/L

(males) and 5.5 x 1012/L (females); Hg >17.5g/dl (males)
and 15.5 g/dl (females); Hct > 55% (males) and 47%
(females). It is caused by several factors such as BM
abnormalities, compensatory erythropoietin production and
inappropriate erythropoietin production, and in relation to
stress, dehydration, plasma loss due to burns and
enteropathy. Other blood cells are usually normal.

1. Polycythaemia Vera

There is a marked increase in all cell lines.

Thought to be caused by chromosomal changes in the
BM.
Clinical Features
Headaches, blurred vision, and night sweats
Ruddy cyanosis, retinal engorgement
Splenomegaly in two thirds of patients
Haemorrhage or thrombosis is very frequent and major
causes of death.
Hypertension in one third of the patients
Gout is common
Laboratory Features
Increased RBC, HB, Hct and TRCV.
Raised WBCs and Plts
139

Increased serum Vitamin B12 and plasma urates

Hypercellular BM.
Increased blood viscosity.
Circulating erythroid precursors

Treatment:
Aimed at reducing blood cell count.
a) Venesection for a rapid reduction red cell volume at start
of treatment. Recommended for young patients and those
with mild disease. The resulting iron deficiency may limit
erythropoiesis.
b) Cytotoxic myelosuppresion- daily high-dose therapy with
busulphan or hydroxyurea.
c) Phosphorus-32 (32P) therapy for older patients with
severe disease.

2. Essential Thrombocythaemia

There is marked megakaryocyte proliferation and

overproduction of platelets (> 450 X 109/L).
Recurrent haemorrhage and thrombosis are principal
features.
Both splenomegaly and splenic atrophy may be present.
Anaemia may result from chronic gastrointestinal or uterine
bleeding or due to the BM disorder itself.
Causes of raised platelet count
Reactive -chronic iron deficiency
-malignancy
-chronic infection
-Post-splenectomy
Endogenous -ET
-PV, CML, MMM

140

Laboratory features:
Large platelets and megakaryocyte fragments in
circulation (blood film).
Marked elevation of platelets.
Abnormal platelet function tests.
Treatment:
1. In severe case hydroxyurea is given to lower platelet
count
2. Platelet pheresis is sometimes recommended in shortterm management.
3. Young patients may be given low-dose aspirin to reduce
the risk of thrombosis.

3. Myelofibrosis

A condition characterized by generalized haematopoietic

stem cell proliferation with splenic and hepatic
involvement.
Circulating stem cells are found and associated with
extramedullary haematopoiesis.
Reactive fibrosis in the BM secondary to hyperplasia of the
megakaryocytes.
Fibroblasts are stimulated by a platelet-derived growth
factor
A third of patients have a history of PV.
Clinical Features:
The onset is insidious in older patients with symptoms of
anaemia.
Splenomegaly is usually the presenting physical finding.
Loss of weight, anorexia and night sweats are common.
Bleeding, bone pain or gout is present in a minority of
patients.
141

Laboratory Features
Anaemia is usual but a normal or high haemoglobin may
be present.
There is high WBC and Plt count at the time of
presentation.
Leukopaenia and thrombocytopaenia appear as the
disease progresses.
Leuko-erythroblastic blood film with characteristic 'tear
drop' red cells.
BM is unobtainable by aspiration.
Trephine biopsy shows a hyperplastic marrow with
increased reticulin-fibre pattern.
Radio-iron studies show extramedullary haematopoiesis.
10-20% of patients transforms into acute myeloid
leukaemia (AML).
Treatment:
1. Supportive blood transfusion and regular folic acid therapy
in severely anaemic patients.
2. Cytotoxic agents, eg. hydroxyurea in patients with gross
myeloproliferation and hypermetabolism.

