Ampath

2014
AMPATH

Training department

HAEMATOLOGY FOR CLINICAL
PATHOLOGY
Haematology is the medical science that deals with blood and the blood‐forming organs.
Course Programme Quality Management System
Developed: June 2012
Written By: Alvine Janse van Rensburg and Shanta Padiachy

Moderated
Dr. Mada Ferreira and Dr. Lelani Pretorius
By:
1st Review
Date:
2nd Review
Date:
3rd Review
Date:
4th Review
Date:
Note: This guide in intended for the exclusive use of Ampath Trust employees and may
not be copied or indiscriminately distributed to persons outside of Ampath.
Page 2 of 336
CONTENTS
Learning Content description Page number

unit
1 Haemopoiesis 8
 Erythropoiesis 12
 Granulopoiesis 14
 Monopoiesis 17
 Lymphopoiesis 19
 Thrombopoiesis 22
Workbook 23
2 Full Blood Count 24
 Analyzers 28
 Analyzer generated data and scattergrams 34
 Calculations 39
 Analytical errors 43
 Reference ranges 45
 Quantitative changes 49
 Reticulocytes 53
 Reticulated platelets 58
Workbook 59
3 Red cells 60
 RBC shape and structure 63
 Function of RBC 64
 Red cell metabolism 65
 Red cell production 66
 Red cell breakdown 67
 Red cell morphology 68
 Clinical implication of abnormal values 73
Workbook 77
4 Anaemias 82
 Iron deficiency anaemia 88
 Anaemia of chronic disease 91
 Sideroblastic anaemia 93
 Lead poisoning 94
 Thalassaemia 96
 Sickle cell syndrome 100
 Megaloblastic anaemia 104
 Haemolytic anaemias 110
 Aplastic anaemia 127
 Hypoproliferative anaemias 130
 Anaemia of pregnancy 131
Workbook 132
Page 3 of 336
unit
5 White cells 135
 Neutrophils 137
 Eosinophils 142
 Basophils 143
 Monocytes 144
 Lymphocytes 145
Workbook 154
6 Acute leukaemias 155
 Acute Lymphoblastic Leukaemia 160
 Acute Myeloid Leukaemia 163
Workbook 167
7 Myeloproliferative disorders 169
 Chronic Myeloid Leukaemia 173
 Polycythaemia Vera 177
 Essential thrombocythaemia 180
 Myelofibrosis 182
Workbook 184
8 Lymphoproliferative disorders 188
 Chronic Lymphocytic Leukaemia 195
 Prolymphocytic Leukaemia 197
 Hairy Cell Leukaemia 198
 Plasma Cell Leukaemia 200
 Large Granular Lymphocytic Leukaemia 201
 Adult T-Cell Leukaemia 202
 Sezary syndrome 203
 Lymphoma 205
 Myeloma 207
Workbook 210
9 Myelodysplastic syndromes 213
Workbook 221
10 Coagulation 222
 Components of haemostasis 225
 Tests of the haemostatic function 235
 Bleeding disorders 244
 Thrombosis 259
Workbook 265
11 Erythrocyte sedimentation rate 267
Workbook 273
12 Blood groups 274
Workbook 284
Page 4 of 336
unit
13 Systemic disease and special situations 286
Workbook 300
14 Methods 301
15 Bibliography 335
Page 5 of 336
Course Programme Acknowledgements
This manual is the exclusive property of Ampath Trust.
It is not intended to replace any of the Standard Operating Procedures,
Work Instructions or Training Manuals implemented within the various
business units in Ampath.
Although it is designed as a reference for student Medical Technicians,

Student Laboratory Assistants and Intern Medical Technologists in order
Use of to meet the requirements of the SMLTSA syllabus guide and facilitate
Manual: competent laboratory performance this guide does not claim to
contain all the Haematology information required. Students/Interns are
required to undertake additional research as needed.
This guide is designed to facilitate self learning. Practical application of
the theory contained in the guide is critical for mastery of the content
and proficiency in Haematology.
The Clinical Pathology Technologist syllabus guide was used as a
guideline for the content contained in this guide.
Welcome. You have started a journey of learning that not only will allow
you to achieve the qualification of your choice but will also help you
cultivate an attitude of responsibility through quality work performance.
Notes to
Each learning unit in this guide forms the foundation of all laboratory
Learner
work.
The “content index” will guide you through the various learning units.
Please refer any discrepancies in the Guide content immediately to the
course facilitator or the Ampath Training department.
At the end of each learning unit, you will see a workbook. This workbook
is intended as a self assessment to measure your understanding
Assessment
continuously throughout the training. It is recommended that you
Detail
complete each workbook under test conditions.
A summative knowledge and performance assessment may also be
administered at the end of the learning program.
Page 6 of 336
ICONS
Assessment Criteria
Indicates what criteria learners will be measured against to prove
competence.
Workbook
Lab diagnosis
Page 7 of 336
HAEMOPOIESIS
Learning Unit 1
HAEMOPOEISIS
Purpose of the Learning Unit
Learning Unit 1 provides you with the underpinning knowledge,

concepts and terminology related to the haemopoesis in the body.
Assessment Criteria
The assessment criteria that you will be able to achieve at the end of
learning unit one:
1.1 Identify the site of haemopoiesis.
1.2 Understand the stages of haemopoiesis.
1.3 Differentiate between the different cells and their life spans.
Page 8 of 336
TERMINOLOGY DESCRIPTION
Haemopoiesis The formation of blood cells
Erythropoiesis The formation of red blood cells
Myelopoiesis The formation of granulocytes and monocytes
Lymphopoiesis The formation of lymphocytes
Thrombopoiesis The formation of platelets
Stem cell Undifferentiated cell that is able to renew itself and
produce specialized cells
Pluripotent Embryonic stem cells can differentiate into almost any cell
type
Multipotent Adult stem cells that can produce specialized cells needed
in a particular tissue or organ in which they arise
During the first few weeks of gestation, blood is produced in the yolk sac. From month
two to seven the liver and spleen take over.
Only from month seven onwards the bone marrow becomes the primary site. In the
normal adult, daily marrow production amounts to approximately 2.5 billion red cells,
2.5 billion platelets and 1.0 billion granulocytes per kilogram of body weight.
During childhood there is a progressive replacement by fat throughout the long bones,
so that in adult life over 70% of the marrow is located in the pelvis, vertebrae and
sternum.
When the liver and spleen resume their fetal haemopoietic role, we call it
extramedullary haemopoiesis or myeloid metaplasia.
Page 9 of 336
http://img.medscape.com/fullsize/migrated/472/097/cc472097.fig1.jpg
Haemopoiesis starts with stem cell division in which one cell replaces the stem cell and
the other is committed to differentiation.
Glycoproteins or growth factors regulate the process.
Page 10 of 336
Page 11 of 336
ERYTHROPOIESIS
The earliest recognizable red cell precursor in the bone marrow is the
proerythroblast; this gives rise to the basophilic (or early) erythroblast, the
polychromatic (or late) erythroblast, the orthochromatic erythroblast (or
normoblast), the reticulocyte and then the mature red cell.
http://legacy.owensboro.kctcs.edu/gcaplan/anat2/notes/Image333.gif
Proerythroblast
Size: 12 – 20 m
Nucleus: Large with fine chromatin strands
Nucleoli: Present
Cytoplasm: Intense basophilic
Basophilic erythroblast
Size: 10 – 16 m
Nucleus: Large with thick chromatin
Nucleoli: Absent
Cytoplasm: Still very basophilic
Polychromatic erythroblast
Size: 8 – 14 m
Nucleus: Large with fine chromatin
Nucleoli: Present
Cytoplasm: Intense basophilic
Page 12 of 336
Orthochromatic erythroblast
Size: 8 – 10 m
Nucleus: Small with coarse pyknotic
chromatin
Nucleoli: Absent
Cytoplasm: Pale pink
Reticulocyte
Size: Slightly larger than a red cell

Nucleus: None, only RNA remnants
Cytoplasm: Polychromatic
Red cell
Size: 7 m
Shape: Biconcave disc
Colour: Eosinophilic
Life span: 120 days
http://images-mediawiki-sites.thefullwiki.org/00/4/0/8/89651162055833176.png 27 Oct. 10
Page 13 of 336
GRANULOPOIESIS
http://upload.wikimedia.org/wikipedia/commons/6/69/Hematopoiesis_%28human%29_diagram.
png (accessed 29/6/11)
The earliest recognisable cell in the myeloid series is the myeloblast which gives
rise to the promyelocyte, myelocyte, metamyelocyte, band form and finally
the mature neutrophil, eosinophil or basophil.
Page 14 of 336
http://img.medscape.com/fullsize/migrated/494/614/coa494614.fig1.gif
Myeloblast
Size: 15 – 20 m
Nucleus: Large, round, occupies about
80% of cell
Nucleoli: 1–3
Cytoplasm: Basophilic, agranular
Promyelocyte
Size: Same size as myeloblast

Nucleus: Coarser chromatin strands
Nucleoli: Present
Cytoplasm: Basophilic with azurophilic or
primary granules
Myelocyte
Size: 25 m
Nucleus: Round, chromatin strands are
Thicker
Nucleoli: Absent
Cytoplasm: Primary or secondary granules
Page 15 of 336
Metamyelocyte
Size: 10 – 18 m
Nucleus: Slightly indented (kidney shaped)
Nucleoli: Absent
Cytoplasm: Fine specific granules
Band
Size: 10 – 15 m
Nucleus: Deeply indented U-shaped
Cytoplasm: Pink with many specific granules
Neutrophil
Size: 12 - 14 m
Nucleus: Lobulated (3-4 lobes)
Cytoplasm: Pink with specific granules
Life span: 6-10 hours in peripheral blood
before moving into tissues
Eosinophil
Size: 12 - 17 m
Nucleus: Lobulated (2-3 lobes)
Cytoplasm: Large, round eosinophilic
granules
Life span: Circulate for 6 hours before
moving into tissues
Basophil
Size: 10 - 14 m
Nucleus: Bi-lobed (2 lobes)
Cytoplasm: Large, bluish-black granules
http://www.cancer.umn.edu/cancerinfo/NCI/Media/CDR0000503952.jpg 27 Oct. 10
Page 16 of 336
MONOPOIESIS
G-CFU, granulocyte colony-forming unit; GM-CFU, granulocyte/macrophage colony-

forming unit; HSC, haematopoietic stem cell; M-CFU, macrophage colony-forming
unit.
http://www.nature.com/nri/journal/v5/n12/images/nri1733-i1.jpg 27 Oct. 10
The earliest recognisable cell in the monocyte series is the monoblast which
gives rise to the promonocyte and the monocyte. Monocytes spend a short
time in the marrow and blood and then leave to enter the tissues where they
mature and carry out their principal functions, and are now called
macrophages.
Page 17 of 336
Monoblast
Size: 10 -20 m
Nucleus: Round, fine lacey chromatin
Nucleoli: 1–3
Cytoplasm: Basophilic to blue-grey
Promonocyte
Size: 14 – 18 m
Nucleus: Oval, may have a fold/fissure
Nucleoli: 1-5
Cytoplasm: Blue-grey, ground glass appear-
ance
Monocyte
Size: 16 - 25 m
Nucleus: Kidney, bean or horshoe shape
with deep indentation
Nucleoli: Absent
Cytoplasm: Blue-grey, ground glass appear-
ance.
Lifespan: Intravascular – several days
Tissues – long-lived macrophages
http://education.vetmed.vt.edu/Curriculum/VM8054/Labs/Lab6/lab6.htm 5/1/11
www.perfusion.com 5/1/11
Page 18 of 336
LYMPHOPOIESIS
http://www.nature.com/nri/journal/v3/n4/images/nri1056-f1.gif 27 October 2010
Lymphocytes are produced by the lymph nodes, spleen, thymus and bone
marrow. They are divided into three functional types: B cells, T cells and
natural killer (NK) cells. B cells differentiate into plasma cells which secrete
antibodies. T cells and NK cells function in cell-mediated immunity. The
earliest recognisable cell will be the lymphoblast which give rise to the
prolymphocyte and then the mature lymphocyte.
Page 19 of 336
Function of T helper cells: Antigen presenting cells (APCs) present antigen on their Class II MHC
molecules (MHC2). Helper T cells recognize these, with the help of their expression of CD4 co-
receptor (CD4+). The activation of a resting helper T cell causes it to release cytokines and
other stimulatory signals (green arrows) that stimulate the activity of macrophages, killer T cells
and B cells, the latter producing antibodies.
http://upload.wikimedia.org/wikipedia/commons/e/ec/Lymphocyte_activation_simple.png
27 October 2010
Lymphoblast
Size: 10 – 20 m
Nucleus: Round, occupy ±80% of cell
Nucleoli: 1–2
Cytoplasm: Basophilic and agranular
Prolymphocyte
Size: 10 – 16 m
Nucleus: Round, chromatin is denser
Nucleoli: 1 prominent
Cytoplasm: More abundant than blast
Page 20 of 336
Lymphocyte (large)
Size: 12 – 16 m
Nucleus: Round, coarse clumped
chromatin
Nucleoli: Absent
Cytoplasm: Sky blue, with fine azurophilic
granules
Lymphocyte (small)
Size: 9 – 12 m
Nucleus: Round, coarse clumped
chromatin
Cytoplasm: Scanty, sky blue, agranular
http://www.websters-online-
dictionary.org/images/wiki/wikipedia/commons/9/90/Lymphoblast.png
http://www.ctcd.edu/mlt/lhansen/images/MLAX1415_images/WBC_series/images/10%20Proly
mphocyte_jpg.jpg
http://www.pathpedia.com/Education/eAtlas/Images/Large-granular-lymphocyte-(LGL)-
%5BCE05H-1%5D.jpeg
Page 21 of 336
THROMBOPOIESIS
http://legacy.owensboro.kctcs.edu/gcaplan/anat2/notes/Image331.gif
Platelets are produced within the vascular channels (sinusoids) of the bone
marrow by fragmentation of the megakaryocyte’s cytoplasm.
SUMMARY
 The bone marrow is the site of blood formation

 Blood production in the spleen and liver is called extramedullary
haemopoiesis
Page 22 of 336
WORK BOOK
Question 1
Tabulate the differences between heparin, sodium citrate and EDTA

anticoagulants using the following headings: Mode of action, uses and
disadvantages. (Hint: use the GLP manual)
Question 2
Where do all blood cells originate from?
Question 3
What stain will be used to show progress of erythropoiesis?
Question 4
What percentage of blasts is normal in peripheral blood?
Question 5
Sketch the following cells and label the contents properly.

i. Promyelocyte
ii. Natural killer cell
iii. Monocyte
iv. Megarkaryocyte
Page 23 of 336
FULL BLOOD COUNT
Learning Unit 2
FULL BLOOD COUNT.

concepts and terminology related to the full blood count, which is a
fundamental requirement for further development.
Assessment Criteria
learning unit two:
1.1 Understand the principles and operation of an automated cell
counter.
1.2 Identify the measured parameters and their normal values.
1.3 Identify and perform the calculated parameters and their normal
values.
1.4 Interpret instrument generated data and scatter grams.
1.5 Understand the role of the reticulocyte count and perform the
relevant calculations.
Page 24 of 336
TERMINIOLOGY DESCRIPTION
Automated Cell These are analyzers that sample the blood, and quantify,
counter classify, and describe cell populations using both electrical
and optical techniques
Measured These are physically counted or determined by measuring
parameter the analyte.
Calculated These are calculated through use of the values stored in
parameter the digital registers (containing measured parameters).
Reference range A reference range or reference interval (or sometimes
referred to as normal ranges) usually describes the
variations of a measurement or value in healthy individuals.
It is a basis for a physician or other health professional to
interpret a set of results for a particular patient. It includes
95% of the values in a Gauss curve.
Before we understand the automated cell counter and it principles, we have

to know what a Full Blood Count is and what it comprises of and what the
relevance is.
The Full blood count (FBC) comprises of the following measured parameters:
 Red Blood Cells (RBC's),
 White Blood Cells (WBC's), plus differential
 Haemoglobin
 Haematocrit ( Hct) (calculated on some analyzers)
 Platelets(Plt)
And the following calculated parameters:

 Mean Corpuscular Haemoglobin(MCH)
 Mean Corpuscular Haemoglobin Concentration (MCHC)
 Mean Corpuscular Volume( MCV) (measured on some analyzers)
 Mean Platelet Volume (MPV)
 Red Cell distribution width (RDW)
Page 25 of 336
RBC A RBC contains haemoglobin, which carries oxygen in the blood
RBC count A RBC count is a measurement of the number of red blood cells
in a specific volume
WBC White blood cells form the basis of the immune system and help
among other functions the body to fight infection
WBC count A WBC count is a measurement of the number of white blood

cells in a specific volume
Hb Haemoglobin is a protein in red blood cells that carries oxygen.
Hct Measures the percentage per volume of whole

blood that is made up of red blood cells
Plt A cell fragment with a role in the clotting mechanism in the body
MCH The MCH is the content (weight) of Hb of the average red cell; it
is calculated from the Hb concentration and the red cell count
MCHC The MCHC is the average concentration of Hb in a given volume

of packed red cells. It is calculated from the Hb concentration
and the hematocrit
MCV It is an estimate of the average size (volume) of a red blood cell
MPV Is a machine-calculated measurement of the average size of

platelets found in blood
RDW This value tells how consistent are the size of the red blood cells
Why is the test done?
The FBC is a screening test, used to recognize and diagnose several diseases.
It can reflect problems with fluid volume (such as dehydration) or blood loss.
It can highlight abnormalities in the production, life span, and destruction of
blood cells.
It can reflect acute or chronic infection, allergies, and problems with clotting.
Page 26 of 336
MCV, MCH, and MCHC values reflect the size and haemoglobin
concentration of individual cells and are useful in the diagnosis of types of
anaemia
When is a Full blood Count Collected?
The Full blood count can be collected anytime, without special requirements
or fasting.
The Blood is collected in an EDTA (Ethylene diamine tetra acetic acid) tube,
gently mixed by inversion, allowing the anticoagulant to mix well with the
blood. (Please note all under filled samples and overfilled samples can
influence the results.)
Page 27 of 336
ANALYSERS
All these measurements are now made on automated and semi-automated

instruments and results are available within minutes.
PRINCIPLES
Automated blood cell counters aspirate and dilute blood samples to

determine variables relating to red cells, white cells and platelets. Except for
haemoglobin, all variables depend on counting and sizing of particles.
Counting and sizing are done by either electrical impedance or by light

scattering. The analyzers have at least two channels. Diluent is present in both
channels, but a lytic reagent is added to one. The lytic reagent will lyse the
red cells, leaving the white cells intact and allow the haemoglobin to be
measured. Further channels are required for differential counts or
reticulocytes.
Note: Flow cytometry (abbreviated: FCM) is a technique for counting and

examining microscopic particles, such as cells and chromosomes, by
suspending them in a stream of fluid and passing them by an electronic
detection apparatus. Therefore all haematology analyzers are flow
cytometers.
Impedance (the Coulter principle)
The sample is first diluted (in a conductive fluid), and then counting is
performed by drawing the cells through an aperture (hole) of the instrument.
Each cell causes a change in electrical resistance as it passes the aperture,
and this pulse is detected and amplified by the instrument. The amplitude of
the pulse is proportional to cell size. Pulse sizes below 30 fl is considered
Page 28 of 336
platelets, pulse sizes between 30fl and 150fl are classified as being red cells
and pulse sizes between 50 – 300 fl is considered white cells.
Optical or light scatter
http://www.idexx.com/view/xhtml/en_us/smallanimal/inhouse/vetlab/lasercyte-hematology.jsf
A diluted blood sample passes in a stream through which a beam of laser light
is focused. As each cell passes through the sensing zone of the flow cell, it
scatters the light. The scattered light is detected by a photodetector and
converted to an electrical impulse. The number of impulses generated is
directly proportional to the number of cells passing through the sensing zone in
a specific time frame.
Forward Scatter(FSc) Approximate cell size
Side Scatter(SSc) Cell complexity or granularity
Fluorescent Labelling Used to investigate cell structure and function.
Page 29 of 336
Immunophenotyping techniques (fluorescent flow cytometry)
http://www.abcam.com/index.html?pageconfig=resource&rid=11446
Cells are stained with a fluorescent compound which can be excited and emit
light at a higher wave length than the light source.
Newer analyzers use this technology for platelet counting as well as where
platelets are labeled fluorescently with a specific monoclonal antibody or
combination of antibodies.
Fluorescent labeling allows investigation of cell structure and function.
FSC correlates with the cell volume and SSC depends on the inner complexity
of the particle (i.e., shape of the nucleus, the amount and type of cytoplasmic
granules or the membrane roughness).
Page 30 of 336
Conductivity
Conductivity is determined using a high-frequency electromagnetic probe

that provides information on the cells' internal constituents (chemical
composition, nuclear characteristics, and granular constituents) by
permeating the lipid layer of a cell's membrane. Conductivity is especially
helpful in differentiating between cells with similar characteristics such as small
lymphocytes and basophils.
Cytochemistry
A methodology unique to the Bayer automated haematology series is the use

of a cytochemical reaction to determine the peroxidase activity of white
blood cells. The mean peroxidase index (MPXI), a measure of neutrophil-
staining intensity, is determined for each specimen. The relative positivity seen
in neutrophils, eosinophils, and monocytes is used in conjunction with data
derived from light scatter to determine the WBC differential.
Method Instrument Impedance Conductivity Light Scatter Cytochemistry

Abbott x x x
ABX x x x
Bayer x x
Coulter x x x
Sysmex x x x
Haemoglobin Determination
Haemoglobin is measured directly using a spectrophotometric reading.
The lysed WBC dilution drains into the Haemoglobin (Hb) cuvette for Hb
measurement.
The lytic reagent rapidly and simultaneously destroys the erythrocytes and
converts a substantial proportion of the haemoglobin to a stable cyanide-
containing pigment while it leaves leucocyte nuclei intact.
A beam of white light goes through the cuvette and then through an optical
filter that has a wavelength of 525nm.
Light passing through the filter falls on a photocell.
The absorbance of the pigment is directly proportional to the haemoglobin
concentration of the sample in g/dL (using a calibration factor).
Page 31 of 336
REAGENTS
Most Full Blood Count analyzers have reagents that perform a certain function.
Below are examples of the types of reagents used in the full blood count
analysis:
Diluent/ Sheath
The diluent is an isotonic solution which acts as a conductive liquid and will
affect the cells minimally.
It rinses the tubing between running different samples.
RBC Lyse
RBC’s are lysed to free the haemoglobin for measurement. It will also convert
a substantial portion of the Hb to a stable cyanide-containing pigment – the
absorbance of which is directly proportional to the Hb concentration.
The lysed RBC’s do no interfere with the WBC measurement.
WBC Lyse
This reagent allows lyses of WBC’s without interference with the RBC
measurement.
Cleaning reagent
Daily cleaning helps to prevent protein build-up in the tubing.
Page 32 of 336
MAINTAINANCE
Haematology analysers require regular maintenance in order for them to work

optimally.
 Daily
This may include the following: performing a shutdown and a start up

procedure, running back ground counts, emptying waste, checking
reagents and supplies required, performing QC and running reproducibility
samples.
 Weekly
On certain analyzers these include bleaching and rinsing the flow cell
chamber, wiping the analyzer and cleaning filters.
 Monthly
This may involve extended cleaning, unblocking and removal of protein

from flow cells, changing tubing, and comparing calibration printouts
(depending on analyzer).
 As required
These will be done as the need occur e.g. “zap” the aperture etc.
Page 33 of 336
ANALYZER GENERATED DATA AND SCATTER GRAMS
SCATTER GRAMS
FORWARD SCATTER (SIZE)
Mono Neutrophils
Eosino
Lymph RBC precursor
SIDE SCATTER (COMPLEXITY)

http://www.antibodies-online.com/images/news/resources_FACS_1_en.jpg
IG – Immature granulocytes
The cells scatter a fraction of the light which is then detected by photomultipliers
(light detectors).
The amount of light measured correlates with the size of the cells and their
complexity.
Granulocytes for example reflect more light than smooth surfaced B- or T-

lymphocytes, due to their rough surface texture and a larger amount of vesicles
inside the cell.
A measurement for the diffraction of light in a flat angle is the forward scatter (FSC),
which depends on the volume of the cell.
A measurement for the diffraction of light in a right angle is the so called side scatter
(SSC). It depends on the granularity, the size of the cells, the structure of its nucleus,
and the amount of vesicles inside the cells. Page 34 of 336
WBC scattergrams/cytograms.
Top left: Abbott CELL-DYN 4000 WBC scatterplot, light scatter vs. volume.
Top right: Sysmex XT 2000i, WBC scattergram, side-scattered light vs. side
fluorescence.
Bottom left: Bayer Advia 120, WBC peroxidase cytogram.
Bottom right: Coulter LH 750, WBC scattergram, light scatter vs. volume
http://itd.unair.ac.id/files/ebook/Henry's%20Clinical%20Diagnosis%20and%20Management%20
by%20Laboratory%20Methods/1393/i4-u1.0-b1-4160-0287-1..50033-1--f8.fig.htm#top
Page 35 of 336
HISTOGRAMS
WBC histogram
WBC: Distribution with three individual peaks and valleys at specific regions
representing the lymphocytes, monocytes, and granulocytes.
All curves normally start and end at baseline (see graph below). This is an
example of a histogram from an impedance count.
http://www.abaxis.com/_media/content/hm2_how_wbc_types.gif
RBC histogram
Red cells are distributed according

to cell size (mean cell volume) and
amount present in blood.
http://medtech.mahidol.ac.th/mtthai/eLearning/AutomateReport/Page51image002.gif
Page 36 of 336
http://ahdc.vet.cornell.edu/clinpath/modules/hemogram/rdw.htm
The Red cell graph above double peak indicates two populations, typically
noted in after blood transfusion or after iron therapy.
The RDW has almost doubled as seen by the much larger width of the
combined curves on the histogram. (Note that both values are increased.
The black curves on the red cell and platelet

histograms indicate expected or normal cell
distributions.
The red curves demonstrate the effect of

microcytic red cells on the histograms.
Microcytic red cells only affect the right end of

the platelet curve; the black and red lines are
superimposed for most of the curve.
http://www.healthcare.uiowa.edu/cme/clia/modules.asp?testID=4#05
Page 37 of 336
Platelet Histogram
Platelets are distributed according to cell size

(x-axis) mean platelet volume) and amount
(y-axis) present in blood.
http://www.polconsultant.com/conteduc/hematology/micro/pat4/histo.gif
The black curves on the red cell

and platelet histograms indicate
"expected" or normal cell
distributions.
The red curves demonstrate the

effect of giant platelets on the red
cell and platelet histograms
http://www.healthcare.uiowa.edu/cme/clia/images/testID04/09.jpg
Left curve shows interferences

(overlapping curves) with giant
platelets and red cell fragments
as seen with impedance method.
With fluorescence methodology,

there is exact separation (right)
Page 38 of 336
CALCULATIONS
The following parameters are calculated on your Haematology analyzer:
 Mean Corpuscular Haemoglobin

 Mean Corpuscular Haemoglobin concentration
 Haematocrit or MCV (depending on the analyzer)
These parameters can be calculated using the equations below:
RED CELL INDICES
In order to understand the conversions, here is a refresher:
Measure Units Conversion

Hct % %/100 = l/l
Hb g/dl Deci = 10-1
RBC 1012/l or 106/µl Micro = 10-6
MCV = Hct / RBC fl Femto = 10-15
MCH = Hb / RBC pg Pico = 10-12
MCHC = MCH / MCV g/dl Deci = 10-1
We use prefixes to indicate decimal fractions or multiples:
1012 109 106 103 102 101 m 10-1 10-2 10-3 10-6 10-9 10-12 10-15 Factor
g
Tetra Giga Mega Kilo Hecto Deka l Deci Centi Milli Micro Nano Pico femto Prefix
mol
T G M k h da d c m µ n p f Symbol
Page 39 of 336
MCH
To convert dl to l = x 10
MCH = Hb(g/dl) x 10 UNITS - pg

RBC x 1012/ l
Example
Hb = 11.5g/dl, RBC = 3.30 x 1012/l
Calculate MCH
MCH = 11.5 x 10 (g/l) UNITS - pg

3.30 x 1012/l
= 34.8 x 10-12g
= 34.8 pg
Decreased MCH (hypochromic) Increased MCH

 Associated with hypochromic,  No such term as hyperchromic.
microcytic anaemias e.g. iron
deficiency, chronic illness,
thalassaemia
MCHC
MCHC = Hb (g/dl) x 100 UNITS – g/dl OR

HCT (%)
MCHC = Hb (g/dl) UNITS – g/dl

HCT (l/l)
X 100 to compensate for haematocrit in %
Example
Hb = 11.5g/dl, HCT = 45 %
MCHC = HB (g/dl) x 100 UNITS – g/dl

HCT (%)
= 11.5 x 100 or 11.5

45 0.45
Page 40 of 336
=25.5 g/dl
Decreased MCHC Increased MCHC

 Associated with hypochromic,  Usually an analytical error due to
microcytic anaemias e.g. iron cold agglutinins, lipaemia or not
deficiency, chronic illness, mixing properly
thalassaemia  Spherocytosis
HCT
HCT = RBC (x 1012/l) x MCV (fl) UNITS - %

10
Example
MCV = 75 fl, RBC = 3.30 x 10 12 /l
HCT = RBC x MCV UNITS - %

10
= 3.30 x 1012/l x 75
10
= 24.75 %
MCV
MCV = HCT (%) x 10

RBC(x 1012/l)
% is 100 and x 10 = x 1000 = 103

Example
HCT = 25%, RBC= 3.30 x 1012
MCV = HCT x 10
RBC
= 25 X 10
3.30 x 1012
= 75 x 10-15l
= 75 fL
Page 41 of 336
Decreased MCV Increased MCV
 Associated with hypochromic,  Round macrocytes are associated
microcytic anaemias e.g. iron with antiretroviral or chemotherapy,
deficiency, chronic illness, alcoholism, liver disease,
thalassaemia hypothyroidism etc.
 Oval macrocytes are associated
with megaloblastic anaemia or
MDS.
Page 42 of 336
ANALYTICAL ERRORS
Type of error Example of error

Sample errors  Inadequate mixing – insufficient mixing or mixing too
vigorously (repeated mixing of samples leads to increased
oxygenation resulting in false low MCV)
 Short sampling – either insufficient sample or probe blocked

e.g. clot from previous sample
Inaccuracy due to  Cold agglutinins, hyperlipidaemia or cryoglobulinaemia

specimen lead to errors in Hb and red cell indices, especially
characteristics increased MCHC
 With some patients, pretreatment of the blood by

ultracentrifugation or enzymatic cleavage is necessary to
remove lipids. Plasma is removed and replaced with saline.
 Very high WBC counts can affect the Hb estimate because

of the turbidity, which also leads to a falsely raised MCHC.
 Storage of blood at room temperature can lead to a falsely

high MCV
 Nucleated red blood cells (NRBC) can lead to falsely high

WBC count as the NRBC is counted as white cells. A manual
differential count will help to correct the white cell count.
Newer analyzers use fluorescence to provide sensitivity and
specificity.
 Partial clotting or aggregation of platelets in the sample is a

common cause of inaccurate platelet counts. Platelet
aggregation occur due to
o Activation of the coagulation system
o Presence of antibodies
 Another in vitro artifact, less common than platelet

aggregation which can also lead to low platelet counts is
platelet satellitism around neutrophils (satellitism has no
clinical significance).
 Red cell fragments can be counted as platelets causing a

falsely elevated platelet count
Page 43 of 336
 Giant platelets can be counted as red cells leading to a
falsely decreased platelet count.
 Non-lysis of red blood cells can be a problem when the

WBC count is measured by light scattering technology.
There can be an elevation in the lymphocyte count.
 Neutrophil peroxidase deficiency is a problem in analyzers

which use peroxidase technology – there is an increase in
monocyte or large unstained cells (LUC) counts.
 Residues in the basophil channel due to NRBC, blast cells,

lymphoma cells, abnormal lymphocytes in HIV subjects can
cause an elevated basophil count.
 Large lymphocytes or large granular lymphocytes can be

counted as monocytes in analyzers using light scattering
technology.
A rough check of red cell parameters is:
Red cell count x 3 =  haemoglobin

Haemoglobin x 3 =  haematocrit
Page 44 of 336
REFERENCE RANGES
Test 95% Range SI Units

Haemoglobin (Hb)
Males 14.3-18.3 (inland)
13 – 17 (sea level)
Females 12.1-16.3 (inland) g/dl
12 – 15 (sea level)
Newborns 13.5-21.5
Child, one year 11.1-14.1
Child, ten years 11.5-15.0
Hematocrit (Hct,PCV)
Males 43 - 54
Females 37 - 49
Newborns 44 - 64 %
Child, one year 30 - 38
Child, ten years 35 - 45
Erythrocyte count (RBC)
Males 4.89-6.11
Females 4.13-5.67
Newborns 4.00-6.00 X 1012/l
Child, one year 3.9-5.1
Child, ten years 4.00-5.40
MCV (mean corpuscular volume)
Adults 79.1-98.9
Newborns 96-116
fl
Child, one year 72-84
Child, ten years 77-91
MCHC (mean corpuscular haemoglobin concentration)
Adults and children 32.0-36.0 g/dl
Newborns 31.0-40.0
MCH (mean corpuscular haemoglobin)
Adults 27-32
Newborns 21-40 pg
Child, one year 25-29
Reticulocyte count (Retic)
Adults and children
Relative 0.5-2.0 /100 RBC
Absolute 20-100 X 109/l
Newborns
Relative 2.0-6.0 100 RBC
Absolute 80-360 X 109/l
Page 45 of 336
Test 95% Range SI Units
White cell count (WCC)
Adults 3.92 – 9.88
Newborns 9.0 – 30.0
X 109/l
One month 5.0 – 19.5
Child, one year 6.0 – 16.0
Neutrophils
Adults 3.0 – 7.5
X 109/l
Lymphocytes
Adults 1.0 – 4.0
X 109/l
Monocytes
Adults 0.2 – 1.0
X 109/l
Eosinophils
Adults 0.0 – 0.5
X 109/l
Basophils
Adults 0.0 – 0.1
X 109/l
Platelet count (Plt)
Adults 150 - 410
Newborns 150 – 450
X 109/l
Two months 210 - 650
Child, one year 200 - 550
Page 46 of 336
Factors influencing reference ranges
Factor Influence
Age  Normal ranges of neonates, infants and children differ
widely from those of adults. During the first few hours of
life, plasma volume decreases so that Hb, RBC and Hct
rise considerably. Numerous NRBC may be present
after birth, but falls rapidly after 24 hours.
 Lymphocyte count is lower in old age
Gender  Males have a higher muscle mass than female, thus

requiring higher oxygen levels in the body with higher
red cell counts and haemoglobin levels.
 The platelet count is higher in women than in men
Ethnic origin  WBC and neutrophil counts are lower in black Africans.
It is not apparent at birth, only from about 1 year.
 The lower RBC count, Hb, Hct and MCV observed in

West Indians and Africans are most likely due to the
high prevalence of thalassaemia trait and
haemoglobinopathies rather than any other ethnic
differences.
 West Indians and African platelet counts are 10-20%

lower than those in Europeans living in the same
environment.
Geographical  Persons living at higher altitudes have a higher Hb level

locations (secondary to decreased oxygen saturation in the
ambient atmosphere) compared to persons living at
sea level.
Blood collection  Variation is plasma volume may occur with different

methodology posture e.g. sitting vs standing; lying vs sitting. The
clinical relevance is undetermined.
Page 47 of 336
Factor Influence
Diurnal variation  There is a slight diurnal variation due to erythropoietin
variation during day and night.
 In patients taking iron supplements, the timing of the

specimen collection must be taken into account.
 The platelet count is higher in the afternoon and

evening.
 WBC and neutrophil counts are higher in the afternoon

than in the morning.
Cigarette smoking  Cigarette smoking reduces oxygen carrying capacity

due to increased levels of carbon dioxide, thus, the
bone marrow compensates, producing higher levels
the red cells. The haemoglobin levels in these patients
are also higher than normal.
 Smokers may have higher platelet counts than non

smokers.
 ESR is higher
Pregnancy  Both plasma and red cell mass increases during

pregnancy. Plasma volume increases proportionally
more than red cell mass resulting in a slight drop in
measured Hb (usually not lower than 10g/dl).
 WBC count is increased during the third trimester as

well as after delivery (post partum). Toxic granulation
and Dohle bodies are common.
 There is a slight increase in MCV from the second

trimester.
 ESR rises
Page 48 of 336
QUANTITATIVE CHANGES
Causes for decreased or increased counts
HAEMOGLOBIN, HAEMATOCRIT AND RED CELL COUNT

Decreased count (anaemia) Increased count
(polycythaemia/erythrocytosis)
 Inadequate production  Primary

o Nutritional like iron, B12 o Polycythaemia vera
o Bone marrow infiltration or  Secondary
fibrosis o Tissue hypoxia like high
o Ineffective production like altitudes, cyanotic heart
MDS disease, lung diseases
o A/ Hypoplastic o Inappropriate synthesis of
 Increased destruction erythropoietin like
o Haemolysis carcinomas
o Blood loss  Relative
 Abnormal distribution o Fluid loss e.g. dehydration
o Hypersplenism o Cigarette smoking
NEUTROPHILS (WBC)
Decreased count (neutropaenia) Increased count (neutrophil
leucocytosis or neutrophilia)
 Viral infections like measles,  Infections (bacterial and miliary

mumps, rubella, influenza, CMV, TB)
HIV  Tissue damage e.g. trauma,
 Overwhelming bacterial surgery, burns
infections  Acute inflammation and severe
 Drugs e.g. chemo- and/or chronic inflammation e.g. gout,
radiotherapy RA etc.
 Bone marrow replacement e.g.  Acute haemorrhage
ALL, myeloma  Myeloproliferative disorders e.g.
 Megaloblastic anaemia CML, PV
 Paroxysmal nocturnal  Certain drugs e.g. adrenaline,
haemoglobinuria (PNH) corticosteroids
 Aplastic anaemia  Some viral infections e.g.
 Auto-immune neutropaenia chickenpox, herpes simplex virus
 Other like cigarette smoking,
exercise, pregnancy
Page 49 of 336
LYMPHOCYTES
Decreased count (lymphopaenia or Increased count (Lymphocytosis)
lympcytopaenia)
 HIV infection  Viral infections including

 Acute stress e.g. trauma, measles, mumps, EBV, hepatitis
surgery, burns, acute infection etc.
 Acute and chronic renal failure  Cigarette smoking
 Hodgkin’s disease  Stress related e.g. myocardial
 Cytotoxic and infarction, cardiac arrest,
immunosuppressive therapy trauma, obstetric complications
 RA, SLE  Some bacterial infections e.g.
 MDS whooping cough, TB, syphilis
 Lymphoproliferative disorders
e.g. CLL, ALL, non-Hodgkin’s
lymphoma etc.
MONOCYTES
Decreased count (monopaenia) Increased count (monocytosis)
 Seldom of clinical significance  Chronic infection

 TB
 Chronic inflammatory
conditions e.g. Crohn’s disease,
RA and SLE
 Long term haemodialysis
 Myeloproliferative and
leukaemic conditions e.g. MDS,
AML
 Carcinoma
EOSINOPHILS
Decreased count (eosinopaenia) Increased count (eosinophilia)
 Seldom of clinical significance  Allergic reactions e.g. eczema,

asthma, hay fever etc.
 Drug hypersensitivity e.g.
penicillin
 Parasitic infections e.g.
schistosomiasis etc.
 Skin diseases e.g. psoriasis
 Recovery from acute infection
Page 50 of 336
 Dialysis related
 Idiopathic hypereosinophilic
syndrome
 Eosinophilic leukaemia
BASOPHILS
Decreased count (basopaenia) Increased count (basophilia)
 No clinical significance  Myeloproliferative disorders

 Reactive e.g. myxoedema,
ulcerative colitis etc.
PLATELETS
Decreased count Increased count (thrombocytosis)
(thrombocytopaenia)
 Inadequate production  Primary

o Congenital bone marrow o Essential
suppression e.g. Fanconi thrombocythaemia
anaemia o CML, Poly vera
o Drugs  Secondary or reactive
o Bone marrow infiltration o Infection, inflammation
e.g. malignancies, o Haemorrhage
myeloma, lymphoma, o Surgery, trauma
leukaemia o Iron deficiency
 Bone marrow destruction or o Post splenectomy and
abnormal release hyposplenism
o Megaloblastic anaemia
o PNH
o MDS
o May-Hegglin anomaly
 Increased destruction
o Idiopathic
thrombocytopaenia
Purpura (ITP)
 Increased consumption
o Disseminated
intravascular coagulation
(DIC)
o Thrombotic
thrombocytopaenia
Purpura (TTP)
 Hypersplenism
Page 51 of 336
 Dilutional thrombocytopaenia
e.g. massive transfusion
Page 52 of 336
RETICULOCYTES
Reticulocytes are immature RBCs that contain remnant cytoplasmic

ribonucleic acid (RNA) and organelles such as mitochondria and ribosomes.
Reticulocytes are stained

with Methylene blue /
Brilliant cresyl blue
These supra-vital dyes (stain

living cells) precipitate the
ribosomes and stain them to
appear as a reticular
network.
An erythrocyte still possessing RNA is referred to as a reticulocyte. The amount

of reticulum in a reticulocyte varies from a large clump in the most immature
cells (group I) to a few granules in the most mature forms (group IV).
The reticulocyte count is a means of assessing the erythropoietic activity of the

bone marrow.
Should a patient present with early stages of anaemia, the bone marrow starts
to compensate and release young red blood cells, called reticulocytes, into
the circulation. It rises within 2-3 days, reaching a maximum in 6-10 days.
RECITICULOCYTE CALCULATIONS
Calculate the percent of reticulocytes as follows:
Reticulocytes % = number of reticulocytes counted ( 10 fields) x 100

1000 RBC
Page 53 of 336
Example
Retics counted = 47 in 1000 RBC
% Retics = 47 x 100
1000
= 4.7%
Absolute Reticulocyte count = Reticulocyte (%) x Red Cell count (x 1012/l)
Example
% Retics = 4.7%
Total RBC = 3.67 1012/l
Absolute Reticulocyte count = 4.7% x 3.67x1012/l

= 4.7/100 x 3.67x1012/l
= 0.172 x 1012/l or
= 172 x 109/l (as per the correct reference units)
Calculation of corrected reticulocyte count
The reticulocyte count is most often expressed as a percentage of total red

cells.
In states of anaemia, the reticulocyte percentage is not a true reflection of
reticulocyte production.
Marrow production should not be overestimated in severely anaemic patients.

A relatively high reticulocyte % is determined if the Hct is low (because of low
number of RBCs).
The corrected reticulocyte count may be calculated by the following formula:
Corrected reticulocyte count = Retic % x patient’s Hct %

average normal Hct*
*Average normal Hct is 45% for men and 42% for women.
Page 54 of 336
Example
Uncorrected retic% = 5.0%
Patient Hct = 25.0% (male)
Average normal Hct for male = 45%
Corrected retic% = 5.0% x 25%

45%
= 2.8%
Calculation of the reticulocyte production index (RPI)
Estimating RBC production by using the corrected reticulocyte count may

yield erroneously high values in patients when there is a premature release of
younger reticulocytes from the marrow (owing to increased erythropoietin
stimulation).
The premature reticulocytes are called

“stress or shift” reticulocytes.
These result when the reticulocytes of

the bone marrow pool are shifted to
the circulation pool to compensate for
anemia.
The younger stress reticulocytes

present with more filamentous
reticulum.
The mature reticulocyte presents with granular dots representing reticulum.

Normally, reticulocytes lose their reticulum within 24 to 27 hours after entering
the peripheral circulation.
The premature stress reticulocytes have increased reticulum and require 2 to

2.5 days to lose their reticulum, resulting in a longer peripheral blood
maturation time.
Page 55 of 336
Maturation Time Hematocrit%
1 day 45
1.5 days 35
2 days 25
2,5 days 15
The peripheral blood smear should be reviewed carefully for the presence of
many polychromatophilic macrocytes, thus indicating stress reticulocytes and
the need for correction for both the RBC count and the presence of stress
reticulocytes.
The value obtained is called the reticulocyte production index (RPI). RPI equal
to or greater than 3 represents an adequate response to anaemia by the
bone marrow, whereas an RPI of less than 2 is considered an inadequate
response of erythopoiesis by the bone marrow to a state of anaemia.
Calculation of the RPI:
RPI = Corrected retic count in %

Maturation time in days
Example
Patient’s corrected retic = 12%
Patient’s hct = 25% (from the table = 2 days)
RPI = 12%
2
=6
Reticulocyte Count Causes

Increased  Haemolytic anemia
 Acute blood loss
 Response to replacement therapy
Decreased  Aplastic anemia
 Marrow suppression by drug, toxin, or viral infection
 Pure red cell aplasia
 Bone marrow replacement (leukemia, lymphoma,
carcinoma)
Normal  Iron deficiency anaemia (can be low)
Page 56 of 336
 Anaemia of chronic disease
 Chronic renal failure (can be low due to lack of
erythropoietin)
 Megaloblastic anaemia (B12 or folate deficiency) (can
be low)
 Myelodysplasia
Page 57 of 336
RETICULATED PLATELETS
Reticulated platelets are immature platelets and can now be measured on

improved methodology analyzers. The quantification is called the immature
platelet fraction (IPF).
The IPF is identified by flow cytometric techniques and the use of a nucleic
acid specific dye in the reticulocyte/optical platelet channel.
The main application is in thrombocytopaenic patients.
An increased IPF will indicate increased destruction, as new platelets are
continuously released from bone marrow. The best examples are Idiopathic
thrombocytopaenic Purpura (ITP) and Thrombotic thrombocytopaenic purpura
(TTP).
If the problem lies within the bone marrow, it cannot release new platelets;
therefore the IPF will be low.
SUMMARY
 The FBC is extremely important in diagnosis of various conditions.

 Different analyzers use different methods.
 Impedance or Coulter principle analyzers use the break of electrical current as the cells
pass through the aperture to size the individual cells.
 Light scattering methods include forward scatter to size a cell and side scatter to assess
the granularity.
 Fluorescent flow cytometry use a fluorochrome dye bound to antibodies, which will
then emit light at specific wavelengths.
 Scattergrams and histograms are plotted according to the data received from the
analyzer and can be useful.
 Sample integrity may influence the readings of the analyzer e.g. lipaemia, cold
agglutinins. Be aware of your analyzer’s “faults”.
 Certain parameters are calculated using RBC, Hct, Hb and MCV counts.
 Each parameter of the FBC has clinical significance if either decreased or increased.
 Reticulocytes are immature red cells which can tell us a lot about bone marrow
function.
 IPF is a useful tool to help establish the reason for thrombocytopaenia.
Page 58 of 336
WORKBOOK
Question 1
Sketch a diff scatter plot of a normal FBC done on your analyzer.
Question 2
Indicate if you will suspect a high or low reticulocyte count in the following
diseases.
a. Iron deficiency
b. Aplastic anaemia
c. Renal disease
d. Megaloblastic anaemia
e. Haemolysis
Question 3
Calculate the red cell indices from the following: RCC = 4.16 x 1012/l
Hb = 12.7 g/dl and Hct = 38.2%
Question 4
Use a one word term that describes the following in a haematology laboratory:
a) Variation in red cell colour

b) Increased mean corpuscular volume
c) Variation in red cells size
d) Increased red cell count and haemoglobin
e) Low mean corpuscular volume
f) Decreased haemoglobin
Question 5
A Full blood count result is obtained with a high MCHC, high MCH and high
MCV. How would you go about determining the presence of cold agglutinins?
Page 59 of 336
RED CELLS
Learning Unit 3
RED CELLS

concepts and terminology related to erythropoiesis
Assessment Criteria
learning unit three:
1.1 Understand the clinical implications of abnormal values.
1.2 Identify inclusion bodies.
1.3 Define terminology and identify abnormal red cell morphology.
Page 60 of 336
TERMINIOLOGY DESCRIPTION
Abnormal Values A value outside of the reference range
Cyanosis It is the appearance of a blue or purple discolouration of
the skin or mucous membranes due to the tissues near the
skin surface being low on oxygen.
Erythropoiesis The process of red blood cells (erythrocytes) production
Erythropoietin Erythropoietin (EPO) is a hormone produced by the kidney
that stimulates the formation of red blood cells by the bone
marrow
Inclusion bodies Nuclear or cytoplasmic aggregates of stainable
substances, usually proteins, that is present inside the cell.
Glycolysis The process of conversion of glucose into energy
Methaemoglobin A form of the oxygen-carrying metalloprotein
haemoglobin, where the iron in the haem group is in the
Fe3+ (ferric) state, not the Fe2+ (ferrous) of normal
haemoglobin.
Morphology It is the identification, analysis and description of the
structure of cells
Stercobilinogen Stercobilinogen is a bile pigment derived from conjugated
bilirubin during transit through the gastrointestinal tract
Transferrin A plasma protein that transports iron through the blood to
the liver
Extravascular Outside the blood vessels
Intravascular Inside the blood vessels
Page 61 of 336
INTRODUCTION
The Red Blood Cell (Erythrocyte):
 are the most abundant blood cells i.e. about 4-6 million/ml
 has no nucleus and survive 120 days.
 is biconcave in shape allowing for flexibility and easy passage through the
vascular system.
Side view Red blood cells are thinner in the

centre and thicker around the
edges.
They are very flexible with the

ability to twist and bend through
Top view the blood vessels, as these
narrow and widen throughout
the body.
http://4.bp.blogspot.com/_HJSoIBTkkEg/S__vytvIkZI/AAAAAAAAAAs/VWAj8DLzSkY/s1600/a.gif
 is rich in haemoglobin, a protein able to bind to oxygen with the aid of iron.
 Is responsible for delivering oxygen (O2) to tissues and partly for recovering
carbon dioxide (CO2) produced as waste.
Page 62 of 336
THE RBC SHAPE AND STRUCTURE
The membrane consists of a skeleton (of structural proteins), pillars of integral

membrane proteins, and covered with a lipid bilayer.
They can be divided into vertical and horizontal connections. Vertical

connections comprise of
 Band 3
 Ankyrin – which binds to β-spectrin chain
 Protein 4.2
Loss of part of this complex leads to hereditary spherocytosis due to loss of
surface-area-volume ratio.
Horizontal connections comprise of

 α and β spectrin
 Actin
 Protein 4.1
They give flexibility to red cells. Loss of part of this complex causes loss of
rigidity and and elliptocytosis is the result.
A change in lipids (e.g. excess cholesterol) causes acanthocytes

This is demonstrated below:
Excess cholesterol found in the outer leaflet of
the red cell membranes, rendering them less
deformable, remodelled as they pass through
the spleen, forming acanthocytes
http://www.wadsworth.org/chemheme/heme/cytoheme/hemepix/slide311B.jpg
http://ahdc.vet.cornell.edu/clinpath/modules/rbcmorph/images/acanthocyte.jpg
Page 63 of 336
FUNCTION OF RED BLOOD CELLS
Haemoglobin
The main function of the red cell is to carry O2 to the tissues and to return CO2
back to the lungs.
The Hb-molecule:
2 Alpha Chains
2 Beta chains
http://3.bp.blogspot.com/_hhUdKwzDmA4/THU6gYTesgI/AAAAAAAAAqY/FpsX72hA1JI/s400/H
aemoglobin.jpg
Adult haemoglobin (Hb A) consists of four polypeptide chains - 2, β2, each
with their own haem group. Normal adult blood contains small quantities of
Hb F and Hb A2.
They contain  chains, with  and  chains respectively, instead of β. Hb

changes from fetal to adult Hb at 3-6 months after birth.
Protoporphyrin combines with iron in the ferrous (Fe2+) state to form haem.
Methaemoglobin
This is haemoglobin present with iron in oxidized state (Fe3+).
Toxic methaemoglobinaemia occurs when a drug or other toxic substance
oxidizes Hb. The patient appears cyanotic.
Page 64 of 336
RED CELL METABOLISM
The red cell carries haemoglobin to tissues for gaseous exchange and must be
able to pass repeatedly through the microcirculation, keep haemoglobin in
reduced (ferrous) state and maintain osmotic equilibrium.
Embden-Meyerhof
The Embden-Meyerhof pathway will generate energy as ATP and NADH which
is needed to keep haemoglobin in the functionally reduced form.
The reducing power is also generated by the hexose monophosphate shunt as
NADPH.
For each molecule of glucose used, two molecule of ATP (energy) are
generated.
Glucoselactate2 x ATP
ATP provides energy to maintain the red cell volume, shape and flexibility.
NADH is needed to have functionally, reduced haemoglobin.
Hexose monophosphate shunt (HMP)

10% of glycolysis occurs through an oxidative mechanism
Glucose-6-phosphate6-phosphogluconateribulose-5-phosphateNADPH
NADPH is needed to keep haemoglobin in the active Fe2+ state
Page 65 of 336
RED CELL PRODUCTION
Erythropoietin
Erythropoiesis is regulated by the hormone erythropoietin, mainly produced in
the kidneys. 10% are produced in the liver or elsewhere. The stimulus for
erythropoietin production is the oxygen tension in the tissues of the kidneys.
Low oxygen saturation (hypoxia) results in increased EPO production with a

subsequent increase in production of red cells
Polycythaemia Vera is a myeloproliferative neoplasm where red cells are

produced without EPO stimulation; the EPO levels may decrease in order to
suppress further red cell production.
Some tumours may produce EPO irrespective of oxygen tension.
In severe kidney damage this mechanism may fail – loss of erythropoietin

production and decreased erythropoiesis and subsequent decrease in red cell
count and haemoglobin (anaemia).
http://www.tarleton.edu/Departments/anatomy/erythro.html
Page 66 of 336
RED CELL BREAKDOWN
http://www.perthhaematology.com.au/haemolysis.html
After 120 days, red cells deteriorate and are removed by the macrophages of
the reticulo-endothelial (RE) system (marrow, liver and spleen). This is then
referred to as extravascular haemolysis.
Red cells are broken down in haem and globin. Haem is further broken down
into iron and protoporphyrin.
Globin is broken down into amino acids and returned to the amino acid pool.
Iron binds to transferrin and is stored as ferritin where it will be used again.
Protoporphyrin is broken down to bilirubin, conjugated in the liver to become
water soluble and is then excreted into the gut via bile. It is excreted in faeces
as stercobilinogen. Some is re-absorbed and excreted by the kidneys as
urobilinogen.
Intravascular haemolysis only occurs in pathological circumstances.
Page 67 of 336
RED CELL MORPHOLOGY
Red blood cells Causes:

Acanthocyte with irregularly
or spaced Beta
Spur cells projections. lipoproteinae
(with the mia and
shape of certain liver
cowboy The projections vary in disorders.
spurs) width but usually
http://bmia.bmt.tue.nl/education/casus/imag contain a sharp end.
es/acanthocytes%2001.jpg
Variations in RBC It is a feature
Anisocytosis diameter of many
anaemias,
Anisocytosis means and other
that the red cells blood
are of unequal size. conditions,
http://meds.queensu.ca/medicine/deptmed/
hemonc/anemia/images/aniso.gif
These round, dark Lead
Basophilic blue granules are poisoning,
stippling precipitated exposure to
ribosomes and some drugs,
mitochondria. severe burns,
anaemias, or
Stained with supra septicaemia.
http://studydroid.com/imageCards/0b/qb/car vital stains
d-12398558-front.jpg
Bite cells result from Glucose-6-
Bite cells attempts of splenic Phosphate
phagocytes literally Dehydro-
trying to "pluck out" genase
the Heinz bodies (G6PD)
Deficiency
Anaemia
http://pathwiki.pbworks.com/w/page/14
673994/HemaSlide14
Page 68 of 336
These are red-purple Seen in severe
Cabot rings staining threadlike anaemia
filaments in the shape including
of a ring or it can megaloblastic
appear as granules in anaemia,
a linear array rather leukaemia
than as complete and lead
rings. poisoning.
http://www.wadsworth.org/chemheme/heme
/cytoheme/hemepix/slide138.jpg
These are thought to
be microtubules from
a mitotic spindle or
remnants of the
nuclear membrane.
Red blood cells with Kidney disease
Echinocytes many blunt spicules or results from
(crenated red faulty drying of
blood cells) Echinocytes contain the blood
or Burr cells adequate smear or from
haemoglobin and the exposure to
spiny knobs are hyper-osmotic
regularly dispersed solutions.
http://apple.objectivepathology.com/moodle over the cell surface,
/file.php/70/hemtech_2_gifs/echinocytes--r.gif
Elliptocytes are red Present in
Elliptocytes blood cells that are various
oval or cigar anaemias
shaped. (predominant
iron
deficiency)
and
Hereditary
elliptocytosis.
/glass/cytopix/slide014ellipt2.jpg
Heinz bodies Seen in G6PD

Heinz bodies appear as small deficiency
round inclusions
within the red cell
body. It is
denatured,
precipitated Hb in
the red cell.
Page 69 of 336
They appear more
http://www.vet.uga.edu/vpp/clerk/tarigo/ind
ex.php clearly stained with
a supravital stain
(new methylene
blue). When they
are ‘eaten’ by the
phagocytes, bite
cells remain.
These are spherical Seen in severe
Howell-Jolly blue-black haemolytic
bodies inclusions of red anemias,
blood cells seen on hyposplenism
Wright-stained or after
smears. splenectomy
(removal of
They are nuclear spleen).
/microscope/pix/howelljolly_nw.jpg fragments of
condensed DNA, 1 to
2 µm in diameter,
normally removed by
the spleen.
RBC larger than the Round:
Macrocytes surrounding ones. It Liver disease,
can be either chemo-,
round or oval in radiotherapy,
shape. ARV therapy,
hypothyroid-
Round ism.
/glass/cytopix/slide001macro3.jpg
Oval: Megalo-
blastic
Oval macrocyte + anaemias and
Hypersegmented neutrophil = MDS
Megaloblastic
http://www.bpac.org.nz/resources/campaign
/cbc/cbc_bt.asp
Page 70 of 336
Small red cells The three main
Microcytes causes are
iron
deficiency,
anaemia of
chronic
disease, and
the
thalassaemia
http://meds.queensu.ca/medicine/deptmed/ syndromes.
hemonc/anemia/images/iron.gif
Also noted in
Sideroblastic
anaemia
NRBC’s or Seen in
Nucleated normoblasts, newborn
red blood cell represent the infants, and in
immature stages patients with
stages of a red haemolytic
blood cell. crises,
megalo-
blastic
http://www.wadsworth.org/chemheme/heme anaemia.
/glass/cytopix/slide011_nrbc1.jpg
These are iron They are seen

Pappenheimer containing granules in severe
bodies in red blood cells. anaemias and
thalassaemias.
It occurs because
the iron is
aggregated with
mitochondria and
ribosomes.
http://imagebank.hematology.org/AssetDetail
They appear as
.aspx?AssetID=1167 faint violet or
magenta specks,
often in small
clusters, due to
staining of the
associated protein.
Page 71 of 336
These are
Poikilocytosis abnormally shaped
red blood cells
http://med2.univ-
angers.fr/discipline/lab_hema/morphogrweb/
5.jpg
Polychromasia may They appear
Polychromatop be defined as under
hilic increased numbers conditions of
erythrocyte / of immature red accelerated
Polychromasia blood cells that red cell
have a blue-gray production.
tint on Wright-
stained smears,
indicating the
presence of
cytoplasmic RNA.
These cells are usually

larger than normal
red cells
This formation Seen in
Rouleaux occurs when red Multiple
blood cells form myeloma,
stacks or rolls. This is macroglo-
due to: bulinaemia,
infection,
Either an artifact or inflammation,
it may be due to HIV infection,
the presence of TB
http://www.mclno.org/webresources/kbase/c
ellatlas/cell%20images/Rouleaux.jpg
high
concentrations of
abnormal globulins
or fibrinogen.
Page 72 of 336
Agglutination is an Observed in
RBC - aggregation of autoimmune
Agglutination erythrocytes in a haemolytic
grape-like cluster. It anaemia.
is the result of
agglutinating
antibody
http://www.kosvi.com/courses/vpat5400cp/rb
c/RBC_agglutination.JPG
Red blood cell Occurs in
Schistocyte/ fragments that microangiopa
red cell frag- result from thic
ments membrane haemolytic
damage anaemia,
encountered severe burns,
during passage uraemia,haem
http://www.wadsworth.org/chemheme/heme through vessels or olytic
/glass/cytopix/slide014schisto3.jpg artificial heart anaemias and
valves disseminated
intravascular
coagulation
(DIC).
Red blood cells Sickle Cell

Sickle cells that have become anaemia
crescent shaped. (HbSS or HbSC
mainly)
When a person with
sickle cell anaemia
is exposed to
dehydration,
infection, or low
http://gossipsports.com/wp- oxygen supply,
content/uploads/2010/09/sickle-cell- their fragile red
100x-website-arrow.jpg blood cells form
liquid crystals and
assume a crescent
shape causing red
cell destruction
and thickening of
the blood.
Page 73 of 336
Red blood cells Haemo-lytic
Spherocytes that appear anaemias.
spherical.
Small
They have no area sphero-
of central pallor as cytes
seen in normal red (micro-
blood cells sphero-
/microscope/pix/spherocytes_nw.jpg
cytes) are
They are formed sometimes
when an antibody is seen in
taken off the surface severe burn
or when excess cases.
membrane is excised
Red blood cells Found in liver
Stomatocyte with an oval or disease,
rectangular area of electrolyte
central pallor; imbalance,
sometimes referred and hereditary
to as a "mouth". stomato-
cytosis.
http://www.fmshk.org/hkabth/em/mar2001fig
3.jpg
Red blood cells that Seen in
Target cells contain a dark red haemolytic
spot in the center anaemias,
encircled by a white especially
ring that makes it sickle cell,
appear like a target. HbC disease,
It is due to extra liver disease
http://www.wadsworth.org/chemheme/heme haemoglobin in the and
/glass/cytopix/slide014_target1.jpg
center. thalassaemia.
Teardrop /leaf Seen in
Teardrop cells shaped red blood myelofibrosis,
cells myeloprolifera
(dacrocytes) tive disorders,
bone marrow
infiltration and
some
/glass/cytopix/slide003tear3.jpg
haemolytic
anaemias.
Page 74 of 336
CLINICAL IMPLICATIONS OF ABNORMAL VALUES
RED BLOOD CELLS

Increased  Decreased 
Primary (problem in the bone marrow) Nutritional deficiencies
 Polycythaemia vera  Iron
 Vitamin B12 and/or folate
Secondary (to other causes)
 Low oxygen tension in the blood Blood loss
 Lung diseases  Trauma/surgery
 High erythropoietin production
e.g. tumours Bone marrow failure
 Congenital cyanotic heart  Infiltration by malignancies (e.g.
disease leukaemia, myeloma etc)
 Pulmonary fibrosis  Chemo- and/or radiotherapy
Relative (water loss) Erythropoietin deficiency

 Dehydration (such as severe  Renal disease
diarrhoea )
 Burns Haemolysis
 Auto-immune
 Allo-immune
 Microangiopathic
 Certain Infections
 Membrane disorders
 Metabolic disorders
 Haemoglobin abnormalities
Page 75 of 336
SUMMARY
 Erythropoietin (hormone) is secreted by kidney in response to hypoxia.

 Normal removal of red cells occur extravascularly
 The different shapes and sizes of the red cells can help with the diagnosis of an
underlying disorder.
Page 76 of 336
WORKBOOK
Question 1
Macrocytosis with numerous target cells and stomatocytes may be seen in:
a) Early iron deficiency

b) Aplastic anaemia
c) Thalassaemia major
d) Liver disease
Question 2
The blood film below (Figure A) was from an adult male patient presenting with
splenomegaly. His haemoglobin was 6.9 g/dl. Latex agglutination test for rheumatoid
arthritis was positive.
Figure A 
Figure
A
1000 X mag
a) Comment fully on the red cell morphology seen in blood film A.
Page 77 of 336
Question 3
Choose the correct answer.

Pappenheimer bodies:
a) Contain iron and stain negative with Perl’s Prussian blue stain.
b) Contain iron and can be found in patients with haemochromatosis.
c) Do not contain iron and are therefore found in patients with iron deficiency
anaemia.
d) Are found in patients with thalassaemia and are sometimes referred to as
golf balls.
Question 4
Study each inclusion carefully and answer the attached questions.
a) Name the inclusion.

b) What do they consist of?
c) In what condition do we see them?
d) Name the specific stain used to identify them.
Page 78 of 336
Question 5
These inclusions appear at the periphery of the cell and stain blue with Wrights or
Giemsa Stain
a) What inclusions are they?

b) What do they consist of?
c) In what condition do we see them?
d) What special stain is used to identify them?
Question 6
a) What are these inclusions?

b) Name the abnormal haemoglobin.
c) What causes the golf ball appearance?
d) Which special stain do we use to identify this?
e) What causes this phenomenon?
Page 79 of 336
Question 7
a) What is this ring?

b) What stain do we use to identify this ring?
c) What causes these rings?
d) In what disorder do we see these rings?
Question 8
Name the hormone that regulates erythropoiesis.
Question 9
What is the function of NADH in the Embden-Meyerhof pathway and why is it

important?
Question 10
The following results are obtained from a patient:
Red Cell Count 3,2 X 1012/L

Haemoglobin 5,8 g/dL
Haematocrit 0,189 L/L
Mean Cell Volume 59 fl
a) Calculate the Mean Cell Haemoglobin and Mean Cell Haemoglobin

Concentration
Page 80 of 336
b) Describe the size and haemoglobin content of these cells.
c) How would you verify this morphology?
Question 11
List 5 common problems that are related to the inability to focus a mounted
haematology slide under the microscope.
Page 81 of 336
Learning Unit 4
ANAEMIAS
Learning Unit 4 provides you with the underpinning knowledge and

terminology related to different types of anaemias.
Assessment Criteria
learning unit four:
1.1 Identify the relevant morphological features associated with the
different types of anaemia.
1.2 Interpret the peripheral blood findings to help with recognition of
the anaemia.
1.3 Demonstrate knowledge of confirmatory tests.
1.4 Differentiate between:
o Hypochromic, microcytic anaemias
o Macrocytic anaemias
o Haemolytic anaemias
o Aplasia
o Anaemia of pregnancy
o Hypoproliferative anaemias
Page 82 of 336
ANAEMIAS
Anaemia Reduction in the haemoglobin concentration in blood
Hypochromic Pale center within red cells, MCH is low
Microcytic Small red cells, MCV low
Macrocytic Large red cells, MCV high
Aetiology The cause of a disease
Heterozygote The pair of genes determining a characteristic is dissimilar
Homozygote The pair of genes determining a characteristic is identical
Anaemia is defined as a reduction in the haemoglobin concentration of the blood.