142

HAEMOSTASIS
A process by which blood loss is prevented by a
series of related and overlapping mechanisms
(vascular, platelet and coagulation) that result in
the formation of fibrin mass enclosing red cells and
platelets.
1. Primary haemostasis - depends upon the
response of blood vessels and platelets to injury:
-Platelets adhere, aggregate, release
contents and retract after injury
-blood vessels undergo vasoconstriction and
release tissue thromboplastin, collagen and
tissue plasminogen activator.
Abnormalities involving primary
haemostasis are characterized by
bleeding from mucous membrane (
epistaxis, menorrhagia, GI bleeding)
and bleeding into the skin (petechiae
and ecchymosis).
143

Laboratory tests for primary haemostatic

disorders:
-platelet count
-tourniquet test
-bleeding time
2. Secondary haemostasis - involves the activation
of coagulation plasma factors enzymatically
through a cascade.
Three pathways are involved:- intrinsic
- extrinsic
- common
Laboratory tests for secondary
haemostatic disorders:
- prothrombin time (PT)
-

activated partial thromboplastin time

(APTT)

- thrombin time (TT)

3. Control mechanisms- haemostasis is a carefully
controlled process, and finely tuned to respond to

144

injury while maintaining a normal blood flow at the

same time.
- the control mechanisms include:
1) localization of fibrin formation ( fibrin and plt
provide reaction surface)
2) consumption of key factors (V and VIII)
3) presence of natural inhibitors:
a) proteins C and S; (Va and VIIIa)
b) extrinsic pathway inhibitors; (VIIa-III)
c) C1 inhibitor; (XIIa, PK, to some extent XIa and
plasmin)
d) Alpha 1 antitrypsin; (thrombin, Xa and XIa)
e) Antithrombin III-heparin;(XIa, XIIa, PK, IXa and
Xa)
f) Alpha2 macroglobulin; (PK and thrombin)
3. Fibrinolysis - involves the digestion of fibrin clots
and fibrinogen in order to keep the vascular system
free of deposited fibrin.
Takes place after the activation of plasminogen to
plasmin.
145

- this is the major haemostatic control mechanism.

A very delicate balance exists between
haemostasis and its control mechanisms.
Any imbalances in the two systems may result in
HAEMORRHAGE or THROMBUS FORMATION.

146

BLEEDING DISORDERS
Recognition of bleeding disorders took place long
before the understanding of haemostatic
mechanisms as shown by:
- avoiding circumcision in males
whose brothers had bled to death.
- first description of haemophilia dating
back in1793 literature.
- recognition of Haemophilia as X-linked
by Otto in 1803.
- Queen Victoria of England was known
to be one of the most prolific carriers of
the haemohiliac genes.
Classification of bleeding disorders is based on four
major components- vascular, platelet, coagulation
and thrombotic
Diagnosis of these disorders is dependent on:

147

i)

Accurate medical history

ii)

good physical examination

iii) use of appropriate laboratory tests

Commonly used terms to describe bleeding
manifestations
1) Ecchymosis: large, irregular areas of blue-black
discoloration of the skin or mucous membrane.

2) Epistaxis: nose or nasal sinuses bleeding from the

3) Haemarthrosis: bleeding into the spaces
surrounding the joints.
4) Haematemesis: vomiting of blood, originating
from the GI or from the pharyngeal tract.
5) Haematoma: bleeding into tissue around a vessel,
resulting in a darkened swelling.
6) Haematorrhea: severe, profuse bleeding.
7) Haematuria: presence of blood in the urine.
8) Haemoptysis: bleeding from the oral cavity,
originating in the larynx, trachea, bronchi or lungs.

148

9) Pertechiae: small flattened red spots on the skin

and mucosal surfaces caused by bleeding from the
capillaries.
10) Purpura: large areas of bleeding into the skin,
tissues and mucous membranes with purple to
brown to yellow discoloration.
11) Embolus: a thrombus or clot that has been
dislodged from the site of formation.
12) Haemangiectasis: dilation of blood vessels.
13) Haemangioma: benign tumor of the blood
vessels.
14) Haemophilia: hereditary defect/deficiency of
coagulation factors VIII, IX and XI.
15) Thrombosis: formation of a blood clot in the
vessels; normal process is to stop haemorrhage,
but pathologic if there is vascular occlusion.

149

A. VASCULAR

DISORDERS

1) HEREDITARY DEFECTS OF THE VESSEL

STRUCTURE

a. Hereditary haemorrhagic telangiectasia

b. Haemangioma-thrombocytopaenia syndrome
c. Connective tissue dysplasia
Hereditary haemorrhagic telangiectasia (HHT) is the
most common of the hereditary vascular disorder.
It is inherited as an autosomal dominant trait.
It is characterized by nose-bleeding.
Small skin lesions (1-3 mm) are found in adults due
to decreased content of elastic fibre.
Laboratory investigations in HHT:
1. Normal bleeding time (3-10 min)
2. Positive Tourniquet test- presence of
petechiae
3. Normal plt count
4. Variable plt function tests
5. Iron deficiency anaemia present
2. ACQUIRED VASCULAR DISORDERS
a) Allergic purpura
b) Senile purpura
c) Secondary to systemic diseases
d) Others; drug-induce, Vitamin C deficiency.
Allergic Purpura is the most commonly acquired forms
of vascular defects.
It is caused by an autoantibody of the IgA type
directed against the vascular endothelium.
150

 Found in children after infection by rickettsial, viral,

bacterial or mycoplasmal agents.
The organism may also damage the endothelium
resulting in vasculitis.
Antibodies produced against the vessels cause
diffuse rashes of petechiae or purpural spots.
The most affected parts are the lower limbs and
the buttocks area.
Appearance may be very rapid and is usually
accompanied by itching.
Allergic origin of AP has been attributed to insect
bites, certain foods, various drugs and vaccinations.
Laboratory investigations- are usually not very
specific:
1) normal bleeding time
2) normal tourniquet test
3) normal coagulation tests
4) increased ESR
5) Increased neutrophil count
6) Proteinuria and haematuria may be
present temporarily.

151

B. PLATELET DISORDERS
They are usually very diverse and complex
reflecting the heterogeneous functions of platelets.
Pathological changes in haemostasis or coagulation
may result from decreased count or dysfunctional
platelets.

1) QUANTITATIVE DISORDERS

a) thrombocytopaenia
b) thrombocytosis and
thrombocythaemia
thrombocytopaenia involves the majority of
platelet disorders.
Platelet count is usually less than 100000 /ul.
There is usually a correlation between bleeding
time and plt count.
It is caused by many conditions:
-impaired platelet production
-increased destruction of circulating platelets
-disorders related to distribution and dilution
Thrombocytosis is a platelet disorder
characterized by increased plt count (may be over
1million/ul):
a) in association with other clinical conditions
(reactive thrombocytosis). In this case the platelets
are normally functioning.
b) it may be associated with myeloproliferative
diseases such as PV, MM and CML.
152

 Thrombocythaemia is a state of uncontrolled

platelet production.
the function of platelets is abnormal.
The count may exceed 10 million/ul.
This condition is defined as essential
thrombocythaemia.
There is often thromboemboli formation and
bleeding associated with this disorder.

2). QUALITATIVE DISORDERS

These involve changes in platelet function and are

grouped into five categories:
i)
abnormalities in plt interaction with
vascular tissue
ii) abnormalities in plt adhesion
iii) abnormalities in primary plt aggregation
iv) abnormalities in secondary aggregation
v) abnormalities in coagulation factor
receptors
Laboratory investigations in platelet disorders
1) Plt count
2) Blood film
3) Bleeding time
4) If count is normal; plt function tests:
i)
clot retraction
ii) Plt aggregation
iii) Plt adhesion
iv) plt factor 3 availability
v) plt antibody screening.