Alterations in the plasma volume can influence the haemoglobin concentration as
well. A reduced plasma volume (e.g. dehydration) may mask anaemia, whereas an
increase in plasma volume (e.g. pregnancy) may suggest anaemia.
Anaemia may not be immediately apparent after acute blood loss because of total
blood volume reduction.
Page 83 of 336
CLINICAL FEATURES
MAJOR ADAPTATIONS:
Cardiovascular
Increased stroke volume and
Tachycardia
Haemoglobin O2 dissociation
curve
Oxygen is given up more
readily to tissues
http://images-mediawiki-sites.thefullwiki.org/04/2/6/4/38041003376270456.png
BLOOD SMEAR
It is essential to screen the blood smear to suggest a specific diagnosis. A mixed

deficiency may be present which will give you normal parameters e.g. mixed iron and
megaloblastic anaemia.
Note the oval

macrocytes
http://i281.photobucket.com/albums/kk202/amiya12/Anaemia_Type.gif
Note the decreased Note the small cell size
number of red cells, with large, pale centre
often macrocytic
Page 84 of 336
CLASSIFICATION OF ANAEMIAS
Anaemias can be classified by two methods: morphological or aetiological.
Morphological classification
Microcytic, hypochromic Normocytic, normochromic Macrocytic

 Iron deficiency  Haemolytic  Megaloblastic
 Thalassaemia anaemias  Non-megaloblastic:
 Anaemia of chronic  Chronic illnesses alcohol, liver disease,
disorders  After acute blood myelodysplasia,
 Lead poisoning loss hypothyroidism,
 Sideroblastic  Renal disease therapy related etc.
anaemia (some)  Bone marrow failure
 Mixed deficiencies
Aetiology classification
Inadequate red cell Increased destruction of Abnormal distribution

production red cells / reduced life
span
 Nutritional  Intrinsic abnormality  Hypersplenism
deficiencies like iron, of the red cells –
B12 or folic acid either inherited or
 Aplastic – either acquired
inherited or acquired (haemolytic)
 Ineffective  Extrinsic factors
production e.g. MDS (haemolytic)
 Bone marrow  Blood loss
infiltration by
malignant cells
 Bone marrow fibrosis
 Hypoplastic -
ineffective formation
of red cells e.g.
chronic
inflammation,
thalassaemia , renal
disease
Page 85 of 336
SUMMARY
 Anaemia is defined as reduced haemoglobin concentration

 It can be classified according to red cell morphology or
aetiology
 Red cell indices and morphology correlate with underlying
disorder
Page 86 of 336
HYPOCHROMIC, MICROCYTIC ANAEMIAS
IRON PROTOPORPHYRIN
Iron deficiency Sideroblastic anaemia

Chronic
Disease
Haem Globin
+
Thalassaemia
Haemoglobin
Page 87 of 336
IRON DEFICIENCY
Iron deficiency anaemia occurs when the body’s stores of iron are insufficient to
maintain erythropoiesis.
Normal iron cycle

Iron is well balanced in the body. One absorbs 1-2 mg iron in the gut; you lose 1-2 mg
in the gut daily. Transferrin acts like the iron taxi. It is a protein that picks up 2 iron
molecules and takes it wherever it needs to go; it can cover about 10 trips a day. It
carries the iron to storage (as ferritin or haemosiderin) and to wherever the need is.
About a third or a fourth of the iron is in storage in the liver and other tissues. Two-thirds
to three-quarter of iron is actively used in red blood cells and in the RE system. A very
small amount is utilized in the muscle as myoglobin and in other enzymes.
Iron is absorbed in the ferrous (Fe2+) form, rather than the ferric (Fe3+) form.
http://www.cdc.gov/ncbddd/hemochromatosis/training/images/iron_cycle.jpg
Page 88 of 336
Causes of iron deficiency
Chronic blood loss (most common cause of iron deficiency)
 Bleeding from the gastrointestinal tract e.g. peptic ulcer, aspirin etc.
 Uterine – menorrhagia
 Rarely – haematuria, haemoglobinuria
Increased demands
 Pregnancy
 Prematurity
 Growth spurts
 Erythropoietin therapy
Malabsorption (rare)
 Gluten-induced enteropathy, gastrectomy
Poor diet (rare)

 A factor in the developing countries
Clinical features of iron deficiency

 Glossitis and angular stomatitis
 Koilonychia (spoon nails)
http://wacky5.com/wp-content/uploads/2010/04/SPOON-SHAPED-NAILS.jpg
 Dysphagia (difficulty to swallow) due to an oesophageal web (Plummer-Vinson

syndrome)
Page 89 of 336
Laboratory diagnosis of iron deficiency
Full blood count indices Blood film Confirmatory tests

 Hb low  Hypochromic,  S-Ferritin low
 MCV low microcytic  Serum iron low
 MCH low  Anisopoikilocytosis  Transferrin saturation
 Retic count low  Pencil cells low
 RDW high  Target cells ±  Transferrin high
 Platelets – can be  Bone marrow iron
high low or absent
Note: In acute/chronic ill patients a complete iron profile is sometimes indicated to

make the diagnosis because s-ferritin can appear normal because of acute phase
response.
Treatment
Oral iron therapy for at least 6 months
Parenteral iron is given with oral treatment failure or in cases of malabsorption or high
iron requirements.
Page 90 of 336
ANAEMIA OF CHRONIC DISORDER (ACD)
This anaemia can results from infection such as tuberculosis, pneumonia, chronic
inflammatory diseases such as rheumatoid arthritis or from malignant diseases such as
carcinoma, lymphoma, sarcoma.
http://www.hakeem-sy.com/main/files/images/Mechanism_anemia_chron_dis.jpg
The mechanism is reduced delivery of iron from reticuloendothelial system to

developing erythroblast together with a shortened red cell survival time and an
inadequate erythropoietin response.
Hepcidin, released by the liver in response to inflammation, inhibits macrophage release
of iron as well as iron absorption.
ACD can develop within two weeks of illness.
Page 91 of 336
Laboratory diagnosis of anaemia of chronic disorders

 Hb low  Initially  Ferritin normal / high
 MCV normal / low normochromic,  Serum iron low
 MCH normal / low normocytic  Transferrin saturation
 RDW normal / high  Later - hypochromic, low
 Platelets and microcytic  Transferrin low
neutrophils – can be  Anisopoikilocytosis ±  Bone marrow iron
high  Difficult to distinguish normal, but number
 ESR high from iron deificiency of erythroblasts with
 Retic low iron is reduced
Treatment
Treat the underlying disorder
SUMMARY
 Blood loss is the most common cause of iron deficiency

 Iron deficiency and anaemia of chronic disorders can be
distinguished by using the iron profile
Page 92 of 336
SIDEROBLASTIC ANAEMIA
Sideroblastic anaemia can be classified as either hereditary or acquired; the common
link is a defect in the haem synthesis.
Classification
Congenital sideroblastic anemia is usually hypochromic, microcytic and
 inherited as an X-linked recessive disorder.
The acquired form is subdivided into primary or secondary causes:

 Primary – Myelodysplasia (refractory anaemia with ring sideroblasts)
 Secondary – Ring sideroblasts also occur in other malignant diseases of the
bone marrow such as other types of MDS, myelofibrosis, myeloid leukaemis,
myeloma, or with drug administration such as TB drugs or other benign
conditions such as haemolytic anaemias, megaloblastic anaemia,
malabsorption, rheumatoid arthritis.
Laboratory diagnosis of anaemia of sideroblastic anaemia

 Hb low  Micro- or macrocytic  Ferritin high
 MCV low  Dimorphic picture  Serum iron high
(congenital) and  Bone marrow iron
high (acquired) present, with ring
 MCH normal sideroblasts
 RDW high (diagnostic)
Iron is deposited in a ring

around the nucleus
Page 93 of 336
LEAD POISONING
Lead inhibits both haem and globin synthesis. In addition it interferes with RNA
synthesis. Denatured RNA is present in red cells and gives the appearance of
basophilic stippling or punctate basophilia. Anaemia may be hypochromic or
haemolytic; bone marrow may show ring sideroblasts.
Basophilic stippling
Page 94 of 336
HAEMOGLOBIN DISORDERS
Normal haemoglobin consists of three types of haemoglobin:

 HbA
 HbF
 HbA2
HbA HbF HbA2

 2β2  22  22
 96 – 98%  0.5 – 0.8%  1.5 – 3.2%
Normal adult blood contains small quantities of HbF and HbA2. The major switch from
foetal to adult haemoglobin happens 3 – 6 months after birth.
Abnormalities result from either synthesis of abnormal haemoglobin or reduced rate of

synthesis of normal - or - globin chains.
Page 95 of 336
THALASSAEMIAS
The thalassaemias are inherited disorders of haemoglobin that result from a reduced
formation of either the alpha or beta chains and are classified accordingly. If - and β-
chains fail to combine, it will lead to lower haemoglobinisation of red cells. When
released into circulation, transport of oxygen is poor. Red cell damage also results from
destruction of inclusion bodies formed by aggregations of unmatched globin chains by
the marrow or spleen i.e. haemolysis.
The geographical distribution of the thalassaemias extends from the Mediterranean

through the Middle East and India to South-East Asia.
Classification
Type Heterozygous Homozygous

  thalassaemias
o 0 (--/) Thal minor (2/3 gene Hydrops fetalis (4 gene
deletion). HbH (-/--) deletion) = Hb Barts(--/--)
o + (-/) Thal minor (1/2 gene Thal minor

deletion)
 β thalassaemias
o β0 Thal minor or trait Thal major
o + Thal minor Thal major or intermedia
Page 96 of 336
Duplicate -globin chain genes are present on each chromosome. Total loss of -
chain production (0 or --/) or partial loss may result from loss of only one gene(+ or -
/).
-thalassaemias are autosomal recessive disorders characterised by reduced (+) or
absent (β0) production of -chains.
The clinical severity of thalassaemia is proportionate to the degree of imbalance
between -globin chain to -globin chain synthesis.
Clinical syndromes of -thalassaemias

 Hb-Barts hydrops syndrome (--/--)
As the -globin chain is essential in foetal as well as adult haemoglobin, this
disorder is incompatible with life and leads to death in utero (hydrops fetalis).
 HbH disease (-/--)

HbH (4) in adults can be detected by haemoglobin electrophoresis (pH6-7) or
reticulocyte preparations. There is a moderate chronic haemolytic anaemia
with splenomegaly.
 -thalassaemia traits
The traits are caused by 1 or 2 gene deletions and are usually not associated
with anaemia, although the MCV and MCH are low.
Clinical syndromes of β-thalassaemias

 Β-thalassaemia major
Severe anaemia is caused by excess  chains leading to ineffective
erythropoiesis and haemolysis. Anaemia becomes apparent when production
of HbF declines (about 3-6 months). Production of  chains helps to ‘mop up’
excess -chains. Compensatory expansion of the marrow space causes the
typical thalassaemia facies (bone abnormalities). There is tendency to fractures,
ulcers and infections. The X-rays have a typical ‘hair-on-end’ appearance.
Page 97 of 336
The child presents with failure to thrive and develops hepatosplenomegaly.
Blood transfusion remains the foundation of treatment but leads to iron
overload. Iron chelation therapy is instituted to minimise iron overload. Iron
damages the heart, liver and the endocrine organs with impaired growth,
absent puberty, diabetes mellitus and hypothyroidism. Splenectomy may be
done to reduce transfusion frequency, but pose a high risk of infections if done
too young. Stem cell transplantation is also an option.
 Β-thalassaemia minor
This disorder is usually asymptomatic. It may be confused with an iron deficiency
picture. There may be a mild anaemia with an elevated red cell count. The
diagnostic feature will be on haemoglobin electrophoresis a raised HbA2.
Laboratory diagnosis of thalasaemia

 Hb low/normal  Hypochromic,  Hb electrophoresis
(depending on microcytic red cells (see below) or HPLC
severity)  Target cells (NB), (high performance
 MCV low polychromasia liquid
 MCH low chromatography)
 RCC high  Supravital stain for
 RDW high HbH
 Retic high
Calculation used to help distinguish between iron deficiency and thalassaemia:

MCV/ RCC =
>13 = iron deficiency
<13 = thalassaemia
Page 98 of 336
Haemoglobin electrophoresis
Origin A2 S F A H
Normal II II II IIIIIIIIIIIII
-thal trait II III III IIIIIII
-thal major II III IIIIIIII
Hb H disease II II II IIIIIIII IIII
HPLC
SUMMARY
 There is a reduced production of either - or -globin chains

 Anaemia is due to ineffective erythropoiesis and haemolysis
 Laboratory diagnosis is based on a high red cell count together
with a hypochromic, microcytic anaemia, usually with target
cells.
 Diagnosis is confirmed with Hb electrophoresis or HPLC
 Treatment includes blood transfusion which leads to iron
overload
Page 99 of 336
SICKLE CELL SYNDROMES
The sickle cell syndromes are a group of haemoglobinopathies where the common
feature is inheritance of an abnormal  chain gene called s. There is a single base
change in the DNA – adenine is replaced by thymine. This leads to an amino acid
change from glutamic acid to valine. Homozygous sickle cell anaemia (HbSS) occurs
when two s genes are inherited. It is a serious disorder. There is no synthesis of normal
 globin and therefore no HbA can be formed. The haemoglobin is predominantly Hb
S with a small amount of Hb A2 and Hb F. It occurs most commonly in West Africans.
Heterozygous conditions e.g. Hb SC and Hb Sthal also cause sickling disease.
Hb S is insoluble and in the deoxygenated state it will aggregate into long polymers
which align to form crystals. The red cells lose their normal discoid form and become
elongated (sickle shaped or boat shaped). The sickled cells block the microcirculation
causing infarcts in various organs. Sequestration of these red cells in the
reticuloendothelial system causes haemolysis.
The abnormal Hb S (carrier state) offers protection against falciparum malaria.
http://learn.genetics.utah.edu/content/disorders/whataregd/sicklecell/images/sicklecell.jpg
Page 100 of 336

Clinical features of sickle cell anaemia (homozygous)
 Haemolytic anaemia
Although the haemoglobin is usually very low, the symptoms are usually mild
because HbS releases oxygen to the tissues more readily than HbA. Parvovirus
infection or folate deficiency can block erythropoiesis and cause an aplastic
crisis.
 Painful vaso-occlusive crises

Cold, infections, dehydration or deoxygenation may trigger acute painful crises
which are potentially disabling. Infarcts occur in a variety of organs as well as
the bones. The most serious complication is vaso-occlusive crises in the brain
which may lead to a stroke. The ‘hand-foot’ syndrome caused by infarcts of the
small bones is frequently the first presentation in infants with the disease.
 Sequestration crises
These are caused by sickling within the organs and pooling of blood, often
intensifying the anaemia. Acute sickle chest syndrome, with occlusion of the
pulmonary vasculature is a feared complication.
 Other
Vascular stasis and local ischaemia is responsible for most of the other
complications:
o Leg ulcers
o Autosplenectomy (ineffective spleen or hyposplenism)
o Pulmonary hypertension
o Kidney damage
o Liver damage
o Proliferative retinopathy (damage to the retina of the eye)
Clinical features of sickle cell trait

 Usually no clinical problems because there is enough Hb A to prevent sickling,
however haematuria can occur due to necrosis within the kidney.
Page 101 of 336

Laboratory diagnosis of sickle cell anaemia

 Hb low(depending  Hypochromic,  Hb electrophoresis
on severity) microcytic red cells (see below)
 Retic high (or low in  Sickle cells  HPLC (high
an aplastic crises)  Target cells, performance liquid
polychromasia chromatography)
 Splenic atrophy  Screening for sickling
(Howell-Jolly’s, when blood is
acanthochytes) deoxygenated
Haemoglobin electrophoresis
Origin A2 S F A H
Normal II II II IIIIIIIIIIIII
Sickle cell trait II II III II IIIIIIII
Sickle cell disease II II IIIIIII IIII
SUMMARY
 Inheritance of two βs genes lead to sickle anaemia (HbSS)

 Laboratory diagnosis include sickle cells on the peripheral blood
smear, Hb electrophoresis, as well as sickling when blood is
deoxygenated (e.g. Na2HPO4)
 It primarily affects West African people
Page 102 of 336

MACROCYTIC ANAEMIAS
Macrocytic (large red cells) anaemias are broadly subdivided into megaloblastic
anaemia and non-megaloblastic anaemia.
Causes include
Megaloblastic Non-megaloblastic
 Vitamin B12 deficiency  Alcohol
 Folate deficiency  Liver disease
 Drugs interfering with DNA  Myxoedema
synthesis ( e.g. methotrexate)  Myelodysplastic syndromes
 Cytotoxic drugs
 Aplastic anaemia
 Pregnancy
 Smoking
 Reticulocytosis
 Myeloma and paraproteinaemia
 Neonatal
Page 103 of 336

MEGALOBLASTIC ANAEMIA
Megaloblastic anaemias show a common characteristic which abnormal delayed

maturation of the nucleus in relation to the cytoplasm. The underlying defect is due to
defective DNA synthesis and is usually caused by deficiency of vitamin B12
(cobalamin) or folate.
Red cells will either die in the marrow or have a reduced survival time – this is known as
intra-medullary haemolysis and the cause of a markedly raised LDH. When the red
cells do enter the bloodstream it appears enlarged.
Vitamin B12
The vitamin is found in foods with animal origin such as liver, meat, fish and dairy
products. The absorption of vitamin B12 occurs in the small intestine and requires
intrinsic factor (IF), secreted in the stomach. The IF-B12 complex can then bind to a
specific IF receptor. B12 is eventually absorbed in the distal ileum and IF destroyed.
B12 then becomes attached to transcobalamin (TC, previously called trancobalamin
II) which delivers B12 to bone marrow and other tissues. TC deficiency can cause
megaloblastic anaemia.
http://thebreakingstory.com/fitness/wp-content/uploads/2011/05/1306069381-98.gif (accessed 23/6/11)
Vitamin B12 deficiency affects all dividing cells in the body, particularly the actively
proliferating cells of the bone marrow which suffer from the impaired DNA synthesis.
RNA synthesis in the cytoplasm are unaffected, therefore the nuclear-cytoplasm ratio is
Page 104 of 336

imbalanced. The chromatin pattern in the nucleus resembles sliced salami. The slow
DNA synthesis leads to cells being released into the blood stream without normal cell
division. Red cells are enlarged and egg-shaped (oval macrocytes) and neutrophils
show hypersegmentation due to excess nuclear material.
Folate
Folate is found in most foods especially liver and green vegetables. Overcooking of
vegetables can destroy Folate.
Folate is needed in a variety of biochemical reactions in the body. No specific protein
enhances uptake of folate into the cells.
Deficiency of B12 leads to impaired conversion of homocysteine to methionine causing
folate to be trapped in the methyl form. Folate is needed as a cofactor in DNA
synthesis.
Causes of deficiency
B12 Folate
 Nutritional  Nutritional
o Vegans (strict vegetarians, no o Old age, institutions,
eggs or dairy products) poverty
 Malabsorption  Malaborption
o Gastric o Tropical sprue, gluten-
 Pernicious anaemia induced enteropathy
 Congenital lack of IF  Excess utilization
 Total/partial gastrectomy o Pregnancy, lactation,
o Intestinal prematurity
 Intestinal stagnant loop o Haematological diseases:
syndrome haem anaemias,
 Chronic tropical sprue myelofibrosis
 Ileal resection o Malignant diseases:
 Crohn’s disease carcinoma, lymphoma,
 Fish tapeworm myeloma
o Inflammatory diseases:
Crohn’s disease, TB, RA,
malaria
 Excess urinary loss
o Liver disease, congestive
heart failure
 Drugs
o Anticonvulsants,
sulfasalazine
 Mixed
o Liver disease, alcoholism,
intensive care
Page 105 of 336

Pernicious anaemia
It is an autoimmune disease of the gastric mucosa leading to atrophy of the stomach.
Most patients have IgG antibodies targeted against gastric parietal cells and IF. IF
secretion is reduced or absent and there is reduced stomach acid.
Helicobacter pylori may trigger autoimmune gastritis. More women than men are
affected and they are usually above 50 years of age.
Clinical features of megaloblastic anaemia

 Jaundice – patients may have lemon yellow tint because of excessive
breakdown of haemoglobin (haemolysis)
 Glossitis (red sore tongue)
http://www.meddean.luc.edu/lumen/MedEd/medicine/pulmonar/pdself/Lymphatics/Glossitis.jp
g (accessed on 23/6/11)
 Angular stomatitis
http://images.paraorkut.com/img/health/images/a/angular_stomatitis-337.jpg (accessed on
23/6/11)
 Mild malaborption symptoms
 Purpura – from thrombocytopaenia
 Neuropathy
The patient may notice a tingling in the feet, difficulty in walking and may fall
over in the dark. Severe psychiatric symptoms are rare.
Page 106 of 336

 Neural tube defect (spina bifida)
Folate deficiency predisposes to neural tube defect in the fetus.
www.sbac.org/SBAC.Org__SB_Facts.htm
Page 107 of 336

Laboratory diagnosis of megaloblastic anaemia

 Hb low  Oval macrocytic red  s-B12 low (if B12
 MCV high cells deficiency)
 Retic low  Hypersegmented  Red cell folate low
 WCC may be neutrophils (folate + B12 deficiency)
low  Erythroblasts with a  Absorption tests e.g.
 Plt may be low sliced salami Schilling where absorbed
appearance radio-labelled B12 is
flushed into 24-h urine. If
malabsorption is
corrected by adding IF
to oral dose, pernicious
anaemia is the likely
cause.
Oval macrocyte Hypersegmented  Parietal cell or anti-IF
neutrophil antibodies
 Bone marrow aspirate
will confirm diagnosis,
but is not always
necessary
Erythroblasts with
primitive nuclei
Images from nursingcrib.com/.../megaloblastic-anemias and Giant band cell

pathy.med.nagoya-u.ac.jp/atlas/doc/node110.html
Page 108 of 336

OTHER MACROCYTIC ANAEMIAS
The exact mechanisms of macrocyte formation are not clear. Increased lipid
deposition on the red cell membrane or alterations of erythroblast maturation time in
the marrow may be implicated.
In the absence of anaemia, alcohol is the most frequent cause of a raised MCV and
round macrocytes.
Round macrocytes are the major characteristic that differentiates the other causes
from megaloblastic anaemia. It is therefore crucial to identify if the macrocytes seen
are round or oval. Clinical history and physical examination is important to establish
the underlying disorder. Anti retroviral or chemotherapy are very common causes of
macrocytic anaemia.
SUMMARY
 B12 and folate are essential for DNA synthesis. If deficient,

megaloblastic anaemia occurs.
 All living cells’ DNA are affected, therefore neutropaenia and
thrombocytopaenia are also common findings.
 The blood smear show oval macrocytes, hypersegmented
neutrophils and ‘sliced salami’ erythroblasts (megaloblasts).
 Confirmation is done with serum B12 and red cell folate levels.
 Other macrocytic anaemias have round macrocytes.
Page 109 of 336

HAEMOLYTIC ANAEMIAS
The haemolytic anaemias are characterized by abnormal destruction of the red cells.
Remember that red cells are normally removed after about 120 days by the
macrophages of the reticuloendothelial system? Normal red cell breakdown occurs
extravascularly (outside - in the liver and spleen).
Haemoglobin is broken down into Heme

and globin.
The globins are broken down to amino

acids, which are then used for protein
synthesis.
The porphyrin ring of heme is oxidized

producing biliverdin and releasing the
iron (Fe3+).
The iron can then be exported into

plasma through iron channels, where it
can be stored within cells as ferritin. With
time, ferritin becomes oxidized and
degrades to form haemosiderin.
Biliverdin is reduced to unconjugated

bilirubin (water insoluble). The
unconjugated bilirubin is released into the
plasma, where it binds to albumin (to
render it water-soluble) and is taken up
by hepatocytes.
It is then excreted in the faeces as

stercobilinogen or via the kidneys as
urobilinogen.
http://ahdc.vet.cornell.edu/clinpath/modules/chem/images/extravascular%20hemolysis%20new.jpg
Page 110 of 336

Intravascular (inside the blood stream) haemolysis occurs in some pathological
disorders and may also occur hand in hand with extravascular haemolysis. Free
haemoglobin is released which binds and thus saturates the plasma haptoglobins
rapidly. The haemoglobin/ haptoglobin complex is removed by the macrophages of
the RE system (liver and spleen). The excess haemoglobin is filtered by the kidney. Free
haemoglobin enters the urine as the reabsorptive capacity is saturated. The renal
tubules become loaded with haemosiderin as iron is released from the haemoglobin.
Methaemalbumin and haemopexin are also formed during intravascular haemolysis.
Intravascular
Extravascular
Haemosiderinuria
http://ahdc.vet.cornell.edu/clinpath/modules/chem/images/intravascular%20hemolysis.jpg
There are three considerations when classifying the haemolytic anaemias:

 Is the disorder inherited or acquired later in life?
 Is the abnormality located within the red cell (intrinsic) or outside (extrinsic)?
 Are the red cells destroyed intravascularly or extravascularly?
Page 111 of 336

CLASSFICATION
Hereditary haemolytic anaemia Acquired haemolytic anaemia
Intrinsic red cell defects Extrinsic (Extracorpuscular)
Environmental change
Membrane Immune
Hereditary spherocytosis Autoimmune
Hereditary elliptocytosis o Warm antibody (EBV, LPD)
o Cold antibody (Mycoplasma, LPD)
Metabolism Alloimmune
Glucose-6-Phosphate Dehydrogenase o Transfusion reactions
deficiency o HDN
Pyruvate kinase deficiency o Allografts – bone marrow
Drug associated
Haemoglobin
Genetic: HbS, HbC, unstable Red cell fragmentation syndromes
Cardiac
o Prosthetic heart valve
o Patches, grafts
Microangiopathic
o TTP-HUS
o DIC
o Malignant disease
o Vasculitis
o Malignant hypertension
o Pre-eclampsia/HELLP
o Renal vascular disorder/HELLP
o Cyclosporin
o Homograft rejection
March haemoglobinuria
Infections
Malaria
Clostridia
Chemical/Physical agents
Drugs, domestic substance, burns
Secondary
Liver and renal disease
Exception
Paroxysmal Nocturnal Haemoglobinuria – acquired disorder with intrinsic defect
Page 112 of 336

Clinical features of a haemolytic anaemia
 Pallor of the mucous membrane
 Mild jaundice (increased breakdown of red cells results in increased bilirubin in

the plasma)
http://www.herbal-ayurveda-remedy.com/home-remedies/images/jaundice.jpg
 Splenomegaly (where the spleen is the major site of red cell destruction)
http://www.nlm.nih.gov/medlineplus/ency/images/ency/fullsize/17212.jpg
Page 113 of 336

Laboratory diagnosis of increased red cell breakdown (haemolysis)
Chemistry Haematology Blood film

 S-Bilirubin high  Hb low Depending on the
 U-urobilinogen high  Retic high (bone abnormality
 Faecal stercobilinogen marrow tries to  Spherocytes
high compensate for
 S-haptoglobin low the losses)
(because it is saturated  Immune –
with haemoglobin) mediated =
 Raised s-LDH positive  Red cell
Coomb’s test fragmentation
Intravascular (additional)
 Haemoglobinuria
 Haemosiderinuria (storage
iron in urine)
 Methaemalbuminaemia
(Schumm’s test)  Polychromasia
 Elliptocytes
http://3.bp.blogspot.com, ahdc.vet.cornell.edu/.../rbcmorph/schisto.htm, med-ed.virginia.edu,

labmed.hallym.ac.kr
Page 114 of 336

INHERITED HAEMOLYTIC ANAEMIAS
MEMBRANE DEFECTS
HEREDITARY SPHEROCYTOSIS
Inheritance may be autosomal dominant or rarely may it be recessive. There may be

alterations in spectrin, ankyrin and other membrane proteins. Some parts of the
membrane are lost and cause the red cells to change shape and become spherical.
These spherocytes are unable to pass through the spleen’s microcirculation and die
prematurely.
The red cells become hyperpermeable to sodium because of the weakened cell
structure. Normally there is sufficient glucose and ATP to expel sodium, but spherocytes
consume glucose at a rapid rate, therefore less glucose means sodium and
subsequently water enter the red cells which causes swelling and haemolysis of these
red cells.
The severity of the haemolysis is variable and can present at any age. Pigment
(bilirubin) gallstones caused by prolonged haemolysis are frequent. A severe anaemia
may develop after a viral infection (usually Parvovirus) and is called an aplastic crisis.
Laboratory diagnosis of hereditary (congenital) spherocytosis

 Hb low  Microspherocytes  Direct Coombs (DAT)
 MCHC high (in some negative
cases)  Osmotic fragility
 Retic count high increased
 Auto haemolysis
increased
www.pathologystudent.com
The Direct Coombs or Direct Antiglobulin Test (DAT) is nearly always positive in immune
haemolysis.
Page 115 of 336

The autohaemolysis test refers to cells being incubated with their own plasma with or
without added glucose.
www.pitt.edu/~super1/lecture/lec35371/022.htm
Osmotic fragility measures red blood cell (RBC) resistance to haemolysis when
exposed to a series of increasingly diluted saline solutions. The sooner haemolysis
occurs, the greater the osmotic fragility of the cells.
Treatment
No treatment is required with mild disease. Spleen (as the major site of destruction) may
be removed in severe cases, but there is a risk of post-splenectomy sepsis, especially in
young children.
HEREDITARY ELLIPTOCYTOSIS
Hereditary elliptocytosis has many clinical and laboratory features similar to hereditary
spherocytosis, except for the appearance of the red cells which are elliptical in shape
and not spherical. Clinically it is a milder disorder and is usually discovered by chance
when looking at the blood film. The basic defect lies within the spectrin dimer formation.
Page 116 of 336

RED CELL METABOLISM DEFECTS
GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENCY
G6PD is a necessary enzyme that protects the red cell from oxidant stress (remember
Hb must be in the reduced form to carry oxygen effectively). The inheritance is sex-
linked, affecting the males; female carriers show half normal G6PD levels. Both the
carriers and the affected males have the advantage of resistance to P.falciparum
malaria. Africans, Middle Eastern and South Asian people are predominantly
affected.
Patients are usually asymptomatic until increased oxidant stress leads to severe
haemolytic anaemia. Triggers can be drugs, fava beans and infections. Intravascular
haemolysis develop rapidly and haemoglobinuria results.
The neonate can also present with jaundice.
Laboratory diagnosis of G6PD deficiency

 Hb low (during crisis)  Bite and blister cells  Direct enzyme assay
or normal (keep in mind that
 MCV normal or retics have a higher
slightly increased enzyme level; thus
(retics) Heinz bodies were testing should not be
plucked out by spleen
 Retic count high done during acute
 Heinz bodies haemolysis (↑↑retics =
(oxidized, denatured false normal test))
haemoglobin) - seen
with retic stain
www.pathwiki.pbworks.com
Page 117 of 336

PYRUVATE KINASE DEFICIENCY
This is inherited as an autosomal recessive disorder where the patients lack an enzyme in
the Embden-Meyerhof pathway. Red cells are unable to generate adequate ATP and
become rigid.
The clinical symptoms are often surprisingly mild for the degree of anaemia, but this is
true because oxygen is released readily from the haemoglobin.
Laboratory diagnosis of PK deficiency

 Hb (very) low  Poikilocytosis  Direct enzyme assay
 MCV normal or  Autohaemolysis
slightly increased increased (not
(retics) corrected by
 Retic count high glucose)
SUMMARY
 Inheretid haemolytic anaemias are usually intrinsic to the red cell

 Hereditary spherocytosis and hereditary elliptocytosis are caused
by defect in the red cell membrane
 G-6-P-D and PK are enzymes in red cell metabolism; deficiencies
may lead to haemolysis
Page 118 of 336

ACQUIRED HAEMOLYTIC ANAEMIAS
AUTOIMMUNE HAEMOLYTIC ANAEMIA (AIHA)
AIHA is caused when a person produced an antibody that this directed against its own
red cells.
Most of the time there is no known reason (idiopathic) for the patient to be making
antibodies. Sometimes there is a known underlying cause – like lymphoma, leukaemia,
another auto-immune disease, infections, drugs (like penicillin, methyldopa).
Whatever the cause, it is not good to have antibodies coating the red cells, because
macrophages of the spleen see them as yummy and will “eat” them.
The class of antibody determine the type of haemolysis e.g. IgM auto-antibodies cause
destruction by agglutination or activation of complement whereas IgG auto-antibodies
bind the macrophages and is “eaten up”. They are characterized by a positive Direct
Antiglobulin Test (DAT), also known as Direct Coombs test.
AIHA can generally be divided into ‘cold’ or ‘warm’ types depending on where the
antibodies react the strongest i.e. 4ºC or 37ºC.
Warm AIHA
The red cells are coated with IgG alone or with complement. Destruction takes place
by the macrophages of the reticulo-endothelial (RE) system, because they have
receptors for the Ig Fc portion. Sometimes the macrophages just “nibble” which
cause the red cell to lose some of the membrane. The red cell then becomes round
to contain the same volume and a spherocyte is formed.
Causes include SLE, CLL, lymphomas, Epstein Barr Virus (EBV) and certain drugs.
There is no age, race or sexual preference. Sometimes the spleen is enlarged

(because of all the eating going on in there).
Treatment involves getting rid of the underlying cause, if possible. Steroids (to induce
immuno-suppression) might help or if it is really severe, the spleen can be removed (to
stop the eating).
Page 119 of 336

Laboratory diagnosis of warm AIHA

 Hb low  Spherocytosis  DAT positive
 Retic high  Bili’s high
 LDH high
 S-haptoglobin low
Spherocytes
Polychromatic cell
http://www.pathologystudent.com/?p=1035 (accessed 19/8/11)
Cold AIHA
In cold AIHA the patient still makes antibodies against his/her red cells and there is
complement fixed to the red cells. The cold type is usually an IgM antibody, which
easily fixes complement. Complement “pokes holes” in the red cells and are
destroyed right in the circulation – therefore intravascular haemolysis occur. There is a
little bit of macrophage action happening as well – complement-coated red cells are
“eaten” – therefore extravascular haemolysis also occurs to some extent.
Complement alone is usually detected on the red cells as the antibody “falls off” in the
warmer parts of the circulation. The antibody is usually directed against the “I”
antigen on the red cells. In infectious mononucleosis it is anti-i.
Causes include idiopathic, lymphoproliferative disorders, infections e.g. infectious

mononucleosis or Mycoplasma pneumonia and Paroxysmal cold haemolobinuria
(PCH).
Remember IgM is a pentamer and they are able to span several red cells, creating big
agglutinates of red cells. These can plug up the small vessels, creating ischemic
conditions in the colder distal parts of the body – like the ear lobe, nose, fingers and
toes. The patient develops acrocyanosis (purple/blue skin colour) wherever a body
part is exposed to the cold. Treatment would therefore involve keeping the patient
warm.
Page 120 of 336

Laboratory diagnosis of cold AIHA

 Hb low  Red cell  DAT positive
 Retic high agglutination  Bili’s high
 MCHC high  Spherocytosis (less  LDH high
(remember we put it obvious)  Haemoglobinuria
in the water bath to  S-haptoglobin low
correct the MCHC)
 Donath-Landsteiner
antibody for PCH
Agglutinated red cells
Page 121 of 336

ISOIMMUNE HAEMOLYTIC ANAEMIA
Here antibodies produced by one person react with another person’s red cells. It is
called iso-antibodies or allo-antibodies. Two very important situations are
incompatible ABO-blood transfusions and transfer across the placenta (Haemolytic
disease of the Newborn). These will be discussed in the Blood group section.
Page 122 of 336

MICROANGIOPATHIC HAEMOLYTIC ANAEMIA
The term ‘microangiopathic” describes intravascular destruction of the red cells in the
presence of abnormal microcirculation. The common feature is red cell
fragmentation.
Mechanical defects
Abnormal surfaces like dysfunctional artificial heart valves or arterial grafts may
physically “cut” the red cells while passing through and cause fragments.
Long distance runners may damage the red cells between the small bones of the feet,
which can cause haemolysis, BUT no fragments are seen on the blood film. This is
known as March haemoglobinuria.
Disseminated intravascular coagulation (DIC)

The deposition of fibrin strands in the small vessels, causes destruction while the red
cells pass through. DIC will be discussed in the Coagulation section.
Thrombotic Thrombocytopenic Purpura (TTP)

TTP can be acquired or familial. The problem with patients with TTP is that there is a
deficiency of a ADAMTS13 metalloprotease (enzyme) which breaks down the ultra
large von Willebrand factor multimers (ULVWF). Platelets form aggregates on the
ULVWF multimeric strings leading to large, occlusive platelet thrombi and consequently
thrombocytopaenia.
http://www.archemix.com/website/products_1779_ttp.php (accessed 14/10/11)
In the familial forms there are several mutations of ADAMTS13 and in the acquired
forms an inhibitory IgG auto-antibody develops, which may be stimulated by infection,
auto-immune disease, certain drugs, or cardiac surgery.
Page 123 of 336

The platelet thrombi cause organ ischaemia. Traditionally TTP was recognized by a
pentad of thrombocytopaenia, red cell fragmentation, neurologic abnormalities,
renal failure and fever.
TTP is now most commonly associated with HIV-infection.
Haemolytic uraemic syndrome (HUS)

HUS resembles TTP, but mainly affects children and organ damage is limited to kidneys.
It is preceded by bloody diarrhoea caused by E.coli 0157 or Shigella infection.
ADAMTS13 levels are normal in HUS.
Laboratory diagnosis of microangiopathic haemolysis

 Hb low  Red cell fragments  DAT negative
 Retic high  Bili’s high
 Platelet low  LDH very high
 Haemoglobinuria
 Coagulation tests –
Normal in TTP NB
Prolonged in DIC
 ADAMTS13
absent/low in TTP
 Clinical info – bloody
diarrhoea, prosthetic
heart valve etc.
accessmedicine.net (accessed 14/10/11)
Page 124 of 336

OTHER HAEMOLYTIC ANAEMIAS
Infections
Meningococcal or pnemococcal septicaemias cause haemolysis. Clostridium
perfringens septicaemia cause intravascular haemolysis with marked
microspherocytes. Malarial haemolysis can occur directly intravascular or
extravascular. Falciparum malaria can cause an acute attack accompanied by
renal failure which is called Blackwater fever.
Chemical and physical

Certain drugs in high doses cause oxidative intravascular haemolysis with Heinz body
formation. Chemical poisoning (e.g. with lead, chlorate or arsine) can cause severe
haemolyis.
Burns will damage red cells causing acanthocytes or spherocytes.
Paroxysmal nocturnal haemoglobinuria (PNH)

Sometimes refer to as Marchiafava-Micheli Syndrome.
PNH is a rare example of acquired haemolysis caused by an intrinsic red cell defect.
GPI-linked proteins (CD55 and CD59) are absent from the cell surface of all cells
(including red cells, white cells and platelets). The cells are sensitive to lysis by
complement and the result is chronic intravascular haemolyis.
Haemosiderinuria is a constant feature which may give rise to an iron deficiency.