153

C. COAGULATION DISORDERS
Coagulation disorders are characterized by deep
tissue bleeding:
-bleeding into the joints (haemathrosis)
-intramuscular bleeding- compartmental
syndromes
-intracranial bleeding
Mucosal membrane bleeding is common in patients
with multiple factor defects/deficiencies.
Some patients develop large ecchymoses
Prolonged coagulation tests; PT, APTT and TT
detect most clinically significant defects.
One or a combination of these tests will be
prolonged.
Most abnormalities are classified as being
hereditary or acquired.
Hereditary forms are almost always single defects
Acquired forms are usually multiple defects
Hereditary
Classic Hemophilia's
Von Willebrand
Defective II, VII, IX and X
Fibrinogen defects
Contact factor defects
Kininogen defects

Acquired
DIC
fibrinolytic syndromes
liver disease
circu'ting anticoa'lants
drug-induced
vitamin K deficiency

154

 Of the hereditary defects, the inheritance is as

follows:
1. Autosomal recessive; most coagulation defects
2. Autosomal dominant; -von Willebrand,
-afibrinogenaemia,
-Hypofibrinogenaemia, and
-Dysfibrinogenaemia
3. Sex-linked recessive; VIII and IX defects

1. FACTOR VIII: C DEFECT

It is one of the most common coagulation defects.

The incidence is about 1/10000.
Haemophilia A is the most classic type
It is inherited as sex-linked recessive disorder
90% of the patients have both VIII:C and VIII:C Ag
defects. They lack some cross reactive antigenic
material (CRM-)
These are truly deficient
10% do not have VIII:C, but have the antigenic
material (CRM+)
these have dysfunctional factor VIII:C
70% of Hemophiliacs show a familial history
30% of the patients show a spontaneous gene
appearance.
Both CRM+ and CRM- show some clinical
manifestations
Women are carriers while men are affected by the
disease.
155

 A carrier mother passes the defect to 1/2 of her

sons (haemophilia A) and 1/2 her daughters
(carriers).
Affected father transmits the defective genes to all
his daughters but none to the sons because sons
inherit the X gene from the mother.
Female haemophiliacs do exist in heterozygous
carriers with very low factor VIII levels. There is no
demonstrable bleeding tendency.
Homozygous female haemophilia has been found in
terms of both parent who were affected (affected
male + carrier female).
Clinical features:
-infants may suffer from post-circumcision
haemorrhage
-recurrent painful haemathrosis and muscle
haematoma
-muscle haematoma dominates the clinical course
of the severely affected patients with progressive
deformity and crippling.
-prolonged bleeding after dental extraction
-haematuria more common than GI bleeding
-clinical severity correlates very well with levels of
factor defect
-operative and post-traumatic haemorrhage are
usually life-threatening
-Intracelebral haemorrhage occurs more
frequently than in the general population.

156

-haemophilia pseudotumors may occur in the

long bones, pelvis, fingers and toes.
-over 50% of haemophiliacs are HIV positive

Laboratory investigations in
Haemophilia:
1. abnormal APTT
2. normal PT (INR)
3. abnormal factor VIII assay
4. bleeding time is normal
5. plt count is normal or decreased
TREATMENT
1. Factor VIII replacement
2. Desmopressin (DDAVP).
3. Local supportive measures used in treating
haemathrosis and haematoma include:
(i) Resting the affected part
(ii) Prevention of more traumas
(iii) Increasing the availability of Factor VIII
by storing in domestic fridges - has reduced
occurrences of crippling condition.
4. Haemophiliacs are encouraged to have regular
dental checks.

157

ACQUIRED COAGULATION DISORDERS

These are more common than the inherited forms
Involve multiple factor defects
They are almost always caused by an underlying
diseases/conditions such as:
1. vitamin K deficiency
2. liver disease
3. DIC
4. Circulating inhibitors; eg. antibodies to coagulation
factors, non-specific inhibitors, antibodies found in RA
and SLE
5. Miscellaneous; eg. heparin therapy, defibrinating
agents, or thrombolytics
A) VITAMIN K DEFICIENCY
Vitamin K is a fat soluble compound found in green
vegetables and biosynthesized by bacteria in the gut.
Deficiency may present with haemorrhagic disease of the
new borne (HDN) in infants or later in life.
It is caused by inadequate dietary intake, malabsorption
or drugs (warfarin), resulting in decreased functional II,
VII, IX and X, protein C and S.
-Immunological assays show normal results
-non-functional proteins are called PIVKA
(proteins formed in vitamin K absence)
Laboratory investigations
1. PT and APTT;
2. platelet count
3. fibrinogen;
4.TT;
5. FDPs or D-dimer assay