Pancytopaenia is a common finding. The patient may develop recurrent thromboses
of large veins.
Laboratory diagnosis of other haemolytic anaemias

 Hb low  No specific  DAT negative
 Retic high characteristic  Clinical info
 PNH –  Malaria parasites  PNH – flow cytometry
pancytopaenia (Hb may be seen in the for CD55 and CD59
low, neutrophils low red cells (absent)
and platelets low)  Ham’s test for PNH
Page 125 of 336

SUMMARY
 Immune haemolytic anaemias are characterized by a positive DAT

test.
 Warm AIHA are associated with IgG antibody and cold AIHA with
IgM and are characterized by spherocytes or agglutination.
 Iso-immune or allo-immune are associated with transfusion
reactions or haemolytic disease of the newborn.
 Microangiopathic haemolysis is associated with red cell fragments.
 PNH is a rare example of acquired haemolysis caused by an
intrinsic red cell defect.
Page 126 of 336

APLASTIC ANAEMIA
[NOTE: Aplastic anaemia is not an anaemia as such, but rather forms part of bone
marrow failure. Because the name causes confusion, it will be discussed in this section.]
Aplastic anaemia is a condition where the bone marrow does not produce sufficient
new cells and will lead to a pancytopaenia. Pancytopaenia describes a reduction in all
the major cell lines – red cells (more specifically haemoglobin), white cells (more
specifically neutrophils) and platelets. This condition needs to be differentiated from
pure red cell aplasia, characterized by reduction in red cells only.
It is classified intro primary (congenital or acquired) or secondary types
Primary Secondary
Congenital (Fanconi and non-Fanconi Ionizing radiation
types)  Accidental exposure (radiotherapy,
Idiopathic acquired radioactive isotypes)
Chemical
 Benzene, organophosphate, DDT
and other pesticides,
organochlorines, recreational drugs
(ecstasy)
Drugs
 BM depression – busulfan,
cyclophosphamide,
 Rarely BM depression –
chloramphenicol, sulphonamides,
gold, anti-inflammatory, antithyroid,
psychotrophic,
anticonvulsant/antidepressant
Viruses
 Viral hepatitis (non-A,B,C) EBV, CMV,
Parvovirus
The underlying cause is the reduction of haemopoietic stem cells, a fault in the
remaining stem cells or an immune reaction against them, which leaves them unable
to divide and differentiate sufficiently to populate the bone marrow.
Fanconi anaemia (FA)
FA is a genetic disease and is often associated with growth retardation and defects of
the skeleton (absent radii or thumbs), short stature and abnormalities of skin, arms,
head, eyes, kidneys and ears.
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The usual age of presentation is 5 – 10 years.
Short stature
Absent thumbs
www.emedicine.medscape.com (accessed 20/10/11)
It is an autosomal recessive genetic disorder, meaning that two genes (one from each
parent) are required to cause the disease.
http://en.wikipedia.org/wiki/Fanconi_anemia
FA affects the DNA repair and therefore patients are more likely to develop acute
myeloid leukaemia (AML), bone marrow failure and myelodysplastic syndromes (MDS).
It is relatively common in South African black and Afrikaans people.
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Idiopathic acquired
This is the most common type of aplastic anaemia.
Secondary
This is often caused by direct damage to the bone marrow by radiation or cytotoxic
drugs.
Signs and symptoms are related to anaemia, neutropaenia and thrombocytopaenia.
Red cell aplasia
This is a rare syndrome which is characterized by reduced or absent erythroblasts in

the bone marrow. The congenital form is known as Diamond-Blackfan syndrome.
Acquired forms can be seen as idiopathic or with autoimmune diseases (especially
SLE), with a thymoma, lymphoma or chronic lymphocytic leukaemia (CLL).
Parvovirus B19 can cause a transient aplasia with a rapid onset of severe anaemia. It
can last for 5 – 10 days.
Laboratory diagnosis of other aplastic anaemia

 Hb low  No specific  Bone marrow evaluation –
 Neutrophils low characteristic hypoplastic with
 Platelets low replacement of fat
 Retic low  Clinical info
 Viral studies
Diagnostic features: Any  Rule out PNH – flow
cytopaenia and low cytometry for CD55 and
reticulocyte count CD59 (absent)
 Fanconi - chromosome
breakage by DEB
(Diepoxybutane)
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HYPOPROLIFERATIVE ANAEMIAS
Anaemia occurs due to a relative or absolute decreased production of erythropoietin

(EPO) or the inability of the bone marrow to respond to erythropoietin.
It is a common mechanism in anaemias of renal disease, endocrine deficiency states
(e.g. hypothyroidism, hypopituitarism) and protein deprivation.
Anaemia of renal disease
There is defective EPO secretion and the result is anaemia. In severe uraemia
(increased urea levels) the red cells show abnormalities like burr cells (echinocytes).
Red cell fragmentation (when kidney endothelium is injured) can also occur, but is less
common.
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ANAEMIA OF PREGNANCY
During pregnancy the blood plasma volume increases which leads to a fall in the
haemoglobin concentration – termed physiological anaemia.
Values below 10 g/dl probably need further investigation.
Iron needs increase during pregnancy; therefore a fall in the mean cell volume (MCV)
can be an early sign of iron deficiency.
The MCV typically rises by approximately 4 fl during uncomplicated pregnancy.
Folate requirements increase two-fold during pregnancy. In some areas a

combination of poor diet and increased need may lead to megaloblastic anaemia.
SUMMARY
 Aplastic anaemia is characterized by pancytopaenia (anaemia,

neutropaenia and thrombocytopaenia).
 Fanconi anaemia is genetic disorder characterized by skeletal
abnormalities
 Hypoproliferative anaemias occur due to decrease EPO
production
 A relative anaemia in pregnancy is normal, unless values are lower
than 10 g/dl.
Page 131 of 336

WORKBOOK
Question 1
A 59-year-old female with a history of aplastic anaemia is admitted to the hospital. On

admission her FBC results were as follows:
WBC 5,5 X10^9/l
RBC 2,87 X 10^12/l
HCT 0.252 l/l
HGB 9.3 g/dl
PLT 94 X10^9/l
The doctor requests a reticulocyte count and as per the practice in the laboratory two
staff count 500 red cells each.
The results are as follows:
Slide 1- 5 reticulocytes per 500 cells
Slide 2 – 3 reticulocytes per 500 cells
Showing all calculations:

a) Calculate the uncorrected reticulocyte percentage
b) Calculate the corrected reticulocyte percentage (the figure of 0.45 is taken
as the normal haematocrit)
c) Calculate the reticulocyte production index (maturation index for
haematocrit 0.25l/l is 2 days)
d) Briefly discuss the reticulocyte results and if these results are to be expected
for this clinical diagnosis.
e) Discuss four causes of aplastic anaemia
Question 2
Name three laboratory factors that affect the results of the osmotic fragility tests.
Question 3
Haemolytic anaemia may be classified into groups based on the defect causing
haemolysis e.g. hereditary (congenital), acquired, intrinsic or extrinsic.
a) Explain what is meant by congenital intrinsic haemolytic anaemia.

b) List 2 conditions related to defective red cell metabolism that can be
classified as a congenital intrinsic haemolytic anaemia.
c) Name one condition that can be classified as an acquired intrinsic
haemolytic anaemia and give one confirmatory test for this condition.
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d) State 2 conditions usually associated with Microangiopathic haemolytic
anaemia.
e) Give 2 causes of warm AIHA.
Question 4
A direct Coombs was performed when investigating a case of auto-immune

haemolytic anaemia (AIHA). Under which category of haemolytic anaemias would
the case be place if:
a) The DAT was positive

b) The DAT was positive with the presence of IgG
c) The DAT was positive and the patient recently received a blood transfusion
Question 5
HbH is associated with ____ Thalassaemia.
Question 6
Fill in the missing word.

a) Sickle cell disease is associated with an increase in Hb____
b) Non-haemoglobin iron is deposited in the red cells causing inclusion bodies
named__________________
c) The normal range for a platelet count is 150 – 450 ____ (supply the unit)
d) __________ is the most common cause of an iron deficiency.
e) Sideroblastic anaemia is diagnosed by ___ _____ in the bone marrow
Question 7
The following red cell indices were obtained from individuals:
Parameter Patient A Patient B

Hb (g/dl) 8.4 7.6
RCC (1012/l) 2.99 5.5
Hct (%) 32 34
MCV (fl) 67 71
MCH (pg) 20.4 21.3
MCHC (g/dl) 30.2 30.4
a) Name possible disease(s) for both patients.

b) Name one confirmatory test per patient and indicate if the result will be
high/low/present/absent etc.
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c) What poikilocyte will be striking in each patient?
Question 8
Draw a labelled histogram showing a dimorphic red cell population and give 3
possible causes.
Question 9
Define the term pancytopaenia and explain why this is a common finding with
megaloblastic anaemia. (4)
Question 10
Write short notes on pernicious anaemia. (6)
Page 134 of 336

WHITE CELLS
Learning Unit 5
WHITE CELLS
Learning Unit 5 provides you with the underpinning knowledge, and

terminology related to white blood cells.
Assessment Criteria
learning unit five:
1.1 Understand the clinical implications of abnormal values.
1.2 Identify inclusion bodies.
1.3 Define terminology and identify abnormal white cell morphology.
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Apoptosis Programmed cell death, which results in the removal of old
cells
Anomaly Any deviation from the normal
Syndrome A combination of signs and/or symptoms that forms a
distinct clinical picture indicative of a particular disorder
Immunity To have sufficient biological defences against infection,
disease or other unwanted invasion
The white blood cells are functionally divided into

 Phagocytes and
 Immunocytes
The phagocytes specialize in the destruction of foreign matter. They can destroy
tumour cells and ingest apoptotic cells or micro-organisms. They marginate to the
endothelium and migrate through to the tissues. During infection, they are rapidly
recruited to kill invading pathogens.
There are two classes of phagocytic cells:

 the polymorphnuclear granulocytes and
 the mononuclear phagocytes
The polymorphnuclear phagocytes have multilobed nuclei and are divided into
neutrophils, eosinophils and basophils.
The mononuclear phagocytes consist of monocytes and macrophages.
The lymphocytes make up the immunocyte population.
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White cells
Phagocytes Immunocytes
Polymorphnuclear Mononuclear Lymphocytes
Neutrophils Monocytes
Eosinophils Macrophages
Basophils
PHAGOCYTES
NEUTROPHILS
The neutrophils are the most numerous of the phagocytes and constitute about 65% of
circulating leucocytes in man. Their life span is short (12-24 hours) after which they
enter the tissues and die after one or two days. They are easily recognized by their
nucleus with 2 – 5 lobes, pale cytoplasm and many granules. These granules are
bactericidal proteins which are released to help with digesting and killing. Primary
(azurophilic) granules appear at promyelocyte stage and secondary (specific)
granules appear at myelocyte stage up to mature neutrophil.
Primary granules contain mainly myeloperoxidase, acid phosphatase and other acid
hydrolases, whereas the secondary granules contain collagenase, lactoferrin and
lysozyme.
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http://classes.midlandstech.edu/carterp/Courses/bio225/chap16/sld006.htm
A raised white cell count (leucocytosis) is often due to elevation of a single lineage.
Elevation of the minor cell population can occur without a rise in the total white cell
count.
Causes of
Neutrophil leucocytosis Neutropaenia
 Infection (mainly bacterial)  Racial
 Inflammation, including auto-  Congenital (Kostmann’s syndrome)

 Cyclical neutropaenia
immune disorders, tissue necrosis
 Marrow aplasia, PNH
and gout  Marrow infiltration
 Metabolic conditions including  Megaloblastic anaemia
 Acute infections like typhoid, miliary
uraemia, acidosis, eclampsia,
TB, protozoal, viral hepatitis
haemorrhage/haemolysis  Drugs
 Corticosteroids  Immune e.g. idiopathic, drugs, HIV,
Felty’s syndrome, SLE
 Marrow infiltration/fibrosis  Hypersplenism
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 Myeloproliferative disorders
Neutrophil morphology
Normal neutrophil
A normal neutrophil has 2-5 lobes with granules.
Hypersegmented neutrophil
Hypersegmentation occur in megaloblastic

anaemia and sometimes in infections
Pelger-Huet neutrophil
This is a bilobed or occasional unsegmented

neutrophil.
Pelger-Huet anomaly is an uncommon
autosomal, recessive condition. These
neutrophils can also be found in Myelodysplasia
Hypogranulated
There is reduced granulation which can be

found in Myelodysplasia.
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Toxic granulation
Increased or toxic granulation may be found in

infections, normal pregnancy or with cytokine
therapy.
Chediak-Higashi syndrome
This is a rare autosomal recessive condition with

abnormally large leukocyte granules resulting
from fusion of lysozymes
Alder-Reilly anomaly
Dense azurophilic granules, resembling toxic

granulation in neutrophils, are seen in leukocytes.
May-Hegglin anomaly
May-Hegglin anomaly is associated with a

thrombocytopaenia, large/giant platelets and
large inclusions resembling Döhle bodies. These
inclusions are spindle-shaped and, randomly
distributed in the cell.
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Döhle bodies
They are small, pale-blue or blue-grey

cytoplasmic inclusions, found towards the
periphery of the cell. They are associated with
infection, inflammation, pregnancy, burns,
chemotherapy, myelodysplasia and in AML.
Auer rods
These clumps of azurophilic granular material

form elongated needles in the cytoplasm.
Auer rods can be seen in the leukaemic blasts of

acute myeloid leukaemia.
Barr body or drumstick
These drumsticks represent the inactive X

chromosome in females.
Vacuolation of the neutrophil
This is usually a feature of severe infection or

following ethanol ingestion.
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Neutrophil with ring-shaped (doughnut) nucleus
These are seen in the myelodysplastic

syndromes.
(Note the trypanosome next to the neutrophil.)
Neutrophil with detached nuclear fragments
Seen in patients with HIV infection,

myelodysplastic syndromes, chemotherapy, etc.
EOSINOPHILS
These cells usually have a bilobed nucleus with large, red granules that pack the
cytoplasm.
Causes of
Eosinophilia Low eosinophil count
 Allergic reactions  No clinical significance

 Parasitic diseases
 Recovery from acute infections
 Certain skin diseases e.g. dermatitis
 Drug sensitivity
Eosinophil morphology
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Normal eosinophil
A normal eosinophil has 2 lobes with red

granules.
BASOPHILS
The nucleus is usually obscured by purple-black granules, which contain heparin and
histamine. Granules can be reduced in numbers in myeloproliferative disorders and in
myelodysplastic syndromes. Mast cells are closely related, but predominate in tissue.
Causes of
Basophilia Low basophil count
 Uncommon  No clinical significance
 Associated with myeloproliferative

disorder
Basophil morphology
Normal basophil
A normal basophil with purple-black granules

that obscure the nucleus
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MONOCYTES
The monocyte is the largest normal peripheral blood cell. They circulate for 20-40
hours; leave the blood to carry out their main functions in the tissues as macrophages.
It has an irregular, often lobulated nucleus and opaque greyish-blue cytoplasm with
fine granules.
Causes of
Monocytosis Monocytopaenia
 Chronic bacterial infections e.g. TB,  Hairy cell leukaemia
brucellosis, typhoid
 SLE, RA
 Hodgkin’s disease
 Myelodysplasia
Monocyte morphology
Normal monocyte
A normal monocyte with a lobulated nucleus

and blue-grey cytoplasm
Monocyte with vacuoles
This is also a normal monocyte with vacuoles in

the cytoplasm
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IMMUNOCYTES
LYMPHOCYTES
The lymphocytes assist the phagocytes in their defence of the body. The bone
marrow and thymus are the primary organs in which lymphocytes develop. The
secondary lymphoid organs in which the specific immune responses are generated
are the lymph nodes, spleen and lymphoid tissues. The immune response depend on
two types of lymphocytes, B and T cells.
B cells develop in the bone marrow, exit the marrow as immature lymphocytes and
circulate in the peripheral blood to the lymph nodes and spleen. In these organs
maturation through antigen presentation into either memory B cells or plasma cells
takes place. Plasma cells reside in the bone marrow and memory B-cells in
lymphoid/other tissue for many years to form an integral part of the immune system.
They comprise about 20% of lymphocytes.
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T cells forms about 80% of lymphocytes and mature during passage from thymus
cortex to the medulla. The natural helper cells express CD4 and suppressor cells
express CD8.
T-helper cells – CD4

T-suppressor cells – CD8
http://www.biologie.uni-hamburg.de/b-online/library/bio201/tcellmat.html
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Natural killer (NK) cells are cytotoxic CD8+ cells that lack the T-cell receptor (TCR). NK
cells play a role in the rejection of tumour or virally infected cells. The granules are
released when in close proximity to the target cells and enter through pores in the cell
membrane to cause apoptosis.
http://arthritis-research.com/content/6/1/8/figure/F1
Immunity
Innate immunity is the defence mechanisms that we are born with e.g. skin and
mucous membranes. Adaptive immunity can either be natural or artificially acquired.
Natural are through disease whereas artificially acquired may be through
immunization. Passive immunity (e.g. from mother to baby) is usually short-lived,
whereas active immunity is induced in the host itself by an antigen.
A further subdivision of adaptive immunity is characterized by the cells involved;

humoral immunity is the aspect of immunity that is mediated by secreted antibodies,
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whereas the protection provided by cell mediated immunity involves T-lymphocytes
alone.
http://www.lionden.com/ap2out-lymph.htm
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Immunoglobulins
Immunoglobulins (antibodies) are proteins produced by plasma cells and B

lymphocytes. They are divided into five subclasses.
IgG: Small, most common, can fix complement

and can cross the placenta, long-term immunity.
IgM: First response to antigen, large (pentamer),
can fix complement and do not cross the
placenta.
IgA: Mainly found in secretions, cannot cross
placenta.
IgE: Involved in hypersensitivity reactions
IgD: Involved in hypersensitivity reactions
Fab fragment
http://pathmicro.med.sc.edu/mayer/igstruct2000.htm
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Causes of
Lymphocytosis Lymphocytopaenia
 Viral infections (EBV, rubella,  Bone marrow failure

 Immunosuppressive therapy
pertussis, mumps etc.)  Hodgkin’s disease
 Chronic infections e.g. TB, syphilis  HIV
 Chronic lymphoid leukaemias  Severe combined immune-
deficiency (SCID)
 Acute lymphoid leukaemias
 Non-Hodgkin’s lymphoma
 Thyrotoxicosis
* *
* Please note that a transient stress related condition e.g. myocardial infarction,
trauma, surgery etc. can cause either a lymphocytosis or a lymphocytopaenia.
Lymphocyte morphology
Normal lymphocytes
Small lymphocyte and large lymphocytes
A normal lymphocyte with scanty cytoplasm
This is also a normal lymphocyte with slightly

more cytoplasm
Large granular lymphocyte (NK cell)
A large cell with abundant cytoplasm and

prominent granules
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Activated lymphocytes
Activated (or reactive) lymphocytes – viral
infection
Reactive changes are seen during a viral

infection
EBV
Reactive changes seen during glandular fever

Monocytoid lymphocyte
Plasmacytoid lymphocyte
Immunoblast
Both T and B cells can transform into

immunoblasts, large cells with a prominent
nucleolus and basophilic cytoplasm
Plasma cells
Plasma cell
B lymphocytes can transform into plasma cells.

These are not commonly seen in the peripheral
blood, but are associated with myelomas or
plasma cell leukaemias or can be reactive to
infections e.g. HIV.
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Plasmacytoid lymphocyte (Turk cell)
These are intermediate stages of B lymphocytes

that transforms in plasma cells.
Not commonly seen in peripheral blood, reside in

the bone marrow.
Mott cell
Plasma cells with abundant globular inclusions

(Russell bodies) composed of immunoglobulin
are called Mott cells, morular cells or grape cells.
These are seen in the bone marrow of patients
with multiple myeloma.
Flame cell
A rare abnormal variant of a plasma cell called

a flame cell is shown here. Not the red tinged
area at the periphery. These cells contain more
immunoglobulin than normal plasma cells and
are seen in bone marrow.
Malignant lymphocytes
Hairy cell
Typical hairy cells with pale grey/blue cytoplasm

with an irregular edge seen with hairy cell
leukaemia
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Sezary cell
Typical morphology with deep nuclear clefting,

similar to ATLL cells.
Smudge cell
Smudge (or smear) cells are seen with CLL. Note

the many mature-looking small lymphocytes.
SUMMARY
 White cells are divided into phagocytes and immunocytes.

 Phagocytes include granulocytes and monocytes
 Immunocytes include the lymphocytes
 The differences in morphology and inclusions can help with the
diagnosis of an underlying disorder.
Page 153 of 336

WORKBOOK
Question 1
In what line of leucocytes will the large abnormal granules of Chediak-Higashi show?
Question 2
Discuss conditions where Döhle bodies might be seen.
Question 3
Prolonged storage of EDTA blood before making a slide can result in artefacts. Discuss
the changes seen in the white blood cells.
Question 4
Discuss the morphological changes as seen with bacterial as well as viral infection.
Question 5
Match the following white blood cells with the relevant function
Column A Column B (Correct Column C

Answer)
1. Neutrophilia a. Allergy
2. Eosinophilia b. HIV
3. Monocytopaenia c. Tissue damage
4. Lymphopenia d. Chonic Myeloid
Leukaemia
5. Basophilia e. Hairy Cell Leukaemia
f. Bacterial Sepsis
Page 154 of 336

ACUTE LEUKAEMIAS
Learning Unit 6
ACUTE LEUKAEMIAS

terminology related to acute leukaemias.
Assessment Criteria
learning unit six:
1.1 Demonstrate knowledge of the FAB and WHO classifications.
1.2 Identify the relevant morphological features associated with myeloid
and lymphoid leukaemias.
1.3 Differentiate between the leukaemias by use of special stains, flow
cytometry or cytogenetics.
Page 155 of 336

Acute Disease of rapid onset, severe symptoms, short duration
Leukaemia Accumulation of malignant white cells in blood and bone
marrow
De Novo Arising spontaneously or anew
Acute leukaemias are usually aggressive diseases in which the malignancy lies in the
haemopoietic stem cells (blasts) or early precursors.
Acute leukaemia is defined by the presence of more than 20% blasts (according to
World Health Organization –WHO) in the blood or bone marrow.
Aetiology
Acquired chromosomal changes as well as some other predisposing factors were

identified.
 Chromosomal translocation (t) – one chromosome breaks and donates a piece
to another chromosome. Philadelphia chromosome is a classic example where
breakages of chromosome 9 and 22 result in a new fusion gene.
 Chromosome deletion (del) or addition – a chromosome is completely or partly
deleted e.g. monosomy 7 in acute myeloid leukaemia (AML). An example of
an additional chromosome is trisomy 8 in AML.
 Point mutations – a change in the base sequence of certain oncogenes.
 Radiation exposure
 Previous chemotherapy
 Benzene exposure
 Down’s syndrome
 Viral infection (HTLV-1)
Each chromosome has two arms: the shorter one is called “p” and the longer one “q”.
When a whole chromosome is lost or gained, a “-“ or “+” is put in front of the
chromosome number. When a part is lost it is prefixed with a “del”. “inv” describes an
inversion where part of the chromosome has been inverted to run in the opposite
direction.
Page 156 of 336

CLASSIFICATION
Acute leukaemias can be further subdivided into myeloid or lymphoid origin. The
traditional classification was based on morphological appearance of the blasts and
was called FAB classification – French, American and British.
Molecular cytogenetic abnormalities are now being incorporated into the
classification as they have prognostic significance. The newer classification is the
World Health Organization (WHO)’s classification.
Acute Lymphoblastic Leukaemia (ALL) Acute Myeloid Leukaemia (AML)

Morphology Morphology
 Blasts show no differentiation  Some differentiate to granulocytes
or monocytes
 Auer rods/granules may be present
Cytochemistry (special stains) Cytochemistry (special stains)

 Myeloperoxidase (MPO) negative  MPO positive
 Sudan black negative
 Non-specific esterase (NSE)
negative
 Periodic Acid-Schiff positive
(coarse block)
 Sudan black positive
Auer rod
 NSE positive for M4, M5 (monocyte)

 PAS positive in M6
Page 157 of 336

ALL (cont) AML (cont)
Flow cytometry Flow cytometry
 CD 19 + (B cell)  CD13 +
 cCD 79a + (B cell)  CD 33 +
 CD7 + (T cell)
 cCD3 + (T cell)
Genetic abnormalities Genetic abnormalities

 11q23 (poor prognosis)  M2: t(8;21), t(6;9) – sharp needled-
 t(9;22) (poor prognosis) shaped Auer rods
 M3: t(15;17)
 M4: inv(16), del(16q) – better
prognosis
Flow cytometry
Flow cytometry is a technology that allows a single cell to be measured for a variety of
characteristics, determined by looking at how they flow in liquid. Instruments used for
this can gather information about cells by measuring visible and fluorescent light
emissions, allowing cell sorting based on physical, biochemical and antigenic traits.
In a flow cytometer, single cells move past the excitation source and the light hitting
the cells is either scattered or absorbed and then re-emitted (fluorescence). This
scattered or re-emitted light is collected by the detector.
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Clusters of differentiation Specificity within haemopoietic lineage
CD34 Blasts – B lymphoblasts, early T precursors,
myeloid precursors, blasts in AML and
undifferentiated acute leukaemia
CD45 Common leucocyte antigen
CD10 Common ALL antigen
When an acute leukaemia is expected, a panel of antibodies is requested.

Depending on the results of the first panel, a second panel will be requested.
First panel
B lymphoid CD19, cCD22, cd79a, CD10
T lymphoid cCD3, CD2, CD7
Myeloid Anti-MPO, CD13, CD33, CD65, CD117
Non-lineage specific TdT, CD34, HLA-DR
TdT: terminal deoxynucleotidyl transferase

c: cytoplasmic
HLA-DR: major histocompatibility complex
Page 159 of 336

ACUTE LYMPHOBLASTIC LEUKAEMIA
As the name implies, ALL is a clonal malignancy of lymphoid precursor cells. In 80% of
the cases the precursors are B-lymphocytes.
ALL is the most common leukaemia in children – about 80% of ALL cases occur in
children between 2 – 10 years. Childhood ALL can often be cured, whereas adult’s
prognosis is not as good.
Clinical features of ALL
These are related to bone marrow failure and organ infiltration.
 Bone marrow failure

Anaemia (pallor, fatigue etc)
Neutropaenia (infections)
Thrombocytopaenia (bruises, purpura, bleeding)
 Organ infiltration
Tender bones
Lymphadenopathy
Splenomegaly
Hepatomegaly
Meningeal symptoms (headache, nausea, vomiting, blurred vision)
Testicular swelling
FAB classification
L1 L2 L3
Best prognosis, most Most frequent in adults Worst prognosis
common ALL in
children
Cell size Small Large, heterogeneous Large, homogeneous
Nuclear Smooth, occasional Irregular, clefts and Smooth, oval-round

shape cleft indents
Nucleoli Invisible-inconspicuous 1 or more prominent 1 or more prominent
Cytoplasm Scanty, slight More, sometimes Abundant, deep

basophilic deep basophilic basophilic, often
vacuolization
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Laboratory diagnosis of ALL

 Hb low  Normochromic,  Bone marrow
 WCC low, normal or normocytic anaemia evaluation
high (>200x109/l)  Blasts (no Auer rods  Lumbar puncture for
 Neutrophils low or granules) CNS involvement
 Platelets low  Cytochemistry
 Flow cytometry
 Uric acid – raised
L1  LDH – raised
L2
Note the vacuoles

L3
(Burkitt
type)
En.wikipedia.org (accessed 21/10/11)

http://www.med-ed.virginia.edu/courses/path/innes/wcd/lymphoid.cfm
Page 161 of 336

Treatment
Treatment includes supportive therapy (e.g. red blood cell and platelet transfusion)
and specific (chemo- and sometimes radiotherapy).
Chemotherapy consists of remission induction (to kill tumour cells and get patient into
remission), intensification (to completely reduce tumour burden), CNS directed
therapy (to prevent CNS disease) and finally maintenance which can extend over 3
years.
Stem cell transplant is reserved for poor prognosis as well as relapsed disease.
Page 162 of 336

ACUTE MYELOID LEUKAEMIA
AML arises out of malignant transformation of myeloid precursor cells. The clone that
develops depends on the level of maturation where the genetic abnormality occurs
e.g. promyelocytic leukaemia (AML M3).
AML occurs in all age groups, but the incidence increase with age.
AML may occur de novo or secondary to previous chemotherapy, myelodysplasia or

myeloproliferative disorders – such cases are referred to as ‘secondary AML’.
Clinical features of AML
It resembles ALL – bone marrow infiltration leads to symptomatic anaemia,

neutropaenia and thrombocytopaenia.
 AML FAB M3 (acute promyelocytic leukaemia) is a medical emergency

because patients are likely to develop disseminated intravascular coagulation
(DIC) with disordered clotting tests and a high risk of spontaneous bleeding into
vital organs.
 AML FAB M5 (acute monocytic leukaemia) has a particular tendency to invade

soft tissue e.g. gums, skin.
FAB classification
FAB Name Criteria Cytogenetics

M0 Undifferentiated
M1 Without maturation 30% or more myeloblasts

3% stain positive for MPO or
Sudan Black
May show Auer rods
M2 With maturation 30% or more myeloblasts t(8;21)

Positive for MPO or Sudan
Black
May show Auer rods
M3 Promyelocytic Abnormal, hypergranular t(15;17)

promyelocytes
Auer rods
Faggot cells
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Granules contain
procoagulants (result in DIC)
M4 Myelomonocytic Granulocytes with a del/inv 16q

monocytic component (eosinophlic
MPO+, ANAE+, component)
t/del 11q
M5 Monoblastic (5a) 30% are monoblasts,
Monocytic (5b) promonocytes or monocytes
ANAE+
M6 Erythroleukaemia 50% or more of all nucleated

marrow cells are erythroid
precursors
M7 Megakaryoblastic 30% or more cells in marrow

are of megakaryocytic
lineage
Pleomorphic blasts
WHO classification
Acute myeloid leukaemia (AML) and related precursor neplasms

AML with recurrent genetic abnormalities e.g. t(15;17), inv(16), t(8;21) etc.
AML with meylodysplasia-related changes (or history of MDS)
Therapy-related myeloid neoplasms (previous chemo- or radiotherapy)
Acute myeloid leukaemia, Not Otherwise Specified

 AML with minimal differentiation
 AML without maturation
 Acute myelomonocytic leukaemia
 Acute monoblastic and monocytic leukaemia As in FAB
 Acute erythroid leukaemia
 Acute megakaryoblastic leukaemia
 Acute basophilic leukaemia
 Acute panmyelosis with myelofibrosis
Myeloid sarcoma (soft tissue tumour consisting of myeloid blasts)
Myeloid proliferations related to Down syndrome
Page 164 of 336

The biggest change is with the inclusion of the genetic abnormalities as well as the
myelodysplasia-related changes. Note that the WHO classifies an acute leukaemia
when there is more than 20% blasts.
Laboratory diagnosis of AML

 Hb low  Normochromic,  Bone marrow
 WCC low, normal or normocytic anaemia evaluation
high (>200x109/l)  Blasts  Cytochemistry
 Neutrophils low  Auer rods or granules  Flow cytometry
 Platelets low if differentiated  Tests for DIC
 Faggot cells in case  Uric acid – raised
of M3  LDH – raised
 See flow chart
Nucleoli
Auer rods
Page 165 of 336

Flow chart
Are the criteria for AML met?
YES NO
AML MDS or another condition
Is there a history of prior

exposure to chemo- or
radiotherapy?
NO
YES
Therapy-related AML Does the patient have t(8;21),
inv(16), t(16;16), t(15;17),
cytological features of M3 or
11q23 rearrangement
YES NO
AML with recurrent genetic Does the patient have

abnormality multilineage dysplasia
NO
YES
AML with multilineage AML not otherwise
dysplasia specified
Treatment
Treatment consists of supportive and specific (chemo) therapy. In addition, AML M3 is
treated with ATRA (all-trans retinoic acid). No CNS prophylaxis is needed.
SUMMARY
 ALL occur mostly in children and AML in all ages, increasing with
age
 ALL is undifferentiated whereas AML can differentiate
 M3 variant is a medical emergency because a life-threatening DIC
can develop
 Bundles of Auer rods are called faggot cells and are a definitive
finding in AML M3.
 WHO differs from FAB in that more than 20% blasts are now
classified as an acute leukaemia and cytogenetics are added.
Page 166 of 336

WORKBOOK
Question 1
A 22 year old woman is referred to the haematology clinic with recurrent infection. A
blood specimen was sent to the laboratory for full blood count (FBC) and white cell
differential. The phlebotomist noticed several bruises on her arm. The FBC results were
as follows:
White cell count: 22.4 X 109/l

Red cell count: 3.0 X 1012/l
Haematocrit: 24.2 %
Haemoglobin: 7.8 g/dl
Mean cell volume: 81.0 fl
Platelet count 42 x 109/l
The following cells, which made up 85% of the differential count, were seen on the
blood film:
1. Calculate the mean cell haemoglobin concentration (MCHC).

2. Name the cell indicated by the arrow.
Page 167 of 336

3. Attempt a diagnosis and describe the morphological features which support your
answer.
4. Name two cytochemical stains that can be performed to confirm the diagnosis
and indicate whether they would be positive or negative.
5. Into which World Health Organization category is this malignancy classified?
6. Which acquired coagulation disorder is associated with this condition?
7. Give the screening tests and the expected results for the disorder mentioned in 6.
Question 2
Match the statement given in column A to the description given in column B. Write
down the number and the corresponding letter of the alphabet.
Column A Column B
2.1 Diagnostically useful in acute promyelocytic A FAB M7
leukaemia
2.2 Blasts show block positivity in acute B Periodic Acid-Schiff
lymphoblastic leukaemia (ALL)
2.3 Its main diagnostic use is in the diagnosis of C Auer rods
hairy cell leukaemia
2.4 Negative reaction in acute lymphoblastic D Tartrate resistant acid
leukaemia (ALL) phosphatase
2.5 French American British (FAB) classification E FAB M6
of acute myeloid leukaemia where 20% or
more of the cells are megakaryoblasts.
F FAB M3
G Sudan Black
Page 168 of 336

MYELOPROLIFERATIVE NEOPLASMS
Learning Unit 7
MYELOPROLIFERATIVE NEOPLASMS

terminology related to the myeloproliferative disorders.
Assessment Criteria
learning unit seven:
different myeloproliferative disorders.
1.3 Differentiate between the disorders by use of special stains, flow
cytometry or cytogenetics:
o Chronic myeloid leukaemia
o Polycythaemia vera
o Essential thrombocythaemia
o Myelofibrosis
Page 169 of 336

Proliferative To grow or multiply by rapidly producing new tissue, cells
etc.
Myeloproliferative These include diseases in which there is excessive
disorders production of blood cells in the bone marrow.
Chronic A disease with gradual onset and long duration, involving
slow changes.
Hypercellular The presence of an abnormal excess of cells
Hypocellular Less than the normal number of cells
Neoplasm A new or abnormal growth, any benign or malignant
tumour
Mutation A change in the genetic material (DNA)of the cell
Translocation A part of the chromosome is transferred to another part of
the same chromosome or to a different chromosome
The myeloproliferative disorders consist of a group of diseases where there is abnormal

proliferation of one of the myeloid cell lines, all of which were derived from a common
haemopoietic cell.
The myeloproliferative disorders are divided into two subtypes: leukaemic and non-
leukaemic disorders. Leukaemic disorders include chronic myeloid leukaemia (CML),
chronic neutrophilic leukaemia and eosinophilic leukaemia. The non-leukaemic
myeloproliferative disorders include Polycythaemia Vera (PV), Essential
Thrombocythaemia and Myelofibrosis (MF).
These conditions are interrelated e.g. CML, PV and ET may transform into a
myelofibrotic phase or may terminate in a leukaemic phase.
Page 170 of 336

This is an overview of the haematologic malignancies. The myeloproliferative disorders
fit into the pink block.
http://alexandria.healthlibrary.ca/documents/notes/bom/unit_5/Unit%205%202005/Lec-
30%20Myeoproliferative%20disorders.xml (accessed 19/6/2012)
CLASSIFICATION
WHO classification
Myeloproliferative neoplasms
 Chronic myelogenous leukaemis, Philadelphia chromosome
 Chronic neutrophilic leukaemia
 Chronic eosinophilic leukaemia
 Chronic idiopathic myelofibrosis
 Polycythaemia vera
 Essential thrombocythaemia
 Myeloproliferative disease, unclassifiable
Page 171 of 336

FAB classification
Myeloproliferative disorders
 Chronic myelogenous leukaemis, Philadelphia chromosome +
 Chronic idiopathic myelofibrosis
 Polycythaemia vera
 Essential thrombocythaemia
Janus kinase 2
Haematopoietic
stem cell
http://www.nature.com/nrc/journal/v7/n9/fig_tab/nrc2210_F1.html (accessed 19/6/2012)
Page 172 of 336

CHRONIC MYELOID LEUKAEMIA
CML is a clonal disorder which occurs at any age, although it is rare below 20 years
with a median age of onset of 40 – 50 years.
The Philadelphia (Ph) chromosome is an acquired genetic abnormality that
characterizes leukaemic cells in CML. It results from a translocation between
chromosomes 9 and 22 and is referred to as t(9;22). It was shown that the ABL proto-
oncogene was normally located on chromosome 9 and was translocated to
chromosome 22 (bcr-abl). At the same time a switch of bcr occurs from chromosome
22 to chromosome 9.
http://www.mayoclinic.com/health/medical/IM03579
The alkaline phosphatase content of the neutrophil cytoplasm is reduced or absent,

therefore giving a low neutrophil alkaline phosphatase (NAP) score. Blast cells in the
chronic phase are usually between 2 – 10%.
Accelerated phase and blast transformation
During the accelerated phase, there is anaemia, thrombocytopaenia and an

increase in basophils, eosinophils or blasts in the blood and bone marrow. The patient
may be in this phase for several months.
Blastic transformation is defined as the presence of more than 20% blasts. The blasts
may be:
Myeloid (AML) (±70% - 80%)
Lymphoid (ALL) (±20%) or
Mixed phenotype (<10%)
Page 173 of 336

Clinical features of CML
 Symptoms related to hypermetabolism (weight loss, night sweats)
 Splenomegaly (frequently a massive spleen which is associated with pain and

discomfort)
 Anaemia (pallor, dyspnoea, tachycardia)
 Bruising, epistaxis or haemorrhage because of abnormal platelet function
 Gout or renal impairment due to hyperuricaemia from excessive purine

breakdown
Laboratory diagnosis of CML

 Leucocytosis (>50  Complete spectrum  Ph chromosome on
x109/L) of myeloid cells conventional
 Basophilia (neutrophils and cytogenetic, PCR or
 Hb low myelocytes FISH
 Plt high, normal or abundant)  Hypercellular bone
low  Basophilia marrow
 Neutrophil alkaline
phosphatase (NAP)
score low
http://dc338.4shared.com/doc/Y0AxlOa8/preview.html
Page 174 of 336

Karyotyping FISH (Fluoresensce in situ hybridization)
http://www.oncolink.org/types/section.cfm?c=8
http://www.ivfclinicindia.com/genetics/haematological_malignancies.asp
Difference between leukaemoid reaction and CML
Leukaemoid reaction CML

 Due to infection  Malignant proliferation
 Distribution from band cells  More neutrophils and
up to blasts myelocytes than the rest of
Band cells
immature cells
Blasts
Myelocytes
Bands
Blasts
 Toxic granulation of  No toxic granulation

neutrophils
 Döhle bodies may be  No Döhle bodies
present
 No basophilia  Basophilia and/or
eosinophilia
 NAP score increased  NAP score low
 Ph chromosome negative  Ph chromosome positive
Page 175 of 336

Treatment
The aim is to inhibit BCR-ABL fusion protein with imatinib (Gleevec). Long-term
remission can be achieved by loss of Ph chromosome. Stem cell transplantation is
reserved for imatinib failures.
Page 176 of 336

POLYCYTHAEMIA VERA
Polycythaemia Rubra Vera (PRV) or more commonly Polycythaemia Vera (PV) means
only a true red cell mass increase and refers to the clonal stem cell disorder. The red
cells, granulocytes and platelets are all derived from the abnormal clone. An increase
in red cells, haemoglobin and haematocrit is the result and characteristic of this
disease. The median age of presentation is 55 – 60 years.
A single acquired mutation, Janus associated kinase 2 (JAK2) is found in almost all
patients with PV. A unique valine to phenylalanine substitution at position 617 (V617F)
occurs in the JAK2 domain. This renders red cell progenitors hypersensitive to
erythropoietin (EPO) due to defective signal transmission.
The JAK-STAT pathway plays a

central role in initiating signal
transduction from the erythropoietin
(Epo) receptor. In most patients with
polycythemia vera (PV) the receptor
is activated constitutively (i.e. all the
time) due to lack of auto-inhibition
of the JAK2 enzyme, because of an
activating mutation involving a
nucleotide substitution of valine to
phenylalanine (V617F):
http://www.angiologist.com/general-medicine/janus-kinase-2-jak2/
Clinical features of PV
 Vascular complications – vascular occlusions and thrombosis
 Haemorrhage (due to acquired von Willebrand disease where the VWF

multimers adsorb onto the platelets in patients with thrombocytosis) –
gastrointestinal, uterine or cerebral. Peptic ulceration may occur.

 Neurological features – headaches, insomnia, memory loss depression, dizziness,
tinnitus (sensation of sounds in the ear) etc. These are due to cerebral
thrombosis or haemorrhages and/or increased blood viscosity.

 Plethoric appearance – rosy face
Page 177 of 336

 Pruritis (itching) – especially after a hot bath. This is probably related to
histamine levels associated with high basophil count

 Urate production – high uric acid levels may be due to high nuclear protein
turnover.

 Splenomegaly
Laboratory diagnosis of PV

 RCC, Hct and Hb  Red cells appear  JAK2 present
high normal, just  NAP score increased
 Neutrophil increased  Bone marrow is
leucocytosis in ½ of  Sometimes hypercellular
patients basophilia  S-EPO low
 Basophila (some)  Large platelets  Uric acid increased
 Thrombocytosis in ½
of patients
NOTE: Diagnosis is made after exclusion of secondary polycythaemia and with help
of JAK2.
Overview of increased RCC, Hb and Hct

Primary Secondary Relative
 Poly Vera  Caused by compensatory increase of  Dehydration
EPO in  Burns
o High altitudes
o Pulmonary disease
o Cardiovascular disease
o Heavy cigarette smoking
 Inappropriate EPO increase e.g. tumours
Page 178 of 336

Treatment
Treatment is aimed at maintaining normal blood count. Venesection will reduce the
haematocrit, but can result in an iron deficiency.
Transition to myelofibrosis occurs in approximately 30% of patients and 5% of patients

may progress to acute leukaemia.
Page 179 of 336

ESSENTIAL THROMBOCYTHAEMIA
Essential thrombocythaemia (or primary thrombocythaemia) is a clonal disorder

involving a multipotent stem cell. It is characterized by megakaryocytic proliferation in
the marrow and raised circulating platelet numbers. The Ph chromosome or BCR-ABL
rearrangement is absent. Half of patients show a positive JAK2 mutation.
ET is mostly discovered doing a routine FBC. Other causes of a persisting
thrombocytosis should first be excluded.
The mean age at presentation is 60 years.
Clinical features of ET
 Assymptomic – ½ of patients experience no symptoms
 Thrombosis
 Haemorrhage – due to abnormal platelet function
 Erythromelalgia (burning sensation felt in hands or feet) – relieved by aspirin
 Splenomegaly - <40% of patients have a palpable spleen
 Splenic atrophy – shrinking of spleen may be due to infarction
Laboratory diagnosis of ET

 Persistent  Large platelets may  Bone marrow shows
thrombocytosis be seen an increase of
abnormal
megakaryocytes
NOTE: Exclude other causes of thrombocytosis
Page 180 of 336

Causes of raised platelet count
Primary Reactive
 ET  Haemorrhage, trauma
 Other myeloproliferative  Iron deficiency
disorders  Malignancy
 Chronic infections
 Connective tissue diseases
 Post-splenectomy
Treatment
The aim is to control the platelet count and to reduce the risk of thrombosis.
The disease may transform to myelofibrosis after a number of years. There is a relative
low risk of transformation to acute leukaemia.
Page 181 of 336

MYELOFIBROSIS
Myelofibrosis (MF) has many names: idiopathic myelofibrosis, myelosclerosis,

agnogenic myeloid metaplasia or myelofibrosis with myeloid metaplasia. Myeloid
metaplasia highlights an essential feature namely extramedullary haemopoiesis
(haemopoiesis in the liver and spleen). This leads to anaemia and a massive liver and
spleen (hepatosplenomegaly). There is an increase in reticulin fibres with progressive
collagen deposition in the bone marrow – normally the marrow has only a few
connective tissue fibres.
Myelofibrosis is a clonal stem cell disease. The fibrosis of the marrow is secondary to
hyperplasia (increased production) of abnormal megakaryocytes. The
megakaryocytes produce fibroblast growth factor which stimulates the fibroblasts to
proliferate with resulting fibrosis.
The JAK2 mutation occurs in approximately 50% of patients.
The disease occurs especially in the middle-aged and elderly. There may be a
previous history of PV or ET.
Clinical features of MF
 Splenomegaly – abdominal discomfort, pain or indigestion
 Hypermetabolic symptoms – weight loss, fever and night sweats
 Bleeding, gout and bone pain occur in minority of patients
Page 182 of 336

Laboratory diagnosis of MF

 Hb low (usually)  Tear-drop cells  Bone marrow usually
 WCC and plt may  Leucoerythroblastic unobtainable by
be high initially and reaction aspiration (dry tap).
decline later in Trephine biopsy
disease show fibrosis
NRBC
Immature white  NAP increased
cell  JAK2 mutation in ½
Tear-drop cell
of patients
 LDH and urate high
http://www.cantechletter.com/2012/02/byron-capitals-loe-things-could-start-to-get-interesting-for-ym-
biosciences/teardrop-cells-myelofibrosis/
Treatment
This is usually palliative (symptom relief).
SUMMARY
 Myeloproliferative disorders are characterized by proliferation of

one or other cell line.
 They are interrelated and can transform into either myelofibrosis or
acute leukaemia
 They are divided into:
o CML (increased white cells)
o PV (increased red cells)
o ET (increased platelets)
o MF (fibrosis of bone marrow)
Page 183 of 336

WORKBOOK
Question 1
Name 2 diseases where you can expect a low NAP score.
Question 2
The translocation t(9;22)is the most common genetic abnormality found in ….
a) Name the disease and the defective chromosome.

b) Write notes on morphology found in these diseases.
c) Name the rest of diseases found in this group.
d) Discuss the different phases seen in this disease.
e) Tabulate differences between this disease and leukaemoid reaction.
Question 3
A 60 year old male patient, with a previous history of polycythaemia vera presented
with massive splenomegaly. His white cell count was 30 X 109/L and platelet count
was 550 X 109/L. A diagnosis of myelofibrosis was made.
a) Describe the peripheral blood film findings that you would expect to see in
myelofibrosis.
b) Complete the table below to differentiate between myelofibrosis and chronic

myeloid leukaemia (CML)
Myelofibrosis CML
Red cell morphology
Nucleated red blood cells
Neutrophil alkaline
phosphatase (NAP) score
Page 184 of 336

Question 4
Describe the relevant haematology laboratory findings of a newly diagnosed,

untreated case of Polycythaemia Vera.
Question 5
Complete the following statements by filling in the missing words or phrases in the
blank spaces.
In the osmotic fragility test (OFT) red blood cells are placed in ____(a)_____ saline
solutions of varying concentrations.
The normal value for the median corpuscular fragility (MCF) is ____(b)_______ g/l NaCl
when the OFT is performed on fresh blood (unincubated).
The Surface area to volume ratio is ___(c)____ in hereditary spherocytosis and

___(d)_____ in thalassaemia.
Hydrops fetalis may occur in alpha thalassaemia when the red cells contain only
tetramers of ____(e)______ chains.
The most significant diagnostic use of the Leucocyte Alkaline Phosphatase (LAP) score
is in ______(f)_____ leukaemia.
The main diagnostic use of Tartrate Resistant Acid Phosphatase (TRAP) is in the
diagnosis of __(g)___.
The platelet count is often ______(h)____ in iron deficiency anaemia.
______(i)_______ is characterised by the presence in neutrophils of basophilic inclusions

resembling Dohle bodies and giant platelets.
Question 6
Which ONE of these is TRUE concerning the translocation that leads to the Philadelphia
chromosome?
a) It leads to increased expression of the c-ABL gene as it brings a strong gene

promoter close to the c-ABL gene
b) It is present in around 60% of cases of CML
Page 185 of 336

c) It is detected on a karyotype as the t(8;21) translocation.
d) It leads to generation of a bcr-abl fusion protein
Which ONE of these clinical features is commonly seen in patients who present with
chronic myeloid leukaemia?
a) Swollen cervical lymph nodes

b) Enlarged spleen
c) Bone marrow failure with reduced peripheral blood cell count
d) Swelling of the gums
Which ONE of the following is NOT a cause of polycythaemia
a) Mutation of JAK-2
b) Renal disease
c) Congenital heart disease
d) Haemoglobin abnormality
e) Iron overload
Which ONE of the following does NOT cause a raised platelet count?
a) Haemorrhage
b) Chronic myeloid leukaemia
c) Mutation of JAK-2
d) Pregnancy
Which ONE of the following is NOT a typical feature of primary myelofibrosis?
a) It cayses a leuco-erythroblastic blood film

b) It may be associated with a raised platelet count
c) Normal serum lactate dehydrogenase level
d) It may be complicated by gout
e) It may cause massive splenomegaly
Page 186 of 336

What is the approximate frequency of the Val617Phe mutation in JAK-2 in
myeloproliferative neoplasms?
a) 99% in Polycythaemia Vera (PV) and 50% in Essential Thrombocythaemia (ET)

and primary myelofibrosis (MF)
b) Approximately 50% in PV, ET and MF
c) 50% in PV, 25% in ET and MF
d) 90% in PV, rare in ET and MF
Page 187 of 336

LYMPHOPROLIFERATIVE DISORDERS
Learning Unit 8
LYMPHOPROLIFERATIVE DISORDERS

terminology related to lymphoproliferative disorders.
Assessment Criteria
learning unit eight:
different lymphoproliferative disorders.
1.3 Differentiate between the disorders by use of special stains, flow
cytometry or cytogenetics:
o T and B cell disorders
o Lymphomas
o Myeloma
Page 188 of 336

Lymphoproliferative These include a spectrum of lymphoblastic and
lymphocytic leukaemias and lymphomas.
Clone A group of identical cells
The lymphoproliferative disorders include a spectrum of B-cells, T and NK cells

neoplasms which also include lymphomas and myelomas. T cells and NK cells share a
lot of properties and are therefore considered together.
[Just remember that the lymphoblastic leukaemias actually form part of the
lymphoproliferative disorders, but were discussed under the section “Acute
leukaemias”.]
Note:
T-cell maturation happens in the thymus
B-cell maturation happens in the bone

marrow
http://www.phoenix5.org/glossary/lymphatic_system.html
Page 189 of 336

B-cell differentiation
B-cell differentiation begins with progenitor B-cells (blasts), which differentiate into pre-
B-cells, then immature B-cells and then mature naïve B-cells. Naïve B-cells are resting
cells, but on encountering antigen that fits their surface Ig receptors, they undergo
transformation and mature into antibody-secreting plasma cells and memory B cells.
Some mature directly into plasma cells and others migrate into the centre of a primary
follicle, proliferate and fill the follicular dendritic cell (FDC) meshwork, forming a
germinal centre (GC).
http://hematopatho.wordpress.com/2009/03/23/classification-of-lymphoid-neoplasms-the-microscope-
as-a-tool-for-disease-discovery/
DLBCL: diffuse large B-cell lymphoma
AG: antigen
CLL/SLL: chronic lymphocytic leukaemia/small lymphocytic lymphoma
Page 190 of 336

T-cell differentiation
T-cell neoplasms correspond with different stages of maturation.

Lymphoid progenitors (blasts) enter the thymus where precursor T-cells develops into
naïve T-cells. The precise maturation path of natural killer cells and δ T-cells are not
fully understood.  T-cells leave the thymus. Upon exposure to antigen they may
undergo blast transformation and develop further into CD4+ and CD8+ effector and
memory T-cells.
Aetiology
 Epstein-Barr virus (EBV) show an association with lymphoproliferative disorders.

 The human retrovirus known as human T-cell leukaemia virus type 1 (HTLV-1) is
linked to adult T-cell leukaemia/lymphoma (ATLL).
Page 191 of 336

CLASSIFICATION
WHO classification of lymphoproliferative neoplasms

(Note: focus on the highlighted sections)
Precursor lymphoid neoplasms
B lymphoblastic leukaemia/lymphoma, not T lymphoblastic
otherwise specified (NOS) leukaemia/lymphoma
B lymphoblastic leukaemia/lymphoma with

recurrent genetic abnormalities
Mature B-cell neoplasms Mature T- and NK-cell neoplasms

Chronic lymphocytic leukaemia/small T-cell prolymphocytic leukaemia
lymphocytic lymphoma
T-cell large granular lymphocytic
B-cell prolymphocytic leukaemia leukaemia
Splenic B-cell marginal zone lymphoma Chronic lymphoproliferative disorder

of NK cells
Lymphoplasmacytic lymphoma
Aggressive NK cell leukaemia
Heavy chain diseases
EBV positive T-cell
Plasma cell neoplasms lymphoproliferative diseases of
 Plasma cell myeloma childhood
Extranodal marginal zone lymphoma of Adult T-cell leukaemia/lymphoma

mucosa-associated lymphoid tissue (MALT)
Extranodal NK/T-cell lymphoma,
Nodal marginal zone lymphoma nasal type
Follicular lymphoma Enteropathy-associated T-cell

lymphoma
Primary cutaneous follicle centre lymphoma
Hepatosplenic T-cell lymphoma
Mantle cell lymphoma
Subcutaneous panniculitis-like T-cell
Diffuse large B-cell lymphoma (DLBCL), NOS lymphoma
DLBCL associated with chronic inflammation Mycosis fungoides
Lymphomatoid granulomatosis Sezary syndrome
Page 192 of 336

Primary mediastinal (thymic) large B-cell Primary cutaneous CD30 positive T-
lymphoma cell lymphoproliferative disorders
Intravascular large B-cell lymphoma Primary cutaneous peripheral T-cell

lymphomas, rare subtypes
ALK positive large B-cell lymphoma
Peripheral T-cell lymphoma, NOS
Plasmablastic lymphoma
Angioimmunoblastic T-cell
Large B-cell lymphoma arising in HHV8- lymphoma
associated multicentric Castleman disease
Anaplastic large cell lymphoma,
Primary effusion lymphoma ALK positive
Burkitt lymphoma Anaplastic large cell lymphoma,

ALK negative
B-cell lymphoma
B-cell lymphoma, unclassifiable, with features

intermediate between DLBCL and Burkitt
lymphoma
B-cell lymphoma, unclassifiable, with feature

intermediate between DLBCL and classical
Hodgkin lymphoma
Hodgkin lymphoma
Nodular lymphocyte predominant Hodgkin lymphoma
Classical Hodgkin lymphoma
Nodular sclerosis classical Hodgkin lymphoma
Mixed cellularity classical Hodgkin lymphoma
Lymphocyte-rich classical Hodgkin lymphoma
Lymphocyte-depleted classical Hodgkin lymphoma
Immunodeficiency-associated lymphoproliferative disorders

Lymphoproliferative disease associated with primary immune disorders
Lymphomas associated with HIV infection
Post-transplant lymphoproliferative disorders (PTLD)
Other iatrogenic immunodeficiency-associated lymphoproliferative disorders
Page 193 of 336

Histiocyte and dendritic cell neoplasms
Histiocytic sarcoma
Tumours derived from Langerhans cells
Interdigitating dendritic cell sarcoma
Follicular dendritic cell sarcoma
Other rare dendritic tumours
Disseminated juvenile xanthogranuloma
FAB classification of chronic lymphoid leukaemias/lymphomas
B-cell T-cell
Chronic lymphoid leukaemias Chronic lymphoid leukaemias
 Chronic lymphocytic leukaemia  Large granular lymphocytic
(CLL) leukaemia
 Prolymphocytic leukaemia (PLL)  T-cell prolymphocytic leukaemia (T-
 Hairy cell leukaemia (HCL) PLL)
 Plasma cell leukaemia
Leukaemia/Lymphoma syndromes Leukaemia/Lymphoma syndromes

 Splenic lymphoma with villous  Sezary syndrome
lymphocytes  Adult T-cell leukaemia/lymphoma
 Follicular lymphoma  Large cell lymphoma
 Mantle cell lymphoma
 Lymphoplasmacytic lymphoma
 Large cell lymphoma
Page 194 of 336

B-CELL DISEASES
CHRONIC LYMPHOCYTIC LEUKAEMIA (CLL)
CLL is the most common form of the chronic lymphoid leukaemias and is a disease of
the elderly; almost all patients are above 50 years of age. The malignant lymphocytes
appear mature morphologically, but are actually arrested at an early stage of
development. Less than 10% are prolymphocytes.
Clinical features of CLL
 Features of anaemia
 Splenomegaly and less commonly hepatomegaly (later stages)
 Lymphadenopathy (swollen lymph nodes)
http://www.documentingreality.com/forum/f149/cervical-adenopathy-lymphadenopathy-
82250/ (accessed 9/11/11)
 Immunosuppression because of hypogammglobulinaemia (too few antibodies)

predisposes to infections
 Two important auto-immune complication

o Auto-immune haemolysis
o Auto-immune thrombocytopaenia
Page 195 of 336

Laboratory diagnosis of CLL

 Hb low (later stages)  Small lymphocytes  Flow cytometry
 Lymphocytosis  Smudge/smear cells (sCD19+, CD5+,
 Platelets low in many  Also look for signs of CD23+)
auto-immune  Cytogenetics for
haemolysis prognosis
Smudge cell
Suspicion: Unexplained  Bone marrow
lymphocytosis in elderly procedure
persons with smudge cells
http://www.med-ed.virginia.edu/courses/path/innes/wcd/lympleuk.cfm
Treatment
Approach to therapy is conservative, aiming for symptom control. Chemo- and
sometimes radiotherapy is given.
Page 196 of 336

PROLYMPHOCYTIC LEUKAEMIA (PLL)
It may initially appear similar to CLL, but diagnosis is made by the presence of >55%
prolymphocytes in the blood. Between 10 – 54% are considered CLL/PLL. The
prolymphocytes are twice the size of small lymphocytes with a central prominent
nucleolus (“punched out”). PLL is usually a disease of the elderly. Only 20% of cases
display T-cell origin.
Clinical features of PLL
 Features of anaemia (poor prognosis)
 Splenomegaly without lymphadenopathy
Laboratory diagnosis of PLL

 Hb low (later stages)  Large  Flow cytometry
 Lymphocytosis prolymphocytes (sCD19+, CD5-,
(usually higher than >55% CD22/FMC7+, CD23-,
CLL and rapidly  Prominent central CD79b+ )
increasing) nucleolus  Bone marrow
 >55%  Abundant, pale procedure
prolymphocytes cytoplasm
http://www.leukemia-images.com/rewrite/show_serie/english/admin/na/stamm/2381unique-
diasammlung/510/na.html
Treatment
Treatment is difficult and the prognosis worse than CLL.
Page 197 of 336

HAIRY CELL LEUKAEMIA
Hairy cell leukaemia is a rare disease which affects more males than females and has
peak incidence at 40 – 60 years.
Clinical features of Hairy cell leukaemia
 Non-specific symptoms including fatigue and weight loss
 Splenomegaly without lymphadenopathy
Laboratory diagnosis of Hairy cell leukaemia

 Pancytopaenia  Unusual large  Flow cytometry
 Neutropaenia often lymphocytes with (CD5-, CD22/FMC7+,
marked (causing villous cytoplasmic CD103+)
infections) projections  Tartrate-resistant
 Monocytopaenia acid phosphatase
(TRAP) stains pos
 Bone marrow
procedure difficult
because of fibrosis
http://lstums.blogfa.com/cat-1.aspx
Treatment
Patients can expect long-term remission with effective treatment.
Page 198 of 336

HAIRY CELL LEUKAEMIA VARIANT
Some cases have clear differences from the typical disease and warrant a different
classification. The white cell count is higher, the hairy cells have a prominent nucleolus
and response to therapy is less satisfactory.
Difference between HCL and HCL-v

Criteria HCL HCL-v
Flowcytometry Pos – CD Pos – CD 11c, 103
11c,19,20,22,25,68,103 No expression of CD25
Cytochemical Stain Positive Negative
(TRAP)
Cell Counts Cytopenia (lymph count Lymphocytosis and
normal) Monocytopenia
Morphology Oval / indented Central round nucleus,
eccentric nucleus, a prominent nucleolus,
Abundant cytoplasm cytoplasmic villi present
and cytoplasmic
projections
Page 199 of 336

PLASMA CELL LEUKAEMIA (PCL)
This is a rare disease characterized by a high number of circulating clonal plasma cells.
Clinical features of PCL
 Pancytopaenia and organomegaly
 Hypercalcaemia, renal involvement, bone disease
Laboratory diagnosis of PCL

 Pancytopaenia  Circulating plasma  Flow cytometry
cells (CD20+, CD38+,
CD138+)
http://www.ask.com/wiki/Plasma_cell_leukemia
Page 200 of 336

T-CELL DISEASES
LARGE GRANULAR LYMPHOCYTIC LEUKAEMIA
Large granular lymphocytic leukaemia is characterized by the presence of circulating

lymphocytes with abundant cytoplasm and large azurophilic granules. It may be T-
cells or natural killer (NK) cells. It is most commonly found is persons over 50 years of
age.
Clinical features of LGL-L
 Infections secondary to neutropaenia
 Associated rheumatoid disease
Laboratory diagnosis of LGL-L

 Lymphocytosis  Abundant large  Flow cytometry
 Neutropaenia granular lymphocytes (CD16+, CD56+,
 Sometime anaemia (between 2-20 x 109/l) – CD57+)
count seperately
http://www.lab.anhb.uwa.edu.au/mb140/corepages/blood/blood.htm
Page 201 of 336

ADULT T-CELL LEUKAEMIA/LYMPHOMA (ATLL)
ATLL is conclusively caused by a human retrovirus, human T-cell leukaemia/lymphoma

virus type 1 (HTLV-1). The virus is endemic in parts of Japan and the Caribbean.
ATLL lymphocytes have a bizarre morphology with a convoluted ‘clover-leaf’ nucleus
and CD4+ phenotype.
Clinical feature of ATLL
 Hypercalcaemia
 Skin lesions
 Hepatosplenomegaly
 Lymphadenopathy
Laboratory diagnosis of ATLL

 Lymphocytosis  Lymphocytes display  Flow cytometry
convoluted nucleus  Lymph node biopsy
 HTLV-1 serology/
molecular testing
http://mltinkemu.yolasite.com/pictures.php
Page 202 of 336

SEZARY SYNDROME / MYCOSIS FUNGOIDES
Sezary syndrome and Mycosis fungoides are T-cell syndromes with primary
manifestation in the skin.
Clinical features
 Exfoliative erythroderma (reddening and flaking of the skin) affecting the palms,
soles and face
Laboratory diagnosis of Sezary syndrome

  Sezary cells with  Skin biopsy –
deep nuclear lymphocytic
clefting infiltration
 Sezary cells
http://www.medicinabih.info/2009/12/26/sindrom-sezary/
Page 203 of 336

SUMMARY
 Lymphoproliferative neoplasms include a variety of T-, B or NK-cell

leukaemias or lymphomas.
 CLL is characterized by an unexplained lymphocytosis with small
lymphocytes and smudge/smear cells. It is often incidental finding.
 PLL shows prolymphocytes with prominent central nucleoli
 Hairy cell leukaemia is TRAP positive and the cytoplasm show hair-
like projections
 ATLL and Sezary syndrome are sometimes difficult to distinguish
from each other because of the similar looking lymphocytes
 HTLV-1 is associated with ATLL
Page 204 of 336

LYMPHOMA
HODGKIN’S LYMPHOMA
The disease is caused by malignant lymphocytes that accumulate in lymph nodes. It

can infiltrate other organs.
The lymphomas can be subdivided into Hodgkin’s and non-Hodgkin’s lymphoma and
is based on the characteristic histological presence of Reed-Sternberg cells in
Hodgkin’s lymphoma.
The Epstein-Barr virus (EBV) has been implicated, but the pathogenesis is unclear. The
disease has a peak incidence in young adults with a slight male predominance.
Clinical features of Hodgkin’s lymphoma
 Painless, non-tender asymmetrical enlargement of lymph nodes
 Hepatosplenomegaly may occur, but is not massive
 Fever, sweating, weight loss, pruritis, fatigue, development of pain at site of

disease after drinking alcohol
Laboratory diagnosis of Hodgkin’s lymphoma

 Anaemia  No characteristic  Diagnosis is made by
 Some neutrophilia/ peripheral blood film histological
eosinophilia finding examination of
 Lymphopaenia excised lymph node
 ESR raised
Reed-Sternberg
cell (owl eye)
http://hematopathology.stanford.edu/
Page 205 of 336

NON-HODGKIN’S LYMPHOMA
These are a large group of lymphoid tumours, most commonly of B-cell origin. They
can sometimes spill over into peripheral blood (leukaemic phase).
The classification is complex, but in simple terms can be divided into ‘high grade’ and
‘low grade’ types.
High grade tumours are composed of large poorly differentiated lymphoid cells and
have an aggressive clinical course. The most common example is Diffuse Large B Cell
Lymphoma.
Low grade tumours are composed of smaller, better differentiated cells. Follicular
lymphoma is an example.
Diagnosis is based on histological examination of a lymph node biopsy.
SUMMARY
 Lymphomas are generally divided into Hodgkin’s lymphoma and

non-Hodgkin’s lymphoma.
 The diagnostic feature in Hodgkin’s disease is the histological
presence of the Reed-Sternberg cells.
 Non-Hodgkin’s lymphomas are divided into high grade and low
grade tumours depending on size and differentiation.
 SOFT TISSUE BIOPSY IS THE MAINSTAY OF DIAGNOSIS. Bone marrow
procedures are only done to check for infiltration and staging.
Page 206 of 336

MYELOMA
Myeloma (or sometimes called myelomatosis) is a malignant disease characterized by

the uncontrolled clonal proliferation of plasma cells in the bone marrow and
monoclonal protein in the serum and/or urine. It is a disease of the elderly with a
median age at diagnosis of about 70 years.
Note:
Just a reminder of
how an
Immunoglobulin
looks.
Heavy chain: G, A,
D, M or E
Light chain: Kappa
or Lambda
http://www.answers.com/topic/immunoglobulin
Some tumours show chromosome translocations involving the immunoglobulin (Ig)

heavy chain and others have an increased number of chromosomes. These cells
secrete a monoclonal Ig or Ig fragments (M-proteins) composed of a single heavy
chain class and a single light chain class (kappa or lambda).
Most myelomas produce IgG or IgA, but light chains alone are also produced in some
cases.
Clinical features of Myeloma
 Bone pain (backache) resulting from vertebral collapse and pathological

fractures
 Features of anaemia
 Recurrent infections due to deficient antibody production (immune paresis)
 Features of renal failure like excessive thirst or urination, vomiting
Page 207 of 336

 Abnormal bleeding because the myeloma protein interferes with platelet
function
 Hyperviscosity syndrome with Purpura, haemorrhages, visual failure, CNS

symptoms
Something to help you remember:

CRAB: calcium (elevated), renal failure, anaemia, bone lesions
Laboratory diagnosis of Myeloma

 Anaemia  Rouleaux formation  Monoclonal protein in
 Neutropaenia and serum/urine(Bence
Plasma cell
thrombocytopaenia Jones protein)
(advanced cases)  Increased clonal
 ESR raised plasma cells in bone
marrow
 Related organ or tissue
involvement (ROTI)e.g.
bone disease
 Flow cytometry to
confirm abnormal
phenotype and
clonality
http://www.med-ed.virginia.edu/courses/path/innes/wcd/myeloma.cfm
Page 208 of 336

Protein electrophoresis showing the abnormal paraprotein in the gamma region
Bone marrow showing an increase in plasma cells
medscape.com
http://healthfiles.net/disease/multiple-myeloma/
Treatment
Treatment generally consists of chemotherapy, management of specific
complications and symptom relief.
WALDENSTROM’S MACROGLOBULINAEMIA
This is an uncommon disease in older men. The malignant cells show features of
lymphocytes and plasma cells and secrete an IgM paraprotein. They present as
lymphoma, myeloma or leukaemia.
SUMMARY
 Myeloma is a malignant proliferation of plasma cells.