158

THROMBOSIS

It is defined as the formation of blood clots in the

circulation that consist of blood constituents such
as platelets, red cells and fibrin. The relative
constitution and distribution of these components
are used to consider the possible mechanism of
thrombosis.
There are two major types of thrombi; arterial and
venous.
Arterial thrombi develop in relationship to platelet
reaction and accumulation in response to vascular
wall damage. It said to contain a platelet rich 'head'
with a small red cell 'tail' in fibrin mesh.
Venous thrombi develop in relationship to the
generation of thrombin in areas of retarded blood
flow. There is a very small platelet content. It is
similar in composition to a clot formed in vitro.
Multiple thrombi in microcirculation results in DIC
syndromes.
Thrombosis is common with age depending on
prevailing risk factors.

159

Risk Factors for Thrombosis

___________________________________________
Arterial
Venous
Positive family history
Smoking
Hypertension
Diabetes mellitus
LDL cholesterol
Increased haematocrit
Left ventricular hypertrophy
Oral contraceptives
Fibrinolytic syndromes
Male sex

obesity
oral contraceptives
varicose veins
infections
trauma
surgery
general anesthesia
pregnancy
fibrinolytic syndromes
malignancy
Immobility
congestive heart failure

nephrotic syndrome

blood protein abnormality

___________________________________________
Laboratory investigations
Thrombotic disorders have attracted much research
attention in their aetiology and prevention.
But, there is no single reliable technique for the
recognition of a thrombotic state.
Multiple, expensive and time-consuming tests are used for
differential diagnosis of these disorders (thrombophilia
screen):

1. full blood count and film

2. PT and APTT
3. TT
4. Fibrinogen assay

160

5. Antithrombin III assay

6. Protein C and S assay
7. Plasminogen assay
8. Plt function tests
9. Plt factor 3 and 4 assays

Treatment
Anticoagulant drugs are widely used to treat
thromboemboli diseases.
Their value in the treatment of arterial thrombosis
is less understood.
1. HEPARIN
Heparin is used to potentiate the action of
antithrombin III to inactivate IIa, IXa, Xa, and XIa.
It also impairs platelet function.
Usually given to treat deep vein thrombosis,
myocardiac infarct, unstable angina pectoris, acute
peripheral arterial occlusion.
Bleeding may result due to antiplatelet effects and
heparin-induced thrombocytopaenia.
This may be prevented by administration of
protamine to inactivate heparin.
Long-term heparin therapy may cause osteoporosis
in pregnancy due to complex formation with
minerals.

2. DIRECT THROMBIN INHIBITORS

161

 Hirudin and its fragments, directly inhibit

thrombin function and has potential as
antithrombotic agent, but it is not yet in clinical
use.
3. ORAL ANTICOAGULANTS
Warfarin derivatives (coumarin) are widely used
and are well absorbed from the gut.
These drugs are vitamin K antagonists.
They decrease biological activity of vitamin K
dependent factors (II, VII, IX and X).
The effects of oral anticoagulant are monitored by
PT/INR.
Bleeding associated with oral anticoagulant is
prevented by drug withdrawal and subsequent drug
adjustment.
4. FIBRINOLYTIC AGENTS
Streptokinase and urokinase
Used to lyse fresh thrombi in patients with major
pulmonary embolism.
5. ANTIPLATELET DRUGS
Aspirin is used to prevent Thromboxane A2
formation by inhibiting activity thereby avoiding
platelet aggregation.

162