 Diagnosis rests on 3 findings: monoclonal protein in serum/urine,
increased plasma cells and related organ or tissue impairment.
 The paraprotein in Myeloma is usually IgG and sometimes IgA or
light chain.
 Bence Jones protein is found in the urine of 2/3 of cases
Page 209 of 336

WORKBOOK
Question 1
Choose the correct answer.
1. Hairy cell leukaemia results from a clonal malignancy of:

a) Myeloblasts
b) Monoblasts
c) T-cells
d) B-cells
2. Cells expressing both CD19 and CD10 flow cytometrically are diagnostic of:
a) Acute myeloblastic leukaemia
b) Chronic myeloid leukaemia
c) Chronic lymphocytic leukaemia
d) Acute lymphoblastic leukemia.
3. The basic structure of an immunoglobulin consists of:

a) Two light and two heavy chains
b) Two light chains with a variable and constant region.
c) Two heavy chains with a variable and constant region
d) Two light chains and two heavy chains with variable and constant
regions.
4. The clinical condition of young adults associated with the Epstein-Barr virus and
heterophile antibodies is commonly referred to as:
a) Rubella
b) Scarlet Fever
c) Toxoplasmosis
d) Glandular fever
Question 2
a. Tabulate the differences between hairy cell leukaemia and hairy cell variant.
b. Describe the morphology and laboratory features of adult T-cell lymphoma
c. What is the malignant cell called in patients with Hodgkin’s disease?
Page 210 of 336

Question 3
A 59 year old man is referred to the haematology clinic after a fall in which he
fractured his femur. A blood sample was sent to the laboratory for a full blood count
and differential count. The full blood count results are as follows:
White cell count: 5.2 X 109/l

Red cell count: 4.16 X 1012/l
Hb: 9.2g/dl
HCT: 0.37l/l
MCV: 83.5 fl
MCHC: 33.4g/dl
Plts: 195 X109/l
ESR: 160mm/hr
Rouleaux formation of red blood cells was seen on the blood smear.
a. Discuss the above results indicating which parameters are normal and which are
abnormal.
b. Attempt a diagnosis and give one reason for your answer.
c. Name the abnormal cells seen in this disorder and describe their appearance in the
peripheral blood and bone marrow.
d. Which tests would you do and what results would you expect in order to diagnose
this disorder.
e. Into which WHO category is this malignancy classified.
Page 211 of 336

Question 4
a) Name the disease.

b) List morphological features.
c) What confirmatory tests are required and give expected results?
Question 5
A 60 year old male patient presented with enlargement of the superficial lymph
nodes. The doctor ordered a full blood count, white cell differential and blood film
examination. The following results were obtained: haemoglobin 11.2 g/dℓ, white cell
count 150 x 109/ℓ, and platelet count 130 x 109/ℓ. The doctor suspected a diagnosis of
chronic lymphocytic leukaemia (CLL).
(a) Describe the white cell differential and the blood film results that would be
consistent with the above diagnosis.
(b) Name any two conditions that may result in a lymphoid leukaemoid reaction.
Page 212 of 336

MYELODYSPLSTIC SYNDROMES
Learning Unit 9
MYELODYSPLASTIC SYNDROMES

terminology related to myelodysplastic syndromes.
Assessment Criteria
learning unit nine:
1.2 Identify the relevant morphological features associated with
myelodysplastic syndromes
1.3 Differentiate between the syndromes.
Page 213 of 336

Cytopaenia Decrease in a cell line either red cells (Hb), white cells
(neutrophils) or platelets
Bi- Denoting two
Pan- Denoting all
The Myelodysplastic syndromes (MDS) have in common characteristic morphological

abnormalities of the blood and bone marrow and a tendency to evolve into acute
leukaemia (previously known as pre-leukaemia). Haemopoiesis is functionally
ineffective which usually result in normo- or hypercellular bone marrow with one or
more cytopaenias. The majority of patients are elderly with a slight male preference.
The term “refractory anaemia” was used to describe anaemic patients who did not
respond to iron, vitamin B or folic acid treatment.
Causes:
Primary Secondary
 Unknown  Ionizing radiation
 Cytotoxic chemotherapy
 Benzene exposure
Clinical features of MDS
 Anaemia related e.g. fatigue, pallor

 Neutropaenia related e.g. infections
 Thrombocytopaenia related e.g. bleeding, purpura
Page 214 of 336

CLASSIFICATION
FAB classification
Type Peripheral blood Bone marrow
 Refractory anaemia  Anaemia; <1% blasts  <5% blasts; ≤ 15%
(RA)/ Refractory ringed sideroblasts
cytopaenia
 Refractory anaemia  Anaemia; <1% blasts  <5% blasts, >15% ring

with ring sideroblasts sideroblasts
(RARS)
 Refractory anaemia  Anaemia; Blasts >1%  Blasts 5 – 19%

with excess blasts and <5%
(RAEB)
 Refractory anaemia  >5% Blasts or Auer  20 – 29% Blasts or

with excess blasts in rods Auer rods
transformation
(RAEB-t)
 Chronic  Blasts up to 20%,

 Monocyte count >1 x
myelomonocytic promonocytes often
109/l, granulocytes
leukaemia (CMML) increased
often increased
Page 215 of 336

WHO classification
Type Peripheral blood Bone marrow
 Refractory anaemia  Anaemia; no blasts  <5% blasts; erythroid
(RA) dysplasia
 Refractory anaemia  Anaemia; no blasts  >15% ring

with ring sideroblasts sideroblasts;
(RARS) erythroid dysplasia
 Refractory cytopaenia  Bi- or  Dysplasia in >10% of

with multilineage pancytopaenia cells in ≥2 myeloid
dysplasia (RCMD) lineages
 Refractory cytopaenia  Bi- or  Dysplasia in >10% of

with multilineage pancytopaenia cells in ≥2 myeloid
dysplasia and ringed and lineages;
sideroblasts >15% ring sideroblasts
 Refractory anaemia  Uni- or multilineage

 Cytopaenias; <5%
with excess blasts-1 dysplasia 5 – 9%
blasts, no Auer rods
(RAEB-1) blasts
 Refractory anaemia
 Cytopaenias or 5 –  Uni- or multilineage
with excess blasts-2
19% blasts or Auer dysplasia 10 - 19%
(RAEB-2)
rods blasts or Auer rods
 Cytopaenias; no  Myeloid or
 Myelodysplasia
blasts or Auer rods megakaryocytic
syndrome unclassified
dysplasia; <5% blasts
(MDS-U)
 Anaemia; normal  Megakaryocytes

 MDS associated with with hypolobated
or increased
isolated del (5q) nuclei; <5% blasts
platelets
Note: Patients with dysplastic features and more than 20% blasts in the bone marrow
are now considered to have acute myeloid leukaemia with multilineage dysplasia.
Page 216 of 336

Morphological abnormalities in MDS
Lineage Peripheral blood Bone marrow

 Erythroid  Oval macrocytosis,  Ring sideroblasts,
acanthocytosis, vacuolated
basophilic stippling, cytoplasm, nuclear
nucleated red blood fragmentation and
cells hyperlobulation,
multinuclearity
Oval macrocyte
Ring sideroblast (iron in
ring form aroundnucleus)
 Granulocytic  Nuclear hypolobulation  Loss of primary and

(Pseudo Pelger-Huet secondary granules,
anomaly), ring nuclei increased blasts
(doughnut), cytoplasmic
hypo- or agranularity
Pseudo Pelger-Huet,
agranular neutrophil
Ring nuclei
(doughnut shape)
 Monocytic  Abnormal nuclear

lobulation, agranular
cytoplasm, nucleoli
Page 217 of 336

 Platelets  Agranular or giant  Micromegakaryo-
platelets cytes, large mono- or
binuclear
megakaryocytes, large
megakaryocytes with
widely dispersed nuclei
http://www.wadsworth.org/chemheme/heme/cytoheme/ans082.htm
http://en.wikipedia.org/wiki/Myelodysplastic_syndrome
http://www.med-ed.virginia.edu/courses/path/innes/wcd/syndrome.cfm
Page 218 of 336

Laboratory diagnosis of MDS

 Cytopaenia or  Sometime oval  Bone marrow
pancytopaenia(50% macrocytes aspirate and biopsy
of cases) i.e.  Granulocyte dysplasia  Cytogenetics
anaemia, e.g. Pelger-Huet
neutropaenia, neutrophils, ring
thrombocytopaenia nuclei, hypo- or
 MCV may be agranularity
increased  Giant platelets
 Monocytosis in some
cases Oval macrocyte
First exclude other causes of

Pseudo Pelger-Huet
macrocytic anaemia e.g. agranual neutrophil
Vitamin B12 or folate
deficiency.
Ring nuclei
(doughnut shape)
Giant platelet
http://en.wikipedia.org/wiki/Myelodysplastic_syndrome
http://www.med-ed.virginia.edu/courses/path/innes/wcd/syndrome.cfm
Page 219 of 336

Treatment
This is often difficult because chemotherapy rarely cures the disease and often makes
it worse. The median survival is 20 months for primary MDS.
MYELODYSPLASTIC/MYELOPROLIFERATIVE DISEASES
These disorders are characterized by the presence of dysplastic features but also
increased number of circulating cells in one or more lineages, indicative of a
proliferative component
WHO classification
Chronic myelomonocytic leukaemia * CMML
Atypical chronic myeloid leukaemia aCML (bcr-abl negative)
Juvenile myelomonocytic leukaemia JMML
Myelodysplastic/myeloproliferative disease Unclassifiable
* CMML was previously part of MDS, but are now re-classified by WHO
SUMMARY
 MDS are characterized by one or more cytopaenias, dysplastic

features and predisposition to evolve to acute leukaemia
 The WHO classification’s major changes involve:
o the exclusion of CMML,
o inclusion of 5q- syndrome,
o definition as acute leukaemia if ≥20% blasts.
 Dysplastic changes in the myeloid lineage include pseudo Pelger-
Huet neutrophils, hypersegmentation, hypo- or agranularity, ring
nuclei, giant platelets.
Page 220 of 336

WORKBOOK
Question 1
Give the WHO classification of Myelodysplastic syndromes and describe how it differs
from the FAB classification
Question 2
The World Health Organisation (WHO) classification of tumours of haemopoietic and

lymphoid tissue resulted in changes when compared to the French American British
(FAB) classification for the following:-
a) Which condition, previously classified as a myelodysplastic syndrome, has now

been classified as an acute leukaemia in accordance with WHO criteria?
b) Which criteria have been included when compiling the WHO classification?
c) Give the minimum number of blasts now required to classify a case as an acute
leukaemia.
Question 3
Discuss all possible morphological findings in MDS using the headings red cells, white
cells and platelets.
Page 221 of 336

COAGULATION
Learning Unit 10
COAGULATION

concepts and terminology related to coagulation.
Assessment Criteria
learning unit ten:
1.1 Understand principles and operation of automated analyzers as well
as manual tests.
1.2 Demonstrate knowledge of the normal values.
1.3 Interpret test results.
1.4 Demonstrate knowledge of both quantitative and qualitative
platelet disorders.
1.5 Demonstrate knowledge of the coagulation disorders.
Page 222 of 336

Haemostasis Arrest of bleeding, involving physiological processes of
blood coagulation and contraction of blood vessels
Thrombosis This is a pathologic process that leads to activation of the
haemostatic system at abnormal time and location
resulting in thrombus formation and vascular occlusion.
Haemorrhage Excessive bleeding due to blood vessel rupture or
abnormalities within blood itself that impairs haemostasis
Fibrinolysis Regulates haemostasis through natural occurring
anticoagulants
In vivo Within the living body
In vitro Outside the body (in a test tube)
The haemostasis system is divided into primary and secondary haemostasis.
Primary haemostasis
Primary haemostasis involves the contraction

of the blood vessels to minimize blood flow
Blood vessels and the adherence of platelets to the
damaged vessels to form a temporary plug.
Platelets
Secondary haemostasis
Page 223 of 336

The secondary haemostasis mechanism is activated
when circulating proteins react with one another in a
complex chain reaction resulting in a fibrin network
which covers the surface of the plug. These proteins are
called coagulation factors and are described by
Roman numerals from I to XIII.
Regulation of haemostasis
Although an efficient and rapid mechanism to stop bleeding is necessary, the
response must be tightly controlled to prevent excessive clotting.
The regulators are natural coagulation inhibitors and the fibrinolytic system.
Direct inhibitors of the coagulation cascade are
 Antithrombin (AT)
 Tissue factor pathway inhibitor (TFPI)
 Thrombomodulin
 Protein C and S
The fibrinolytic system is activated in cascade fashion with the end result: plasmin
degrades fibrin.
Components of haemostatic response

The five major components involved are
 Blood vessels
 Platelets
 Coagulation factors
 Coagulation inhibitors
 Fibrinolysis
Page 224 of 336

COMPONENTS OF HAEMOSTASIS
BLOOD VESSELS
http://medicinembbs.blogspot.com/2011/02/normal-hemostasis.html
Initial slowing of blood flow in the area of injury occurs through

 Vasoconstriction (a local neural response)
 Release of endothelin (from injured endothelium)
Page 225 of 336

PLATELETS
Thrombopoietin is the major regulator of platelet production and is produced by the

liver and kidneys. The normal platelet lifespan is 7 – 10 days. Up to 1/3 of platelets
pool in the spleen. Following a break in the endothelial lining, the underlying collagen
is exposed. Platelets are quickly recruited to the area. The platelets adhere to the
exposed connective tissue (collagen) through binding by VWF.
Adhesion
Glycoprotein Ia facilitates adhesion to collagen
GPIIb/IIIa binds subendothelium en thus von Willebrand factor (VWF) and fibrinogen
which is important in platelet-platelet aggregation.
Shape change
Platelets become more spherical and extrude long pseudopodia which enhance
platelet vessel wall and platelet-platelet interaction. The end result is a flattened,
spread-out platelet with granules and organelles in the centre.
This shape enhances platelet-platelet and platelet-vessel wall interaction.
Page 226 of 336

Granules
The platelet contains three types of storage granules: dense,  and lysosomes.
-granules contain among others fibrinogen, VWF and other clotting factors.
Dense granules are less common and contain adenosine diphosphate (ADP) and
calcium.
Lysosomes contain hydrolytic enzymes.
ADP promotes platelet activation.
The ongoing platelet activation by ADP and TXA2 results in a platelet plug. In this
platelet plug the platelets are completely degranulated and adjacent to each other.
COAGULATION FACTORS
The coagulation cascade is essentially a series of enzymatic conversions, turning

inactive proenzymes into activated enzymes culminating in the formation of thrombin.
Thrombin then converts the soluble plasma protein fibrinogen into the insoluble fibrous
protein fibrin. Fibrin forms a mesh in the platelet aggregates and converts the unstable
primary platelet plug to a firm, stable plug.
Each reaction in the pathway results from the assembly of a complex composed of:
enzyme (activated coagulation factor),
a substrate (proenzyme form of coagulation factor),
a cofactor (reaction accelerator).
These components are assembled on a phospholipid complex (e.g. platelet, exposed

subendothelium) and held together by calcium ions.
The generation of thrombin in vivo is a complex network of amplification and negative

feedback loops to ensure a localized and limited production.
Page 227 of 336

Coagulation is initiated by the interaction of Tissue Factor (TF) with plasma factor VIIa.
The factor VIIa-TF complex activates both factors IX and X. Factor Xa forms small
amounts of thrombin from prothrombin, but is insufficient to initiate significant polymer
formation. It activates the coenzymes, factor V and factor VIII, platelets and factor XI.
The extrinsic pathway is rapidly inactivated by Tissue factor pathway inhibitor (TFPI)
which forms a complex of VIIa, TF, Xa and TFPI.
Thrombin generation is now dependant on the intrinsic pathway which has been
primed by the small amounts of thrombin. IXa and VIIIa on phospholipids surface in
the presence of Ca2+ activated sufficient Xa which then in combination with Va, PL
and Ca2+ forms a prothrombinase complex and results in the explosive generation of
thrombin which acts on fibrinogen to form the fibrin clot.
The following pages will show how the different parts of the cascade model functions
and is mainly how haemostasis takes place in vitro.
Page 228 of 336

Platelet activation Tissue factor released
by injured cells
Tissue factor is released as a co-factor and

binds factor VII in the presence of Ca2+ to
activate factor X. This complex activates
small amount of factor IX. As factor Xa is
formed, it will activate factor VII.
Tissue thromboplastin
Extrinsic system (PT)

(III)
VII  VIIa
PL,
Ca2+
V  Va
X  Xa
Takes place on
the surface of PL, Ca2+
activated
platelets Prothrombin (II)  (IIa) Thrombin
Common pathway
(I) Fibrinogen  Fibrin (Ia)
Fibrin polymers
weave through
platelet plug
Frbin stabilizing factor XIII  XIIIa
The common pathway is marked as the Stable fibrin clot,

conversion of prothrombin to thrombin. fibrin strands
Factor V acts as a co-factor and it takes crosslinked
place on the surface of the activated
platelet. Thrombin converts fibrinogen to
fibrin. Factor XIII, activated by thrombin and
calcium ions, converts fibrin to a stable cross-
linked fibrin clot.
Page 229 of 336

Injury to blood vessel Platelet activation
Contact activation
Factor IX is activated by factor XIa in the

presence of calcium ions. Conversion of factor
X to factor Xa takes place on the surface of the
platelet and is catalyzed by a complex
XII  XIIa composed of factor IXa, factor VIII, calcium ions,
and phospholipid (from the platelet). Factor VIII
acts as a cofactor. The common pathway
Intrinsic system
(measured by PTT)
Ca2+
XI  XIa continues as above.
IX  IXa
VIIII  VIIIa
X  Xa
Takes place on
the surface of
activated
Common pathway
Fibrin polymers
weave through
platelet plug
Stable fibrin clot,

fibrin strands
crosslinked
Page 230 of 336

Injury to blood vessel Platelet activation Tissue factor released
Contact activation by injured cells
Von Willebrand factor adheres
platelets to damaged cells
Platelet aggregation and

formation of loose platelet plug
XII  XIIa
Intrinsic system (measured

(III)
Ca2+
XI  XIa
by PTT)
TFPI inhibits
Xa + VIIa VII  VIIa
IX  IXa
PL,
Ca2+
VIIII  VIIIa V  Va
X  Xa Protein C  Ca
Takes place on + Protein S,
the surface of PL, Ca2+ degrades Va + VIIIa
activated
Heparin activates
Antithrombin which
inhibits IIa, XIa, IXa,
Common pathway
Xa (I) Fibrinogen  Fibrin (Ia)
Fibrin polymers
weave through
platelet plug
Stable fibrin clot,

Coagulation factor inhibitors: fibrin strands
crosslinked
It is important that thrombin formation is limited to the
site of injury.
The first inhibitor is tissue factor pathway inhibitor (TFPI).
It inhibits Xa and VIIa and TF.
Antithrombin directly inactivates thrombin. Heparin
potentiates its action markedly.
Protein C and S are inhibitors of cofactors V and VIII.
Page 231 of 336

FIBRINOLYSIS
Fibrinolysis is a normal response to vascular injury.
Tissue plasminogen activator (tPA) is released from the endothelial

cells and binds fibrin. It enhances the conversion of thrombus-
bound plasminogen to plasmin. Plasmin is capable of digesting
fibrinogen, fibrin, factor V and VII and many proteins. A variety of
split products or degradation products form. The smallest
fragments D and E can be detected in plasma. Activated protein
C stimulates fibrrinolysis by destroying plasma inhibitors of tPA.
Inhibition
tPA is inactivated by plasminogen activator inhibitor (PAI).
2-antiplasmin inhibits any local free plasmin.
Stable fibrin clot,

Tissue-type plasminogen fibrin strands
crosslinked
activator (tPA – bound to fibrin)
Fibrinolytic system
Plasminogen  Plasmin
Clot breaks into soluble
fragments
Fibrin degradation products

Page 232 of 336
Platelet activation
Injury to blood vessel Tissue factor released
Contact activation by injured cells
Von Willebrand factor adheres
platelets to damaged cells
Platelet aggregation and

formation of loose platelet plug
XII  XIIa
Intrinsic system (measured

(III)
Ca2+
XI  XIa
by PTT)
TFPI VII  VIIa

IX  IXa
PL,
Ca2+
VIIII  VIIIa V  Va
X  Xa Protein C  Ca
Takes place on
the surface of + Protein S,
activated PL, Ca2+ degrades Va + VIIIa
platelets
Prothrombin (II)  (IIa) Thrombin
Heparin activates
Antithrombin which
inhibits IIa, XIa, Ixa, Xa
Common pathway
Fibrin polymers
weave through
platelet plug
Stable fibrin clot,

fibrin strands
Tissue-type plasminogen
crosslinked
activator (tPA – bound to fibrin)
Fibrinolytic system
Plasminogen  Plasmin
Clot breaks into soluble
fragments
Fibrin degradation products
Page 233 of 336

THE CELL-BASED MODEL OF FIBRIN FORMATION
New understanding of haemostasis incorporates the role of cells. This model suggests
that coagulation actually occurs in vivo in distinct overlapping phases. It requires
participation of 2 different cells types: a cell-bearing tissue factor (TF) and platelets.
Initiation phase:
Once injury occurs and blood is exposed to a
TF-bearing cell, FVIIa rapidly binds to exposed
TF. The TF-FVIIa complex then activates small
amounts of FIX and FX. Although it occurs
slowly, FV can be activated directly by FXa.
FXa and FVa form the prothrombinase complex
which cleaves prothrombin and generates
small amounts of thrombin. Any FXa that
dissociates from the membrane surface is
inactivated by TFPI or AT.
Amplification phase:
The small amount of thrombin generated on
the TF-bearing cell activates platelets which
results in shape change, shuffling of membrane
phospholipids and release of granule contents
to provide additional “fuel for the fire”. In
addition it also cleaves FXI to FXIa, activates FV
to FVa and cleaves VWF off of FVIII.
Propagation phase:
The release of granule contents recruits more
platelets to site of injury. Once the intrinsic
tenase complex forms (FIXa-FVIIIa) on the
activated platelet surface, it rapidly begins to
generate FXa. It then binds to FVa and cleaves
prothrombin to thrombin. This prothrombinase
activity results in a burst of thrombin generation
leading to cleavage of fibrinogen to form a
mass of fibrin.
Page 234 of 336

TESTS OF HAEMOSTATIC FUNCTION
Defective haemostasis and abnormal bleeding may result from:
 A vascular disorder
 Platelet disorders, either functional or numerical (thrombocytopaenia)
 Defective blood coagulation
TESTS FOR VASCULAR DISORDERS
In some cases of vascular disorder, the bleeding time will be abnormal.
Page 235 of 336

TESTS FOR PLATELET DISORDERS
Platelet disorders can be divided into platelet function problems or low platelet
numbers (thrombocytopaenia).
Blood count and blood smear

A thrombocytopaenia is a common cause of bleeding. The smear should be assessed
in order to establish a possible cause of thrombocytopaenia; red cell fragments
indicate the presence of micro-angiopathic haemolysis e.g. disseminated
intravascular coagulation (DIC) or thrombotic thrombocytopaenic purpura (TTP);
target cells, stomatocytes, round macrocytes may indicate liver disease as possible
cause; red cells infected with Plasmodium species indicate malaria; etc.
Bleeding time
This test is used to assess platelet function including diagnosis of VWF disease. The test
involves the application of pressure to the upper arm with a blood pressure cuff, after
which a small incision is made in the forearm. The time lapsed is recorded when
bleeding stop. It usually stops between 3 – 9 minutes. The bleeding time is influenced
by vascular abnormality, platelet abnormality and VWF abnormality.
Platelet function analysis-100 (PFA-100)

The PFA-100 is a more sensitive screening test than the manual bleeding time. It is
useful in the diagnosis of von Willebrand disease (types 1, 2A, 2B, and 3) and may be
useful for other specific platelet function disorders. The test uses stimulants of platelet
aggregation and adhesion in an environment that simulates an injured blood vessel
wall. The time until complete occlusion of blood flow through an aperture (closure
time) is the measured end point.
Well-known agonists of this process include epinephrine, adenosine diphosphate
(ADP), collagen, thrombin and thromboxane A2.
Page 236 of 336

http://www.platelet-research.org/3/pfa.htm
Interpretation
The following table summarizes some of the abnormalities that have been reported
with the PFA-100.
Disorder CT Collagen-ADP CT Collagen-EPI
Normal N N
Aspirin and NSAIDs N ↑
VWD ↑ ↑
Platelet-Type VWD ↑ ↑
Gray Platelet Syndrome ↑ ↑
MYH9-related Disorders N ↑
MDS N or ↑ N or ↑
Liver Disease ↑ [possibly as a result ↑ [possibly as a result
of ↓Hb] of ↓Hb]
Uraemia ↑ [possibly as a result ↑ [possibly as a result
of ↓Hb] of ↓Hb]
Reported reference ranges for closure times are:
 78 - 199 seconds for the CEPI cartridge

 55 - 137 seconds for the CADP cartridge
Platelet aggregation study

It measures the decrease in light absorbance in platelet-rich plasma as platelets
aggregate. Initial aggregation is caused by an external agent (ADP, collagen,
ristocetin, arachidonic acid and adrenaline) and secondary response is to
aggregating agents released from the platelets themselves. The pattern of response
to each agent helps to make the diagnosis.
Page 237 of 336

In the patient shown below, the only abnormality is a lack of agglutination with
ristocetin. Possible diagnoses are therefore, Von Willebrand Disease or Bernard Soulier
Syndrome.
http://www.practical-haemostasis.com/Platelets/platelet_function_testing_lta.html
Page 238 of 336

Summary of platelet aggregation patterns
Disorder Defect Platelet Smear ADP ADP (5- Collagen AA (100 Adrenaline Ristocetin
count (2µM) 10 µM) (1 µg/ml) µM) (10 µM) (1.2
mg/ml)
Normal None N None 1◦ + 2◦ 2◦ only N N N N
wave
aggr
seen
Glanzmann’s Defect in N None A A A A A N
Thrombasthenia GPIIb/ IIIa
receptor
Bernard Soulier Defect in ↓ Giant N N N N N A
syndrome GPIb-V-IX platelets
receptor
Aspirin / NSAIDs Inhibits N N 1◦ wave 1◦ wave ↓ or A A ↓ N
cyclo- only only
oxygenase
Gray platelet Deficiency Often ↓ Gray plt N or ↓ N or ↓ Variable, N N N
syndrome of - or usually ↓
granules platelet
ghosts
VWD Dysfunctio ↓ TSP N N N N N N
nal VWF
Uraemia Acquired May ↓ N ↓ N or ↓ N or ↓ ↓ N or ↓
Liver disease Multiple May ↓ TSP 1◦ wave 1◦ wave ↓ ? 1◦ wave N or ↓
only only only
DIC Multiple May ↓ TSP ↓ ↓ ↓ ↓ ↓ ↓
N – normal
A – absent
↑ - increased
↓ - decreased
1◦ - primary
2◦ - secondary
TSP - thrombocytopaenia
Page 239 of 336

TESTS FOR COAGULATION DISORDERS
Screening tests assess the extrinsic pathway, intrinsic pathway and the conversion of
fibrinogen to fibrin.
Prothrombin time (PT)

The PT measures the extrinsic and common pathway, therefore factor VII, X, V,
prothrombin and fibrinogen. Tissue thromboplastin (a brain extract) and calcium are
added to citrated plasma. Factor VII reacts with tissue factor (thromboplastin) to
activate factor X.
Normal values range between 8 – 12 seconds, depending on the analyzer and/or
reagent used.
Common causes of a prolonged PT are vitamin K deficiency, liver disease, DIC and
oral anticoagulant therapy (Warfarin).
The International Normalized Ratio (INR) is used in monitoring Warfarin therapy. The
ratio of the patient’s PT versus a normal control PT was used; however different
thromboplastin reagents will not have the same sensitivity. The method and reagent
have to be taken into consideration. An international reference thromboplastin
reagent has been developed for use in standardizing the PT ratio. Manufacturers of
thromboplastin calibrate each lot number of their reagent against the standard WHO
reagent. The results are used to develop the International Sensitivity Index (ISI) for
each batch. This is then used in the calculation of the INR.
Patjent’s PT_ ISI
INR = Normal PT
The therapeutic range for INR is between 2.0 – 3.5. The “normal” (not on
anticoagulants) INR would then be 1.0.
Activated partial thromboplastin time (APTT)

The APTT is a useful procedure for screening of coagulation disorders in the intrinsic
system, for detecting the presence of circulating anticoagulants (inhibitors) and for
monitoring heparin therapy. It measures factors VIII, IX, XI, XII in addition to factors X,
V, prothrombin and fibrinogen. The calcium in whole blood is bound by the sodium
citrate anticoagulant to prevent coagulation. Therefore, calcium, a phospholipids
substitute for platelets (partial thromboplastin) and an activator (e.g. kaolin) are
added to plasma. The time required to clot, is the activated partial thromboplastin
time.
Normal values range between 26 – 33 seconds, depending on analyzer and/or
reagent used.
Common causes of a prolonged APTT are liver disease, DIC, Haemophilia, Christmas
disease, Warfarin and heparin therapy.
Page 240 of 336

Correction studies
Prolonged clotting times in the PT and APTT because of factor deficiencies are
corrected by the addition of normal plasma to the test plasma (50:50 mix). If there is
no correction, the presence of an inhibitor is suspected. If there is correction it
indicates factor deficiencies.
Thrombin Time (TT)

The TT measures the availability of functional fibrinogen. It is sensitive for a deficiency
of fibrinogen, dysfribrinogenaemia or inhibition of thrombin, such as heparin. Thrombin
is added to citrated plasma and the length of time for a fibrin clot to form is recorded
as the thrombin time.
Specific assays of coagulation factors

The factor assays are based on the PT or APTT principles where all factors are present in
the substrate plasma, except the one to be measured.
Tests for fibrinolysis

Fibrin or fibrinogen degradation products (including D-dimers) can be demonstrated in
the blood of patients with primary fibrinolysis. The presence of crosslinked D-dimer
indicates a stable fibrin clot has been lysed and will be found in pulmonary embolism,
deep vein thrombosis (DVT), DIC, arterial thromboembolism, sickle cell disease etc.
Page 241 of 336

THROMBOELASTOGRAPHY
Near patient testing became important to reduce the turn around time between
sampling and resulting of coagulation tests. Thromboelastography (TEG) provides a
complete picture of the formation and dissolution of the clot, more closely reflecting in
vivo activity. It is sensitive to all components of clot formation, including platelets and
coagulation factors and it can provide a quick diagnosis.
The TEG is produced by placing a sample of whole

blood into a pre-warmed (37°C) cup which is
oscillated through an angle of 4°45′, rotating once
every 10 s. This simulates a low shear environment
resembling sluggish venous flow. A plastic pin is
suspended in the blood sample by a torsion wire and
monitored for motion. As a clot forms, the cup and pin
become coupled by fibrin-platelet bonds, and the
torque of the rotating cup is transmitted by the torsion
wire to a mechano-electrical transducer. The resulting
electrical signal is converted (nowadays by computer)
into a characteristic cigar-shaped graphical output
representing a function of shear elasticity against time;
this is termed the TEG
http://www.haemoscope.com/index.html
Page 242 of 336

BLEEDING DISORDERS
Abnormal bleeding can result from:
 Vascular disorders
 Thrombocytopaenia
 Defective platelet function
 Defective coagulation
VASCULAR BLEEDING DISORDERS
Vascular disorders are characterized by easy bruising and spontaneous bleeding from
small vessels. The bleeding time may be prolonged, but the platelet function testing is
normal.
Page 243 of 336

THROMBOCYTOPAENIA
Abnormal bleeding associated with thrombocytopaenia are characterized by

purpura, mucosal haemorrhage and prolonged bleeding after trauma. Before
investigating further, it is important to establish if the low platelet count is genuine or an
artefact. A clot in the sample, platelet clumping and/or platelet satellitism around the
neutrophils as it appears on the peripheral blood film can lead to a false low platelet
count.
Causes of thrombocytopaenia
Failure of production
 Selective megakaryocyte depression
o Rare congenital defects
o Drugs, chemicals, viral infections
 Part of general bone marrow failure
o Cytotoxic drugs
o Radiotherapy
o Aplastic anaemia
o Leukaemia
o Myelodysplasia
o Myelofibrosis
o Marrow infiltration
o Multiple myeloma
o Megaloblastic anaemia
o HIV infection
Shortened life splan

 Immune
o Autoimmune
o Idiopathic (ITP)
o Associated with SLE, CLL or lymphoma
o Infections like HIV, malaria, virus
o Drug induced
o Heparin
 Non-immune
o Disseminated intravascular coagulation (DIC)
o Thrombotic Thrombocytopaenic Purpura (TTP)
Sequestration (abnormal distribution of platelets)

 Splenomegaly (platelets pool in enlarged organ)
Page 244 of 336

Causes of thrombocytopaenia (continued)
Dilution
 Massive blood transfusion
Clinical features of thrombocytopaenia
 Purpura and more extensive petechial haemorrhages
http://www.netdoctor.co.uk/diseases/facts/purpura.htm (accessed 7/2/12)
 Conjunctival haemorrhage, nose and gum bleeding as well as menorrhagia
 Intracranial bleeding is rare
CLINICAL SYNDROMES
IMMUNE THROMBOCYTOPAENIC PURPURA (ITP)
It was formerly known as idiopathic thrombocytopaenic purpura and can be divided

into acute and chronic forms.
Acute ITP
It is most commonly seen in children. It typically follows vaccination or a viral infection
such as chickenpox or infectious mononucleosis (EBV). Most cases resolve
spontaneously and only about 10% becomes chronic. The diagnosis is one of
exclusion and it is debatable if a bone marrow investigation is needed.
Page 245 of 336

Chronic ITP
It is relatively common disorder, mostly occurring in young women. The cause is usually
unknown (idiopathic), but can be seen in association with SLE, HIV, CLL, Hodgkin’s
disease or autoimmune haemolytic anaemia.
Platelet auto-antibodies (usually IgG) target the epitopes (antigen sites) on platelets,
commonly glycoprotein IIb/IIIa. These sensitised platelets are then removed by the
macrophages of the liver and spleen. The platelet’s lifespan is reduced to a few hours.
http://www.medscape.org/viewarticle/420904_6 (accessed 7/2/12)
Laboratory diagnosis of ITP

 Plt low (10-50 x109/l)  Reduced platelet  Diagnosis usually by
numbers, often large exclusion
“stress” platelets  Marrow shows
normal or increased
megakaryocytes
Page 246 of 336

Treatment
A platelet count above 50 x 109/l generally does not require treatment.
Corticosteroids form the first line of therapy.
Splenectomy is considered when steroid therapy is not effective (to remove the place
of destruction).
THROMBOTIC THROMBOCYTOPAENIC PURPURA (TTP)
TTP can be acquired or familial. The problem in patients with TTP is that there is a
deficiency of a ADAMTS13 metalloprotease (enzyme) which breaks down the ultra
large von Willebrand factor multimers (ULVWF). Platelets form aggregates on the
ULVWF multimeric strings leading to large, occlusive platelet thrombi and consequently
thrombocytopaenia and microangiopathic haemolysis.
http://www.archemix.com/website/products_1779_ttp.php (accessed 14/10/11)
In the familial forms there are several mutations of ADAMTS13 and in the acquired
forms an inhibitory IgG auto-antibody develops, which may be stimulated by infection,
auto-immune disease, certain drugs, or cardiac surgery, among others.
The platelet thrombi cause organ ischaemia. Traditionally TTP was recognized by a
pentad of thrombocytopaenia, red cell fragmentation, neurologic abnormalities,
renal failure and fever.
TTP is now, in South Africa, most commonly associated with HIV-infection.
Haemolytic uraemic syndrome (HUS)

HUS resembles TTP, but mainly affects children and organ damage is limited to kidneys.
It is preceded by bloody diarrhoea caused by E.coli 0157 or Shigella infection.
ADAMTS13 levels are normal in HUS.
Page 247 of 336

Laboratory diagnosis of TTP

 Hb low  Red cell  S-Urea and s-Creat
 Plt low fragmentation to confirm renal
failure
 LDH high
 Clinical info to
neurological
abnormalities
 HIV status
 Normal coagulation
test (in contrast with
DIC)
 ADAMTS13 low or
absent
http://www.clevelandclinicmeded.com/medicalpubs/diseasemanagement/hematology-
oncology/disorders-platelet-function/ (accessed 7/2/12)
DIC will be discussed with the coagulation disorders.
SUMMARY
 It is important to confirm a low platelet count by inspecting the

sample for a clot or the blood film for clumping, satellitism or
fragments.
 Causes included failure of production, shortened life span, pooling
in the spleen and dilution.
 ITP is characterized by immunological destruction of platelets.
 TTP is characterized by anaemia, thrombocytopaenia, red cell
fragmentation, neurological abnormalities and renal failure.
Page 248 of 336

DEFECTIVE PLATELET FUNCTION
Platelet function disorders are suspected in patients with bleeding problems,

prolonged bleeding time and normal platelet count. These disorders may be
hereditary or acquired.
HEREDITARY DISORDERS
These include Glanzmann’s Thrombasthenia, Bernard Soulier syndrome and Storage

pool disease.
Glanzmann’s thrombasthenia
There is a deficiency of membrane GPIIb-IIIa which leads to failed primary platelet

aggregation. It presents in the neonatal period. The platelets fail to aggregate in vitro
to all agonists, except ristocetin.
Bernard Soulier syndrome
There is a deficiency of GPIb and is characterized by large platelets. There is also a

combination of thrombocytopaenia with the dysfunction and abnormal morphology.
There is defective binding to VWF, defective adherence to exposed subendothelial
connective tissues. Platelets do not aggregate with ristocetin.
Page 249 of 336

Storage pool disease
In the rare grey platelet syndrome, there is an absence of the -granules with
deficiency of their proteins. The platelets are grey and larger than normal.
Deficiency of the dense granules are seen in the more common δ-storage pool
disease.
ACQUIRED DISORDERS
Antiplatelet drugs
Aspirin is the most common cause of abnormal platelet function. The bleeding time is
prolonged and there is associated gastrointestinal haemorrhage. The cause of the
defect is inhibition of cyclo-oxygenase with impaired thromboxane A2 synthesis. There
is impairment of the release reaction and aggregation with adrenaline and adenosine
diphosphate (ADP). The defect lasts for the life of the platelet, i.e. 7 – 10 days.
Hyperglobulinaemia
Multiple myeloma or Waldenstrom’s disease can cause interference with platelet

adherence, release and aggregation.
Myeloproliferative (MPD) and Myelodysplastic disorders (MDS)
Intrinsic disorders of platelet function occur in many patients with MPD or MDS as well
as PNH. Acquired Von Willebrand Disease is sometimes seen in MPD due to adsorption
of the ULVWF multimers onto the platelets especially when the platelet count is raised.
Chronic renal failure
Ureamia is associated with various platelet abnormalities. Epistaxis (nose bleeds),

gastrointestinal haemorrhage and menorrhagia (heavy menstruation) are common
problems.
Page 250 of 336

DEFECTIVE COAGULATION
Coagulation disorders can be subdivided into hereditary and acquired disorders.
HEREDITARY DISORDERS
Hereditary deficiencies of each of the coagulation factors have been described, but
Haemophilia A (factor VIII deficiency), Haemophilia B (factor IX deficiency) and von
Willebrand disease are the most common.
HAEMOPHILIA A
The inheritance is sex-linked (or X-linked) recessive disorder meaning the problem lies
on the X chromosome. Thus, all men with the defective gene have haemophilia, all
sons of haemophiliac men are normal (because they inherited a normal X
chromosome from their mother) and all daughters are carriers.
http://www.haemophilia.org.au/bleedingdisorders/cid/25/parent/0/pid/1/t/bleedingdisorders/title/adul
ts (accessed 7/2/12)
Up to 30% of all new cases of haemophilia are a result from spontaneous mutations.
Clinical features of Haemophilia
 Joint and soft tissue bleeds and excessive bruising as soon as the child becomes
active.
Page 251 of 336

http://fn.bmj.com/content/92/4/F324.extract (accessed 7/2/12)
 Recurrent, painful haemarthroses (joint bleeding especially elbows or knees).
 Spontaneous haematuria and gastrointestinal haemorrhages can occur.
 Operative and post-traumatic haemorrhages are life-threatening.
 A serious complication of haemophilia is the development of antibodies

(inhibitors) to infused factor VIII. This makes further replacement therapy difficult
Laboratory diagnosis of Haemophilia A
Coagulation Clinical info Confirmatory tests

 APTT prolonged  Deep tissue, muscle,  Factor VIII low
 PT normal joint bleeds
 Bleeding time normal  Family history
Page 252 of 336

Treatment
The basis of treatment relies on replacement of factor VIII.

The treatment is complex, and severe disease is managed by a multidisciplinary team
dedicated to their care.
HAEMOPHILIA B
The inheritance and clinical features of Haemophilia B or Christmas disease are

identical to Haemophilia A. The only difference is that the deficiency is factor IX.
Antibodies to factor IX (or inhibitors) are less common.
VON WILLEBRAND DISEASE (VWD)
In this disorder, there are either reduced levels of von Willebrand Factor (VWF) or
abnormal function of VWF resulting from a point mutation or major deletion. VWF is
produced in endothelial cells and megakaryocytes. It promotes platelet adhesion to
damaged endothelium and is a carrier molecule for factor VIII. This will also explain
the low factor VIII seen in VWD.
http://www.orthosupersite.com/view.aspx?rid=26898 (accessed 4/2/2012)
Page 253 of 336

Classification of VWD
Type Name Description

 Type 1  Partial quantitative  Decreased concentration
deficiency
 Type 2  Qualitative  Functional abnormality

(further sub- deficiency
divided)
 Type 3  Complete deficiency  Rare
Laboratory diagnosis of VWD
Coagulation Additional info Confirmatory tests

 Bleeding time  Blood group O  VWF levels low
prolonged individuals tend to  Defective plt
 APTT prolonged - have lower VWF aggregation with
due to low F VIII levels ristocetin. Other
 PT normal agents normal.
 Platelet count  Multimer analysis –
normal, except for different subtypes
type 2B
Treatment
Mild bleeding requires little intervention. More significant bleeding responds to DDAVP
which stimulates release of VWF from stores. Patients are also treated with
intermediate purified factor VIII concentrate (similar to haemophilia A).
Page 254 of 336

SUMMARY
 Haemophilia A & B are sex-linked disorders which are characterized

by factor VIII and IX deficiency respectively.
 Bleeding often occurs in the joints.
 VWD is a relatively common disease.
 Deficiency of VWF causes abnormal platelet function and low
factor VIII activity.
 PTT prolonged in all cases, but bleeding time is only prolonged in
VWD. PT normal in all cases.
ACQUIRED COAGULATION DISORDERS
These are more common than the inherited disorders and multiple clotting factor
deficiencies are usual.
Causes
Deficiency of vitamin K dependant factor
 Haemorrhagic disease of the newborn
 Biliary obstruction
 Malabsorption of vitamin K (e.g. tropical sprue)
 Vitamin K antagonist therapy (Warfarin)
Liver disease
Disseminated intravascular coagulation
Inhibition of coagulation
 Specific inhibitors (e.g. against factor VIII)
 Non-specific inhibitors (e.g. SLE, RA)
Miscellaneous
 Diseases with M-protein production
 Therapy with heparin
 Massive transfusion syndrome
Vitamin K deficiency
Vitamin K is derived from green vegetables and intestinal flora.

Deficiency is caused by
 Inadequate diet
 Malabsorption
 Inhibition of vitamin K by drugs such as warfarin
Page 255 of 336

 Liver immaturity, low quantities in breast milk (haemorrhagic disease of the
newborn)
Vitamin K is stored in the liver and then acts as a cofactor for gamma-glutamyl
carboxylation of coagulation factors II (prothrombin), VII, IX and X as well as proteins C
and S. Non functional proteins, factors II, VII, IX and X, are called PIVKA (proteins
formed in vitamin K absence).
Drugs like Warfarin block Vitamin K epoxide reductase (VKORC), thereby inhibiting
maturation of clotting factors.
Liver disease
The liver produces all the factors of the intrinsic and extrinsic coagulation pathways. In
advanced liver disease, there are often multiple haemostatic abnormalities including
 Reduced synthesis of clotting factors
 Increased consumption of clotting factors (DIC)
 Low platelet counts (because of decreased thrombopoietin production)
 Low levels of fibrinogen as well as functional abnormalities
 Accelerated clot lysis
DIC is a combined thrombotic and bleeding disorder.

DIC begins as a result of procoagulant material that is released into the circulation. An
inappropriate widespread deposition of fibrin with consumption of coagulation factors
and platelets occur. The main clinical presentation is bleeding, but some patients
have microthrombotic lesions. Once the coagulation factors have been consumed,
disseminated bleeding occurs.
The key factor is the release of tissue factor into the circulation by damaged tissues,
malignant cells or injured endothelium. This leads to the generation of thrombin which
causes formation of fibrin, activation of platelets and fibrinolysis.
Causes of DIC
Entry of procoagulant material
 Severe trauma especially head injury
 Amniotic fluid embolism
 Premature separation of placenta
 Widespread mucin-secreting adenocarcinoma
 Acute promyelocytic leukaemia (AML M3)
 Liver disease
 Falciparum malaria
 Snake bites
Page 256 of 336

Widespread endothelial damage and collagen exposure
 Gram negative septicaemia
 Meningococcal septicaemia
 Septic abortion
 Certain virus infections (HIV, CMV, Varicella, etc)
 Severe burns
 Hypothermia
Endothelial Tissue damage Red cell/ platelet

damage damage
Activation of Activation of Release of

intrinsic pathway extrinsic pathway phospholipids
Formation of Platelet
thrombin consumption
Red cells over fibrin

strands
Fibrinogen Fibrin clot FDP/D-dimer
Depletion of
clotting factors
and platelets
HAEMORRHAGE
Page 257 of 336

Clinical features of DIC
 These are dominated by bleeding, oozing from venepuncture sites.
 Less frequently, microthrombi may cause skin lesions, gangrene of the fingers or
toes.
Laboratory diagnosis of DIC
Coagulation FBC and slide Confirmatory tests

 PT, APTT prolonged  Hb low  TT – grossly
 Thrombin time  Platelet count low prolonged
prolonged  Red cell fragments  Clinical picture
 Fibrinogen on slide
concentration low
 FDP/D-dimer high
 Antithrombin low
http://aliasgharkiani.blogfa.com/8911.aspx (accessed 7/3/2012)
Treatment
The underlying cause is treated. Replacement of coagulation factors is important.
SUMMARY
 Acquired deficiencies include liver disease, vitamin K deficiency,

DIC.
 DIC is characterized by red cell fragmentation, prolonged PT, PTT
and TT and increased D-dimers.
Page 258 of 336

THROMBOSIS
Thrombi are solid masses formed in the circulation from blood constituents. Thrombi
are involved in the pathogenesis of myocardial infarction, cerebrovascular disease,
peripheral arterial disease and deep vein occlusion. Risk factors include increasing
age, immobility, obesity, trauma/surgery, pregnancy, smoking, hypertension, family
history, hyperlipidaemia, diabetes mellitus etc.
http://www.nature.com/nm/journal/v17/n11/full/nm.2515.html (accessed 7/3/2012)
The term thrombophilia is used when the major mechanism for thrombosis is enhanced
coagulation.
Thrombophilia
Thrombophilia can be subdivided in hereditary disorders (familial) or acquired risk

factors.
Page 259 of 336

Familial thrombophilia could be caused by a genetic defect of the inhibitors of
coagulation or fibrinolytic system. The following abnormalities are risk factors:
 Factor V Leiden gene mutation (activated protein C resistance)
 Prothrombin G20210A gene mutation
 Protein C and S deficiencies
 Antithrombin deficiency
Factor V Leiden or activated protein C resistance (APCR)
The anticoagulant property of activated

protein C (APC) is the ability to inactivate
cofactors Va and VIIIa by limited proteolysis.
The mutation (an exchanged protein) is

located in an important blood clotting factor
(factor V). As a result of this mutation, factor V
is resistant to cleavage by activated protein C,
as the factor V cleavage site has changed.
http://www.ismaap.org/index.php?id=61 The patients are therefore more coagulable
called “hypercoagulability”.
“APC resistance” is a term used to denote this

state. APC resistance is therefore due almost
entirely to this qualitative change in factor V,
which is genetically pre-determined.
Factor V Leiden mutation can be determined

with PCR techniques.
Page 260 of 336

Protein C and S deficiencies
Hereditary deficiency of protein C is an autosomal dominant disorder found in 2 – 5 %

of patients with thromboembolic disease. An acquired deficiency can occur in liver
disease, DIC, warfarin treatment, pregnancy, oral contraceptives, auto-antibodies etc.
Protein S is a cofactor for protein C and the clinical features are similar to protein C
deficiency.
Antithrombin deficiency
Antithrombin is the major physiological inhibitor of thrombin and clotting factors IXa,
Xa, XIa and XIIa. Inheritance is autosomal dominant. (It can also be decreased in
DIC, liver disease, heparin use etc.).
Prothrombin G20210A
Prothrombin G20210A is a variant that leads to increased plasma prothrombin levels

and increased thrombotic risk.
Acquired risk factors include

 Post surgery (major abdominal or hip operations)
 Immobility
 Malignancy
 Inflammation
 Blood disorders (increased viscosity, thrombocytosis)
 Oestrogen therapy
 Antiphospholipid syndrome
 Obesity
Investigation for thrombopilia
 FBC – to detect elevation in haematocrit, white cell count, platelet count

 Blood smear – to find evidence of myeloproliferative disorder, malignant
disease etc.
 PT and PTT – APTT is often shortened. Prolonged APTT, not corrected by normal
plasma, may indicate lupus anticoagulant
 Anticardiolipin antibodies
 Thrombin time and reptilase time – if prolonged it suggest abnormal fibrinogen
Page 261 of 336

 Fibrinogen assay
 APCR or Factor V Leiden PCR
 Antithrombin assays
 Protein C and S assays
 Protein G20210A gene analysis
SUMMARY
 Thrombophilia is the term used for increased risk of thrombosis due

to enhanced coagulation.
 It can be either inherited or acquired.
Page 262 of 336

ANTICOAGULANT DRUGS
Anticoagulant drugs are widely used to treat venous thromboemolic disease.
Heparin
Two forms of heparin are widely used, unfractionated heparin and low molecular
weight heparin (LMWH). Both exert anticoagulant properties by binding antithrombin
and potentiating its activity. Antithrombin inhibits the actions of factor Xa and
thrombin. LMW heparin, in contrast to unfractionated heparin, has a greater anti-Xa
than antithrombin activity.
Unfractionated heparin is given by continuous intravenous infusion and is monitored

with the APTT. The objective is to keep the APTT ratio at 1.5 – 2.5. Heparin is continued
until oral anticoagulation is therapeutic.
Unfractionated heparin can inhibit platelet function and a heparin induced
thrombocytopaenia may develop.
LMW heparin (e.g. Clexane) has a longer half-life and can be given once per day by
subcutaneous injection. In comparison to unfractionated heparin, LMW heparin has a
more predictable dose response. Therefore routine monitoring by laboratory testing is
not necessary. Laboratory monitoring includes anti-Xa activity measurement and not
APTT. LMW heparin has a lower risk of induced thrombocytopaenia.
Warfarin
Warfarin is an oral anticoagulant which is a vitamin K antagonist. There is thus

decreased activity of the vitamin K-dependant factor II, VII, IX and X.
After warfarin is given, factor VII levels fall considerably within 24 hours, but factor II
(prothrombin) only falls to 50% of normal after 3 days. The dosage can therefore only
be adjusted after 3 days of use.
The INR is used to monitor therapy. Therapeutic range is usually between 2 and 3.5.
Indication Target INR

Deep vein thrombosis (DVT)
Pulmonary embolus
Atrial fibrillation 2.5
Cardioversion
Recurrent venous thromboembolism

Mechanical prosthetic heart valves 3.5
Thromboembolism in antiphospholipid syndrome
Page 263 of 336

Several drugs interfere with the actions of warfarin e.g. alcohol, antibiotics, aspirin,
corticosteroids, oral contraceptives, antihistamines etc.
Warfarin is usually stopped 3 days before surgery in order to get the INR <1.5. LMW
heparin is given and continued till INR >2.0 again after re-starting warfarin.
SUMMARY
 Unfractionated heparin is monitored with the APTT.

 LMW heparin requires no monitoring, but when it is necessary anti-
Xa activity is measured.
 The INR is used to monitor Warfarin therapy.
Page 264 of 336

WORKBOOK
Question 1
Different patients present with symptoms of abnormal bleeding. Their laboratory results
are given in column A. Examine them and identify the appropriate clinical
condition/cause from column B.
Column A Column B
PT APTT TT FIB PLT Condition
1 N N N N N A:Disseminated intravascular coagulation
2 Long N N N N B: Disorder of platelet function
3 N Long N N N C: Factor VIII deficiency
4 Long Long N N N D: Acute leukaemia
5 N N N N Low E: Vitamin K deficiency
6 Long Long Long Low Low F: Factor VII deficiency
PT = Prothrombin time APTT = Activated partial thromboplastin time

TT = Thrombin time FIB = Fibrinogen N = Normal coagulation times in seconds
PLT = Platelet count Long = Prolonged coagulation times in seconds
LOW = Coagulation time in seconds less than normal range
Question 2
Compare and contrast von Willebrand’s Disease and Haemophilia A by completing

the expected results for the tests listed.
Haemophilia A Von Willebrands

Disease
Platelet count
Bleeding Time
Prothrombin time (PT)
Partial thromboplastin time
(PTT)
Question 3
What tests would you include in an inherited thrombotic profile for a young patient
being investigated for a deep vein thrombosis in the lower leg?
Page 265 of 336

Question 4
1. What test is used to monitor Heparin therapy?

2. Which coagulation pathways are measured by the test referred to?
3. What other causes may also result in a prolonged test?
Question 5
1. What test is used to monitor Warfarin therapy?

2. Which coagulation pathways are measured by the test referred to?
3. What other causes may also result in a prolonged test?
Question 6
a) A patient has severe hepatocellular disease (liver disease) with congestive

splenomegaly. Complete the table below indicating whether the results of the
investigations listed will be normal, prolonged, shortened, high, or low and
explain the reason for each result.
Laboratory investigation Result Reason for result

Prothrombin time
Partial thromboplastin time
Platelet count
Bleeding time
b) A patient has a coagulopathy involving the fibrinogen molecule, which is

functionally abnormal. What term is used to describe this coagulopathy?
c) Which test is useful in the diagnosis of the above-mentioned coagulopathy?
Page 266 of 336

ERYTHROCYTE SEDIMENTATION RATE
Learning Unit 11
ERYTHROCYTE SEDIMENTATION RATE

terminology related to the erythrocyte sedimentation rate.
Assessment Criteria
learning unit eleven:
1.1 Demonstrate knowledge of the test indication.
1.2 Apply abnormal values.
1.3 Understand the factors that influence the ESR.
Page 267 of 336

Inflammation The body’s response to injury – it involves pain, heat,
redness, swelling and loss of function of the affected part.
Infection Invasion (and multiplication) of harmful organisms
(pathogens) in the body.
Inflammatory response to tissue injury includes alteration in serum protein

concentration, especially increases in fibrinogen, haptoglobin, immunoglobulins and
C-reacitve protein (CRP) – this is called the acute phase response.
The changes occur in acute infection, chronic inflammation, malignancy, acute tissue
damage and following physical injury. Measurement of the acute-phase response is a
helpful indicator of the presence and extent of inflammation or tissue damage. The
usual tests used to assess this response are the (CRP) and ESR.
The ESR is slower than the CRP to respond to acute disease and is less specific. The ESR
is influenced by immunoglobulins (which are not acute-phase reactants) and by
anaemia. Therefore the ESR rarely reflects the current disease activity and clinical
state of the patient as closely as the CRP.
The ESR is a useful screening test and the manual method is cheap, simple and
independant of power supply.
Westergren developed the method which is essentially the measurement of the

sedimentation of red cells in diluted blood in an open-ended tube of 30 cm length
mounted vertically on a stand, after one hour.
MECHANISM OF ERYTHROCYTE SEDIMENTATION
The rate of fall depends on the difference in specific gravity between the red cells and
plasma. This is influenced greatly by the extent to which rouleaux-formation (coin
stacking of red cells) occurs, more rapid sedimentation than single cells. Red blood
cells are negatively charged and will therefore repel each other. Serum proteins when
positively charged change the charge on the red blood cells causing them to stick to
each other along their flat surfaces thereby causing rouleaux.
The rouleaux formation and red cell clumping that are associated with increased ESR
are mainly controlled by the concentration of fibrinogen and other acute-phase
proteins (e.g. haptoglobin, ceruloplasmin and CRP). Rouleaux formation is also
enhanced by the immunoglobulins. The process is skewed by albumin.
Page 268 of 336

Anaemia, by altering the ratio of red cells to plasma, encourages rouleaux formation
and accelerates sedimentation.
Sedimentaion can be observed to take place in three stages:

 A preliminary stage (or aggregation stage or lag phase), then the
 Sedimentation stage
 Packing stage
The Aggregation phase
In the first ± 10 minutes the red cells come together in rouleaux formation (like stacked
coins). The more the rouleaux, the faster will be the next stage.
Coin-stacking
http://www.sciencephoto.com/image/304956/large/P2420459-Red_blood_cells,_SEM-SPL.jpg
The Sedimentation phase
In this stage, the red cells settle at a constant rate.
The Packing phase
During this stage, sedimentation slows as the cells pack at the bottom of the tube.
Laboratory diagnosis
Page 269 of 336

Manual ESR
Blood can either be collected directly into a specific citrate tube or EDTA blood can
be diluted in the proportion of 1 volume of citrate to 4 volumes of blood. The test
should then be carried out in the diluted sample without delay. The tube must be
placed vertical and left undisturbed for one hour, free from vibrations and draughts
and not exposed to direct sunlight.
It is then read to the nearest 1 mm of the height of the clear plasma above the
column of sedimenting cells. The result is expressed in mm/hour.
http://www.clinicallabwarehouse.com/polymedco-westergren-sediplast-esr-sys-tubes-3-8-sod-cit-100-
bx-s-1000/ http://train-srv.manipalu.com/wpress/?p=113089
Automated methods
Sedimentation is measured after aggregation has occurred and before the cells start
to pack, usually at 18-24 min. The rate during this time period is extrapolated to the
sedimentation that would have occurred after 60 minutes and converted to the
conventional ESR equivalent through an algorithm.
Page 270 of 336

http://www.indiamart.com/cpc-diagnostics/products.html
Technical sources of error affecting the ESR include:

 Specimen is too old (EDTA > 4 hours) results in crenation and sphering which
alters the shape and rigidity of the cells and therefore prevent stacking of red
cells, thereby decreasing the ESR
 Improper filling of ESR tube – bubbles will cause a falsely increased ESR
 Inaccurate timing – less than or greater than 1 hour
 Inaccurate reading
 Temperature – an increase in temperature, increases the ESR, a decrease in
temperature decreases the ESR (due to expansion and contraction of the tube)
 ESR tube not vertical – results in an increased ESR
 Vibration of ESR tubes – results in an increased ESR
Physiological and pathological factors increasing the ESR include:

 Increased levels of plasma proteins in infection, inflammation and malignant
conditions (including fibrinogen and IgM)
 Alpha2-macroglobulins – affect RBC aggregation
 Anemia – decreased numbers of RBCS
 Autoagglutination – clumps of red cells (not coin-stacks), clumps fall faster
 Macrocytes – increased surface area (heavier), RBCS sediment faster
 Haemolysis – destruction of the RBCS (decreased number)
REFERENCE RANGES
There is a progressive increase with age. Pregnancy increases the ESR, especially in
the later stages.
Adults Range (mm/hr)

Male 0 – 15
Female 0 – 20
Page 271 of 336

CLINICAL IMPLICATIONS OF ABNORMAL VALUES
Decreased Increased
No clinical significance Infection
Rheumatoid arthritis
Tuberculosis
Multiple myeloma
Pregnancy
Myocardial infarction
HIV infection
Anaemia
Systemic lupus erythematosis (SLE)
PLASMA VISCOSITY
In general the plasma viscosity and ESR increase in parallel with each other. Plasma
viscosity is, however, primarily dependant on the plasma proteins, especially fibrinogen
and is not affected by anaemia.
There are significant differences in plasma viscosity between men and women as well
as in pregnancy.
SUMMARY
 The ESR is a useful screening test for infection and chronic
inflammation.
 CRP is generally a better marker, as it is not affected by
anaemia or immunoglobulins.
 Sedimentation occurs in three stages:
o Aggregation
o Sedimentation
o Packing
Page 272 of 336

WORKBOOK
Question 1
Indicate if the following statement is True or False
The erythrocyte sedimentation rate increases during inflammation as the

concentration of plasma proteins increases.
Question 2
What is the principle of an ESR?
Question 3
What is the clinical significance of a raised ESR results?
Question 4
List sources of technical errors affecting the ESR.
Page 273 of 336

BLOOD GROUPS
Learning Unit 12
BLOOD GROUPS

terminology related to blood groups.
Assessment Criteria
learning unit twelve:
1.1 Differentiate between the ABO and Rhesus blood groups.
1.2 Demonstrate knowledge of the test principles.
1.3 Demonstrate knowledge of the direct and indirect Coombs tests.
Page 274 of 336

Genotype The genetic constitution of an individual (which we cannot
necessarily observe)
Phenotype The observable characteristics
Blood group antigens exist on the red cell membrane surface. The most important
blood group systems are the ABO and Rh systems.
ABO
Red cells can be group based on the presence or absence of three antigens A, B and
H. From these 4 blood groups can be distinguished i.e. A, B, AB or O (A and B absent).
Natural occurring antibodies (IgM) against A and/or B are found in the plasma of all
people whose red cells lack the corresponding antigen.
The antigens are present on most body cells including white cells and platelets. About
80% of the population possess secretor genes which allow them to secrete antigens in
body fluids e.g. saliva, semen and sweat.
http://www.beltina.org/health-dictionary/blood-type-abo-rh-types-a-b-ab-0.html
Phenotype/Blood Genotype Antigens Natural Frequency

group antibodies (IgM) (approximate)
O OO H Anti-A, Anti-B 45%
A AA or AO A Anti-B 40%
B BB or BO B Anti-A 10%
AB AB AB None 5%
Page 275 of 336

Blood donors with blood group O are considered universal donors, because there are
no AB-antigens. AB blood group is considered as the universal recipient, because
there are no antibodies against the blood group A or B in the plasma.
RHESUS (RH) SYSTEM
The term Rh refers to a complex blood group system that comprised up to 50 different
antigenic specificities, with the major one D or Rh0. Rh positive refers to the presence
of the D antigen on the red cells and Rh negative refers to the absence of the D
antigens. Rh antigens are highly immunogenic; meaning that they will elicit an
antibody response if exposed to the D-antigens. Its clinical relevance relates to
haemolytic disease of the newborn (HDN), an often fatal condition.
Fisher-Race
This theory involve the presence of 3 separate genes D, C and E (capital D,C,E) and
their alleles c and e and the absence of D (small c,d,e). No d has been found, and is
therefore considered a silent allele in the absence of D. The three genes are closely
linked on the same chromosome and are inherited as a group of 3 e.g. DCe or dCE
etc. The phenotype (the antigens expressed on the red cell that can be detected
serologically) of a red cell is defined by the presence or absence of D, C, c, E and e.
Weak D
The term “Du” is used to describe some weak reactions with the anti-D reagent.
Sometimes testing must be carried through the antiglobulin phase of testing to
demonstrate presence of D antigen.
The genetic inheritance of these antigens (that are complete but few in numbers) is
most frequently seen in the black population.
A second mechanism that may result in a weakened expression of D antigen is
described as a position effect. The arrangement of C in relation to D appears to
interfere with the expression of the D antigen – (DCe/dCe). The C in the opposite
chromosome suppresses the expression of D.
The mosaic D is the third mechanism where one or more parts of the D antigens are
missing.
Donor blood for transfusion is considered Rh positive if either the D or Du test is positive.
If blood types Rh negative, it must be confirmed with an indirect antihuman globulin
technique.
In possible transfusion recipients the application of Du is controversial. Because they
have the D antigen and cannot make alloanti-D, Rh positive blood may be transfused.
Very rarely the D-mosaic individuals can form alloanti-D when exposed to D-positive
red cells. It is therefore custom to rather transfuse a Du individual with Rh negative
blood.
Rh antibodies
Page 276 of 336

Unlike the ABO antibodies, most Rh antibodies are IgG and react at 37C. They do not
occur naturally, but are produced after exposure to foreign red cells, either through
pregnancy or transfusion. Exposure to less than 1 ml of Rh-positive red cells can
stimulate antibody production. Rh antibodies do no bind complement. Therefore
when an Rh antibody coats the red cells intravascularly, complement-mediated
haemolysis cannot occur. Haemolysis occurs usually extravascularly.
Because Rh antibodies are usually IgG and because the Rh antigens are well
developed early in fetal life; antibodies can cross the placenta of an Rh-negative
pregnant female and can coat the fetal red cells, leading to haemolysis of the fetal
red blood cells and subsequent anaemia. This condition is known as Haemolytic
disease of the newborn (HDN).
In HDN the direct antiglobulin test (DAT) is positive.
Image from http://www.aafp.org/afp/2004/0601/p2599.html (accessed on 2/3/11)
To prevent formation of anti-D it is prudent to:

 Always give Rh-negative females of childbearing age Rh-negative blood
 Administer anti-D during pregnancy in Rh negative females
Page 277 of 336

BLOOD GROUPING TESTS
Blood grouping testing traditionally relies on the visual identification of agglutination of

red blood cells induced by the presence of antibodies against antigens present on the
cell surface. Newer technologies include the use of gel tubes/columns.
ABO typing
Tube technique
The red cells suspended in saline, agglutinate

in the presence of anti-A or anti-B. Positive
reactions show as agglutinates; in negative
reactions the cells are dispersed.
Forward grouping: patient cells against

known antisera.
Reverse grouping (not shown): patient’s

serum against known cells.
http://www.homehealth-uk.com/medical/bloodgroup.htm
Page 278 of 336

Gel technique
In the 4 columns on the left, erythrocytes from the patient are added to the columns
which contain antibodies to the red cell membrane antigens. The patient's blood
group is B (Rhesus positive), so agglutination occurs and the resulting complex does
not pass through the gel column on centrifugation. On the 2 rightmost columns, serum
from the patient is added to the columns which contain antigens
http://www.ganfyd.org/index.php?title=Coombs'_test
Rh antigen typing
The presence or absence of the D antigen is demonstrated by testing the red cells (in
suspension) with serum anti-D. A positive reaction is demonstrated by agglutination of
the red cells and indicates Rh positive status.
If negative, it must be confirmed with an indirect antihuman globulin technique.
The test is performed with strict adherence to manufacturer’s instructions and with the
use of positive and negative controls.
Causes of false reactions with typing reagents

False positive False negative
Cold agglutinins Cell suspension too heavy
Incubated too long Forget to add reagent
Rouleaux Re-suspension too vigorous
Fibrin interference Incorrect reagent selected
Bacterial contamination Reagent deterioration
Incorrect reagent
Antibody testing
The patient’s serum is tested against screening red cells of known antigenic type; this
test is referred to as indirect antiglobulin test. This is to detect immune antibodies (non-
ABO) which may destroy donor red cells. Clinically relevant antibodies are generally
reactive at 37 ◦C. Agglutination may be enhanced by enzyme treatment of red cells
or the use of low ionic strength saline (LISS).
Page 279 of 336

The first step involves incubation of test red cells with serum and in the second step the
red cells are washed and antihuman globulin (AHG) reagent added. Agglutination
implies that the serum contained antibodies which have coated the red cells in vitro.
Direct Coombs
The direct Coombs or direct antiglobulin test (DAT) is used to detect antibodies or
complement on the red cell surface where sensitization has occurred in vivo. AHG
reagent is added to washed red cells. A positive test occurs in haemolytic disease of
the newborn (HDN), immune haemolytic anaemias and haemolytic transfusion
reactions.
TRANSFUSION REACTIONS
Haemolytic transfusion reactions may be immediate or delayed.

Massive intravascular haemolyis can occur as the result of complement-activating
antibodies of IgM or IgG classes, usually with ABO specificity. The DAT test will be
positive on a post-transfusion sample. The patient may have fever, bilirubinaemia,
anaemia, decreased haptoglobin and haemoglobinuria.
Page 280 of 336

The D antigen is the most immunogenic antigen apart from the ABO system.
Circulating antibodies appears within 120 days after primary exposure and within 2-7
days after secondary exposure.
Rh-mediated transfusion reaction usually results in extravascular haemolysis.
The patient may have fever, mild bilirubinaemia, anaemia and a decreased
haptoglobin. The direct antiglobulin test is positive and the antibody test may
demonstrate circulating antibodies.
Other immunogenic antigens include Kell, c, Duffy etc.
HAEMOLYTIC DISEASE OF THE NEWBORN (HDN)
HDN or erythroblastosis fetalis involves a reaction where Rh-antibodies produced by

the mother, cross the placenta and cause destruction of fetal red cells.
The mother was sensitized to form antibodies either through previous pregnancies or
transfusion, only a small number occurs during pregnancy itself, whereas ABO
incompatibility needs no prior stimulus. The incidence of the disease decreased with
the introduction of Rh immune globulin (Rhesugam).
Image from http://www.pennmedicine.org/health_info/pregnancy/000203.htm (accessed on 2/3/11)
Blood group antigen-alloantibody implicated:

Severe HDN Moderate HDN
ABO (<1%) ABO (<10%)
Rh-D (20%) Rh-D (30%)
Rh-c (7%) Rh-c (23%)
Kell - K1 (38%) Kell - K1 (30%)
Duffy - Fya (4%) Duffy - Fya (2%)
Page 281 of 336

The macrophages of the reticuloendothelial system remove the antibody-coated red
cells of the fetus. Depending on the amount of antibody, the amount of fetal red cells
destroyed can cause anaemia. The fetal bone marrow will produce more red cells,
releasing immature red cells into the circulation, from there the term erythroblastosis
fetalis. In severe cases, the fetus may die in utero. Extramedullary haemopoiesis
occurs in the liver and the spleen.
The term Hydrops fetalis is used to describe severe oedema, effusions (pleural and
pericardial) and ascites from the extremely enlarged liver and spleen, causing portal
hypertension and liver damage.
The destruction of red cells continues even after delivery because the antibody persists
in the circulation. The haemoglobin from the destroyed red cells is metabolized to
bilirubin. During gestation, it crosses the placenta and is eliminated by the mother.
After birth, the immature liver of the newborn cannot conjugate this bilirubin efficiently
and it can reach levels toxic to the infant’s brain. If left untreated, it can cause
kernicterus (bilirubin deposit in the basal ganglia) or permanent brain damage.
Exchange transfusion as well as administration of intravenous immunoglobulin is the

main treatment options.
Intrauterine transfusion can be performed by injecting red blood cells into the
umbilical artery. Early delivery to interrupt the crossing of maternal antibody can be
considered.
After delivery, phototherapy with ultraviolet lights to help with the breakdown of
bilirubin can be used.
Administration of Rh immune globulin (Rhesugam) as preventative measures occurs at

28 weeks and within 72 hours after delivery or a risk event (abortion, ectopic
pregnancy, etc.).
A Kleihauer-Betke test can be performed to estimate the amount of fetal cells in

maternal circulation. The chance of developing antibodies is related to the number of
fetal cells found. If the Kleihauer is positive, flow cytometry for more accurate results
can be performed. The amount of anti-D to be administered can be calculated from
the results of these tests.
Page 282 of 336

The presence of fetal cells in maternal circulation can be determined with a Kleihauer-Betke
test. Fetal cells contain fetal hemoglobin, HbF, which is resistant to acid elution. Adult Hb,
which contains little to no HbF, is not resistant, and will be lost from the cell. After incubation in
acid solution, the fetal cells will stain brightly with the pink dye, but adult cells will be ghostly
pale. The image shows about 10% fetal cells.
http://tulane.edu/som/departments/pathology/training/hematopathology_images_21.cfm
SUMMARY
 A or B blood group antigens exist on red cell membranes.
 Rh positive refers to the presence of the D antigen.
 Transfusion reactions can occur if blood is not carefully selected
and tested for antibodies.
 HDN refers to a severe disease where antibodies to D-antigen
cross the placenta and destroys the baby’s red cells.
 Clerical errors are the reason for most incompatible blood
transfusions.
Page 283 of 336

WORKBOOK
Question 1
You have received blood from a Rh negative woman who has just given birth to her
second Rh positive child.
1.1 What test would you perform to determine whether trans-placental haemorrhage
has occurred?
1.2 Describe the principle of the test.
1.3 What further testing would you recommend on the mother and why?
Question 2
2.1 Explain the difference between a Direct and Indirect Coombs test.
2.2 List possible causes of falsely negative Coombs reactions.
Question 3
The table below shows agglutination results obtained from four different donors.
Identify the ABO blood groups labelled 3.1 to 3.4.
Agglutination of test cells Agglutination by test Blood Group

with serum of
Anti-A Anti-B Anti-AB A cells B cells O cells
+ + + - - - 3.1
- + + + - - 3.2
- - - + + - 3.3
+ - + - + - 3.4
Page 284 of 336

Question 4
You are given 4 parents blood groups and 4 baby blood groups. Match the correct
parent/child group. Explain why.
Parents Baby
A Mother A- 1 A+
Father B-
B Mother B- 2 Twin 1: B-
Father A+ Twin 2: A+
C Mother A+ 3 O-
Father A-
D Mother A- 4 A-
Father A-
Page 285 of 336

Learning Unit 13
SYSTEMIC DISEASE OR SPECIAL SITUATIONS

terminology related to systemic disease or in special situations.
Assessment Criteria
learning unit thirteen:
1.1 Identify and describe abnormal morphological features
associated with specified clinical conditions
o Iron overload
o Non haematological malignancies
o Liver disease
o Renal disease
o Infections
o Parasites
o Down’s syndrome
o Splenic atrophy
o Pregnancy
o Neonatal haematology
Page 286 of 336

SYSTEMIC DISEASE OR SPECIAL
SITUATIONS
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Atrophy Wasting away of an organ due to degeneration of cells
IRON OVERLOAD
There is no mechanism to eliminate excess iron; therefore absorption is usually

regulated to avoid accumulation.
Causes
 Increased iron absorption
o Hereditary (primary) haemochromatosis
o Ineffective erythropoiesis e.g. thalassaemia, sideroblastic anaemia
o Chronic liver disease
 Increased iron intake

o African siderosis (dietary and genetic)
 Repeated red cell transfusions

o Transfusion siderosis
Hereditary haemochromatosis
There is an excessive absorption of iron from the gastrointestinal tract leading to an

overload of the liver, endocrine organs and sometimes heart. The most common
gene involved is HFE.
Clinical features
 Hepatic (liver) disease

 Endocrine disturbances such as diabetes mellitus or impotence
 Skin pigmentation
 Arthropathy (joint disease)
 In severe cases – cardiac failure or arrhythmia
Page 288 of 336

http://thesecretoftheblood.blogspot.com/
Laboratory diagnosis of iron overload

Diagnosis is suspected by increased serum iron, increased serum transferrin saturation
and ferritin.
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Treatment
Treatment is regular venesection.
Iron chelating therapy may also be used to treat iron overload during transfusions.
http://www.tree.com/health/iron-disorder-hemochromatosis-treatment.aspx
http://blogs.nejm.org/now/index.php/iron-chelating-therapy/2011/01/14/
Page 290 of 336

MALIGNANCIES (NON HAEMATOLOGICAL)
Anaemia is commonly seen in malignancies other than haematological tumours. The

cause is often anaemia of chronic disease, but can also include chemotherapy,
blood loss, haemolysis and bone marrow infiltration.
Invasion of the bone marrow by tumours can result in a pancytopaenia and a

characteristic leucoerythroblastic reaction (immature white cells as well as nucleated
red blood cells). In the bone marrow, clumps of the malignant cells may be seen.
Thrombocytosis (with an increased incidence of deep vein thrombosis) and

thrombocytopaenia (bleeding) are both associated with malignant disease.
Disseminated tumours (particularly mucin-secreting adeno-carcinomas) can cause
DIC.
LIVER DISEASE
A macrocytic (round) anaemia with target cells and occasionally stomatocytes is

associated with liver disease. Portal hypertension often results in splenomegaly.
Thrombocytopaenia occur due to the hypersplenism or inability to produce
thrombopoietin. The PT is usually prolonged due to deficiencies in factors II, VII, IX and
X and in severe cases factor V and fibrinogen.
RENAL DISEASE
Due to impaired erythropoietin production, there is almost always a normochromic

anaemia in chronic renal disease. There is variable shortening of the red cell lifespan
and burr cells (ecchinocytes) occur. The reticulocyte count is normal or slightly low.
Platelet and coagulation abnormalities are also associated with chronic/acute renal
failure. Thrombotic Thrombocytopaenic Purpura (TTP) and Haemolytic uraemic
syndrome (HUS) are discussed in the coagulation section.
INFECTIONS
Bacterial infections cause a neutrophil leucocytosis. A left shift (immature

granulocytes), toxic granulation and Dohle bodies may be present. Leukaemoid
reactions occur in severe infections. The ESR and C- reactive protein (CRP) are usually
moderately to severely elevated. DIC is associated with certain infections e.g.
meningococcal infections.
Viral infections cause a lymphocytosis with reactive lymphocytes. The CRP is usually
low or mildly elevated. Atypical lymphocytes are seen with infectious mononucleosis
caused by the Epstein-Barr virus (EBV). These lymphocytes are activated T-cells.
Page 291 of 336

HIV infection is associated with a wide range of haematological changes. These are
caused by a combination or direct effect of the virus, opportunistic infections and side
effects from the drugs used in treatment.
A steady decline in lymphocyte count is seen. Thrombocytopaenia and neutropaenia
may be immune or secondary to bone marrow dysfunction. Dysplastic features are
common. Anaemia is common and become more severe as the disease progresses.
Round macrocytes are associated with anti retroviral treatment.
Haematological problems associated with HIV infection are immune
thrombocytopaenic purpura (ITP) and lymphomas.
PARASITES
Malaria infections are associated with a degree of haemolysis. The most severe
abnormalities are seen with Plasmodium falciparum. In the worst case scenario, DIC
occurs with intravascular haemolysis and haemoglobinuria. Thrombocytopaenia and
anaemia are commonly found in malaria infections. Neutropaenia may also be the
result of the hypersplenism.
Plasmodium falciparum invades erythrocytes of all ages, whereas P.vivax and P.ovale
invade only reticulocytes and P. malariae only mature cells. Infections caused by
P.vivax and P.falciparum are the most common, and the latter is much more likely to
be life threatening.
http://www.parasitesinhumans.org/plasmodium-falciparum-malaria.html
Page 292 of 336

Malaria is carried by Anopheles mosquitoes. Of the over 400 Anopheles species, only
30–40 can transmit malaria. The infection starts, when a female mosquito injects (in her
saliva) "sporozoites" (one form of P. falciparum) into human skin while taking a blood
meal. A sporozoite travels (in the bloodstream) into the liver where it invades a liver
cell. It matures into a "schizont" (mother cell) which produces 30000–40000 "merozoites"
(daughter cells) within six days. The merozoites burst out and invade red blood cells.
Within two days one merozoite transforms into a trophozoite, then into a schizont and
finally 8–24 new merozoites burst out from the schizont and the red cell as it ruptures.
Then the merozoites invade new red cells. P. falciparum can prevent an infected red
cell from going to the spleen (the organ where old and damaged red cells are
destroyed) by sending adhesive proteins to the cell membrane of the red cell. The
proteins make the red cell to stick to small blood vessel walls. This poses a threat for the
human host since the clustered red cells might create a blockage in the circulation
system.
A merozoite can also develop into a "gametocyte" which is the stage that can infect
a mosquito. There are two kinds of gametocytes: males (microgametes) and females
(macrogametes). They get ingested by a mosquito, when it drinks infected blood.
Inside the mosquito's midgut, male and female gametocytes merge into "zygotes"
which then develop into "ookinetes." The motile ookinetes penetrate the midgut wall
and develop into "oocysts." The cysts eventually release sporozoites, which migrate
into the salivary glands where they get injected into humans. The development inside
a mosquito takes about two weeks and only after that time can the mosquito transmit
the disease. P. falciparum cannot complete its life cycle at temperatures below 20 °C.
http://www.parasitesinhumans.org/plasmodium-falciparum-malaria.html
The four types may be distinguished by their characteristic appearances within the red
cells. Please see table that follows.
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Stages in Erythrocytes Parasite Picture
blood
Ring Normal; multiple Delicate cytoplasm; 1-2 small

infection more chromatin dots; appliqué
common (accolle) forms
Trophozoite Normal; rarely, Seldom seen; compact

P. falciparum
Maurer’s clefts cytoplasm; dark pigment
Schizont Normal; rarely, Seldom seen; 8-24
(Meront) Maurer’s clefts merozoites; dark pigment
Gametocyte Distorted by Crescent or banana shape;
parasite chromatin in a single mass

(macro) or diffuse
(microgametocyte)

blood
Ring Normal to Large cytoplasm with

enlarged; fine occasional pseudopods,
Schuffner’s dots large chromatin dot
P. vivax
Trophozoite Enlarged; distorted; Large ameboid cytoplasm;

fine Schuffner’s dots large chromatin; yellowish-
brown pigment
Schizont Enlarged; distorted; Large, almost fill RBC; 12-24

(Meront) Schuffner’s dots merozoites, yellowish brown
pigment
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Gametocyte Enlarged; distorted; Round to oval; compact;
Schuffner’s dots almost fill RBC

blood
Ring Normal to Sturdy cytoplasm, large

enlarged; chromatin
occasional
Schuffner’s dots
Trophozoite Normal to Compact with large

enlarged; round to chromatin; dark-brown
oval; some pigment
fimbriated;
P. ovale
Schuffner’s dots
Schizont Normal to 6-14 merozoites; clustered

(Meront) enlarged; round to around mass of dark-brown
oval; some pigment

fimbriated;
Schuffner’s dots
Gametocyte Normal to Round to oval, compact,

enlarged; round to almost fill RBC,
oval; some
fimbriated;
Schuffner’s dots
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blood
Ring Normal Sturdy cytoplasm, large

chromatin
Trophozoite Normal; Ziemann’s Compact with large

P. malariae
stippling chromatin; occasional band

forms; coarse, dark-brown
pigment
Schizont Normal; Ziemann’s 6-12 merozoites; clustered

(Meront) stippling around mass of dark-brown
pigment
Gametocyte Normal; Ziemann’s Round to oval, compact,

stippliong almost fill RBC,
http://www.dpd.cdc.gov/dpdx/html/frames/m-r/malaria/body_Malariadiagfind2.htm
Trypanosoma brucei and T. brucei gambiense are responsible for East African and
West African variants of trypanosomiasis (sleeping disease) respectively. Both are
transmitted by the tsetse flies. The clinical problems are related to the central nervous
system.
T. cruzi cause an American trypanosomiasis or Chagas’ disease.
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http://www.parasitesinhumans.org/trypanosoma-brucei-sleeping-sickness.html
DOWN’S SYNDROME
Children with Down syndrome (DS) have a tenfold to twentyfold increased risk of
leukemia compared to children without DS. During the first 3 years of life there is an
increased risk for AML, particularly the megakaryoblastic subtype.
SPLENIC ATROPHY OR POST SPLENECTOMY
Hyposplenism can be the result of surgical removal or can also occur in sickle cell
anaemia, essential thrombocythaemia etc.
During the post-operative period, platelets often rise and peak at 1 – 2 weeks.
Thrombocytosis may persist. Howell-Jolly bodies, acanthocytes and target cells are
commonly seen on the blood film. Patients with hyposplenism are at a lifelong risk of
infection.
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PREGNANCY
Anaemia
Better O2 delivery
Neutrophilia Changes in
Main changes Main effect
pregnancy Optimising clotting
Thrombocytopenia
↑Clotting protein
Blood plasma volume increases by approximately 45% above the non-pregnant level.
It begins to rise early in pregnancy with most of the escalation taking place in the
second trimester. The red cell mass also increases, but to a lesser extent.
Erythropoietin levels increase throughout pregnancy. The overall effect of all these
changes is a slight drop in haemoglobin and haematocrit. Values below 10 g/dL are
probably abnormal and require investigation.
In uncomplicated pregnancy, the mean corpuscular volume (MCV) typically rises in
the second trimester.
The effect of pregnancy on the platelet count is somewhat controversial; some studies
show a mild decline in platelets, whereas others do not. Values less than 140 x 109/L
require investigation.
The white cell count rises during pregnancy with the occasional appearance of
myelocytes or metamyelocytes in the blood.
The levels of many procoagulant factors increase during pregnancy whereas activity
of the fibrinolytic system diminishes in preparation for the haemostatic challenge of
delivery.
Plasma levels of vWF, fibrinogen, factors VII, VIII, IX and X all increase markedly while
factors II, V and XII are essentially unchanged and factors XI and XIII decline.
Levels of protein C and antithrombin remain stable whereas protein S falls with
increasing gestation. Fibrinolysis is also impaired by increases in plasminogen activator
inhibitors I and II, the latter a product of the placenta.
Iron deficiency
There is an increased demand for iron during pregnancy because of

 the increased red cell mass,
 transfer of iron to the fetus and
 blood loss during delivery.
These requirements exceed the iron storage of most young women and in general
cannot be met by the diet. Even in cases of maternal iron deficiency the fetal
requirements are always met and therefore there are no correlation between the
haematocrit of the fetus and that of the mother.
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Iron deficiency is associated with a risk of preterm delivery or delivery of a low-birth-
weight infant.
A fall in the MCV is the earliest sign of iron deficiency.
Folate deficiency
Folate deficiency is the next most frequent nutritional deficiency leading to anaemia
in pregnant women. This is because of a combination of poor diet and exaggerated
folate requirements. Given the protective effect of folate against neural tube defects
(spina bifida), folic acid should be taken periconceptually and throughout pregnancy.
Food are also nowadays being fortified with folate.
Bleeding disorders and thrombocytopaenia
DIC is thought to arise from the procoagulant properties of amniotic fluid containing
vernix and fetal squamous epithelial cells in the pulmonary circulation followed by a
secondary fibrinolytic response. Life-threatening bleeding is seen with some
pregnancy-unique complications (placenta abruptio, retained dead fetus, and
amniotic fluid embolism) resulting in DIC.
Gestational and immune thrombocytopaenia
It is difficult to differentiate between gestational thrombocytopaenia and idiopathic

thrombocytopenic purpura (ITP).
Gestational thrombocytpaenia occurs later in pregnancy (second and third trimester)
and is less severe than ITP. The platelet count is always > 70 x 109/L and recovers within
6 weeks after delivery. No treatment is necessary and the infant is not affected.
ITP can occur during any point of the pregnancy and the thrombocytopaenia can be
severe. The problem is that the antibody can cross the placenta and the fetus may
become thrombocytopaenic.
In general patients with platelet counts < 10 x 109/L will require treatment, between 10
– 30 x 109/L who are in their second or third trimester and are bleeding will require
treatment and >50 x 109/L do not need treatment. Treatment can include steroids,
intravenous immunoglobulin and splenectomy. Newborns of mothers with ITP should
be monitored for up to a week after delivery to ensure that the platelet count does
not drop.
Eclampsia and HELLP syndrome
The spectrum of hypertensive disorders of pregnancy ranging from pre-eclampsia to

severe pre-eclampsia and HELLP syndrome to eclampsia may also result in
thrombocytopaenia. Pre-eclampsia is recognized by high blood pressure and
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proteinuria. HELLP (haemolysis, elevated liver enzymes and low platelet count) is
recognized by arterial hypertension, headaches, blurred vision, malaise, nausea, water
retention and a band pain around the upper abdomen. HELLP can lead to lung and
heart failure, liver and kidney damage, internal bleeding, stroke and other serious
complications in the mother.
The only effective treatment is delivery of the baby. Magnesium sulphate helps to
decrease the risk of seizures and progression to eclampsia.
Thrombosis
Pregnancy is a prothrombotic state with the consequent risk of thromboembolism.

There is an association between thrombophilic conditions in the mother and with
recurrent fetal loss. This is presumed to result from placental thrombosis and infarction.
Warfarin should not be used during pregnancy as it crosses the placenta and is
teratogenic (causes abnormalities in fetus). Low molecular weight heparin is the
treatment of choice because it does not cross the placenta and has a lower risk of
osteoporosis and heparin-induced thrombocytopaenia.
NEONATAL HAEMATOLOGY
Normal values
Cord blood haemoglobin varies between 16.5 – 17 g/dl with a progressive fall to ± 10
or 11g/dl at 8 weeks (termed nadir meaning low point) from which point it recovers
again to ± 12.5 g/dl at 6 months. Nucleated red blood cells are seen on the blood film
for the first 4 days and will persist in preterm infants for 1 week.
The MCV is initially very high (±120 fl) and decrease by about 9 weeks to adult levels.
At 1 year age, the MCV has fallen to around 70 and rises throughout childhood again.
Neutrophils are high at birth and decrease after about 4 days and from this point on
the lymphocyte count is higher than the neutrophils throughout childhood.
Abnormal values
Premature infants have a more marked fall in haemoglobin after birth and are more
prone to iron and folate deficiencies.
In Haemolytic disease of the Newborn (HDN), the baby has anaemia, jaundice with a
positive direct antiglobulin test.
Polycythaemia in neonates can be caused by placental transfusion (delayed
clamping of the cord), intrauterine hypoxia, endocrine disorders and genetic disorders
(e.g. Down’s syndrome).
Some causes of thrombocytopaenia in neonates include DIC in severe systemic
disorders, intrauterine infections, platelet antibodies, congenital disorders (e.g. TAR
syndrome) and others.
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WORKBOOK
Question 1
An adult male patient presents to the HIV clinic with signs of chronic infection. The
rapid HIV test was positive. Describe the haematological abnormalities (full blood
count and peripheral blood film) that may be found.
Question 2
Match the infection, disease or condition to the most suitable laboratory test
method in the list below to obtain a diagnosis. (Each test method can only be
used once)
1. Infectious Mononucleosis a. Serum/Urine Proteins
2. Neutrophil leukocytosis b. Prothrombin
3. Haemolytic Disease of the c. Kleihauer
New Born
4. Iron Overload d. Monospot/Paul Bunnell
5. Bernard Soulier Syndrome e. CD55/CD59
6. Liver Disease f. Perl’s Prussian Blue
7. Paroxysmal Nocturnal g. Myeloperoxidase
Haemoglobinuria
8. Acute Myeloid Leukaemia h. Neutrophil Alkaline
Phosphatase
9. Multiple Myeloma i. Haemoglobin
10. Polycythaemia j. Glycoprotein Ib
Question 3
Compare and contrast the infected red cells and the early trophozoite stages of P
falciparum, P.vivax, P.malaria and P ovale infections by using the following headings:
Size of infected red cells, shape of infected red cell, red cell inclusions, chromatin.
Question 4
A splenectomy was performed on a patient with haemolytic anaemia that failed to

respond to steroid therapy. Describe the peripheral blood smear picture you would
expect at 10 days post-splenectomy
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Learning Unit 14
METHODS
Learning Unit 14 provides you with the principles and methods and
methods of all test procedures relevant to haematology.
Assessment Criteria
learning unit fourteen:
1.1 Demonstrate knowledge of the principles of all test procedures.
1.2 Interpret the test results.
o Thin and thick stains
o Supravital stains
o Wright’s stains
o Special stains like Myeloperoxidase, Sudan Black, Periodic Acid
Schiffs, Non-specific esterase, Chloroacetate esterase
o Perl’s Prussian blue stain
o Leucocyte/Neutrophil Alkaline Phosphatase stain
o Prothrombin time, Partial Thromboplastin time, Fibrinogen
o D-dimer and Fibrin degradation products
o Bleeding time
o Osmotic fragility
o Kleihauer test
o Sickle cell test
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METHODS
o Haemoglobin electrophoresis
o Ham’s test
o Blood groups
o Direct and Indirect Coombs
o Donath Landsteiner
o Vitamin B12
Page 303 of 336

STAINS
ROMANOWSKY STAINS
Among these stains are the Giemsa and Wrights stain. Automated slide stainers or
rapidiff techniques are included.
Principle
The polychromatic stain consists of a mixture of:
 Methylene blue. (azure B) This colours the acid components of the cell, e.g. the
nucleus.
 Eosin Y. This colours the basic components of the cell, e.g. the cytoplasm.
Staining reagents are dissolved in absolute alcohol and are used as a fixative for the
preparation on the slide.
THIN AND THICK STAINS
Thin smears may be stained with the normal Wright’s stain.

Thick smears for parasites can be stained with either Field’s stain or Giemsa stain.
The pH (7.2) is critical for staining Schuffner dots.
MYELOPEROXIDASE STAIN (MPO)
The stain is mainly used to distinguish between AML and ALL. A lysosomal enzyme
localised to the azurophilic granules of neutrophils and monocytes are stained.
Developing granulocytes are always positive, strongly in promyelocytes and
myelocytes but may be NEGATIVE in early myeloblasts. Almost all mature neutrophils
give positive reactions as do eosinophils, basophils, promonocytes and monocytes.
SUDAN BLACK STAIN
Parallels MPO, staining the lipid membrane around the granules, but it takes much
longer.
ESTERASE STAIN
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Principle
Monocytes and macrophages and to some extent megakaryocytes, platelets and
certain lymphocytes will stain when exposed with alpha naphthyl acetate as substrate
with either Fast Garnet GBC or Fast Blue BB as diazonium salt.
The monoblasts stain positive with the non-specific esterase stain.
Specimen
Freshly prepared bone marrow smears of patient. For a positive control use a fresh
bone marrow smear.
Reporting results
Red brown granules in most normal monocytes and in leukaemic monoblasts.
Monocytes have fluoride sensitive enzyme. Granulocytes are usually negative.
Lymphocytes vary in positivity. Erythroblasts negative or only weakly positive.
Megaloblasts and erythraemic erythroblasts are often strongly positive.
Platelets show positivity.
IRON STAIN
Principle
The stain is based on the well-known Prussian-Blue (Perl’s) reaction. Ionic iron reacts
with an acid-ferrocyanide solution to give a blue colour. Iron stains of the marrow are
very useful in the diagnosis of iron deficiency anaemia. Developing normoblasts,
which contain iron granules, are known as sideroblasts. These are found normally, but
they are absent or their number is greatly diminished in patients with iron deficiency
anaemia. In sideroblastic anaemia on the other hand, the number of iron granules in
the normoblasts is greatly increased. Iron granules are greatly increased in peripheral
blood of patients who have been splenectomized. Haemosiderin is present in urinary
sediment of patients with intra-vascular haemolysis and may be demonstrated as free
granules or as granules within epithelial cells.
NEUTROPHIL ALKALINE PHOSPHATASE (N.A.P)
Principle
Several azo-dye methods using phosphates of naphthol or of naphthol derivatives as
substrates and various diazonium salts as capture agents are used satisfactorily.
Specimen
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Fresh finger prick smears of patient. For a positive control use fresh finger prick smears
taken from a pregnant lady. Fix the smears and keep unstained smears wrapped
individually in tissue paper in the freezer.
Reporting results
Scoring Results
Alkaline phosphatase activity is indicated by a precipitate of bright blue granules; cell
nuclei are stained red. Based on the intensity of staining and the number of blue
granules in the cytoplasm of the neutrophil, individual cells are rated as follows:
0: Negative, no granules
1: Positive but very few blue granules
2: Positive with few to a moderate number of granules
3: Strong positive with numerous granules
4: Very strong positive with cytoplasm crowded with granules
The score in an individual smear consists of the sum of the scores of 100 consecutive
neutrophils.
Calculation
A = 0 x number of neutrophils 0
B = 1 x number of neutrophils 1
C = 2 x number of neutrophils 2
D = 3 x number of neutrophils 3
E = 4 x number of neutrophils 4
A+B+C+D+E = NAP score in%
Normal value:
40 - 120 %
PERIODIC ACID SCHIFF’S (P.A.S)
Principle
The Periodic Acid Schiff's (P.A.S) reaction depends on the liberation of
carbohydrateradical from combination with protein and their oxidation to aldehydes
by Schiff's reagent. In blood cells a positive reaction usually denotes the presence of
glycogen.
Periodic acid oxidizes the 1-2 glycol group of various compounds - chiefly glycogen in
blood and marrow cells to produce a dialdehyde, which then gives the Schiff's s spot
reaction with leuco fuchsin (Schiff's) to release the strongly magenta - coloured
fuchsin. The reactivity of glycogen can be removed by pre-treatment of the cells with
salivary amylase.
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RETICULOCYTE COUNT
Brilliant Cresyl Blue stain or New Methylene Blue
Principle
Retics are young red blood cells; they contain remnants of the basophilic
ribonucleoprotein, which was present in large amounts in the cytoplasm of the
nucleated precursors from which they were derived. This basophilic material has the
property of reacting with certain dyes such as Brilliant Cresyl Blue to form a blue
precipitate of granules or filaments. As the number of reticulocytes in the peripheral
blood is a fairly accurate reflection of the erythropoietic activity, a reticulocyte count
is one of the essential procedures of the diagnostic Haematology. Reticulocytes are
greatly increased in the case of a haemorrhage or response to therapy in anaemias.
If the haemoglobin level is < 8.0 g/dl more blood should be added, and if the Hb > 18
g/dl less blood should be added.
The reticulocyte count assesses the amount of effective erythropoiesis taking place in
the marrow.
CYTOCHEMICAL STAINS (PICTURES)
Cytochemical stains are useful for the subclassification of acute myeloid leukemia.
Leukaemic blast cells stained by

myeloperoxidase showing a
brownish-black deposit in the
cytoplasm. This was a case of
acute myeloid leukaemia of FAB
M2.
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Leukaemic blast cells stained for α-
naphthyl acetate esterase (ANAE)
activity using fast RR as the dye. This
was a case of acute myeloid
leukaemia of FAB M5.
Composite photograph of a patient with AML-M1 who had a 95% PAS-positive blasts
(left image) and SBB (Sudan Black B) positive blasts (right image).
Composite photograph of the

peripheral blood film of a patient
with AML-M2, the blasts showed
unusual nuclear lobulation, these
blasts contained SBB positive
granules and Auer rods.
Page 308 of 336

Composite photograph showing Leishman staining (left) and SBB, cytochemical,
staining (right) diagnosis of the variant form of AML-M3
Leukaemic blast cells stained for

chloroacetate esterase (CAE)
activity, using Corinth V as the
dye. This was a case of acute
myeloid leukaemia of FAB M2.
Page 309 of 336

Iron in the form of haemosiderin is normally present in developing normoblasts and in
the RE cells of the bone marrow. The presence of iron is demonstrated by the blue to
blue-green granules.
NAP score
These slides point out the subjective nature of the scoring process ranging from a 4 to a 3 to a
2 to a 1. I am sure you can appreciate the inexact nature of this scoring process.
Page 310 of 336

COAGULATION
PROTHROMBIN TIME
Principle
The prothrombin time (PT) measures the extrinsic and common pathways. It is
measured by adding tissue thromboplastin, human or rabbit origin and calcium ions to
platelet poor plasma resulting in the activation of the extrinsic clotting factors,
thrombin generation and formation of a fibrin clot. Tissue factor can be released from
a wide range of cell types following rupture or damage and forms a calcium
dependant complex with factor VII. This test measures the activity of the extrinsic
pathway and will be abnormal when any of the following clotting factors are
abnormal or decreased, Factors VII, V, X, II (prothrombin), and I (fibrinogen). This test
is always performed when testing the effect of oral anticoagulants such as Warfarin.
The International Normalized Ratio (INR) is used as an international standard to which
effective anticoagulant therapy can be monitored. This makes it possible to regulate
anticoagulant therapy no matter where the test is done or which method is used.
Normal values for prothrombin time are between ±10 and ±14 seconds.
(For the Prothrombin test, tissue thromboplastin, which contains phospholipid plus a
tissue factor, is used as the activating agent. The Prothrombin Time measures factor VII
and the common pathway, i.e. Factors II, V, X and fibrinogen).
Results
Each batch of thromboplastin is standardized against the Commission of European
Communities Reference Thromboplastin. This allows the determination of the
International Sensitivity Index (ISI), which is a measure of the sensitivity of the reagent
and is used in the standardization of PT results.
The ISI of a batch of thromboplastin is indicated on the box, or package insert. This
value is used to convert a Prothrombin ratio obtained with this reagent to a
Prothrombin ratio that would have been obtained had the reference thromboplastin
been used.
This is done by raising the test Prothrombin ratio to the power of the ISI. The normalized
ratio thus obtained is termed the International Normalized Ratio (INR).
ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT)
In this test an activator such as ellagic acid triggers the intrinsic coagulation pathway
by activating the contact factors. In the presence of calcium ions and phospholipid
(partial thromboplastin), the contact factors cause a cascade of enzyme reactions
culminating in the generation of thrombin and fibrin clot formation.
Page 311 of 336

Principle
Ellagic acid binds to factor XII and alters its conformation exposing the active site.
Factor XIIa takes part in a positive feedback loop activating prekallikrein to kallikrein in
the presence of high molecular weight kininogen. This kallikrein feeds back onto
factor XII generating more factor XIIa. In a second kininogen catalysed reaction,
factor XIIa activates factor XI, which starts a series of reactions dependant on calcium
ions and phospholipids, with factor VIIIa and and factor Va as catalysts. Automated
APTT is used to test all factors in the intrinsic pathway (VIII, IX, XI & XII) including Factors
II, V, X and fibrinogen of the common pathway. It is also sensitive to the presence of
heparin, but it cannot be used to monitor oral anticoagulant therapy since it is not
sensitive for Factor VII nor is it sensitive to platelet dysfunction.
Plasma is incubated with pre-warmed APTT reagent for a standard period of time,
known as the contact activation time. The contact phase of coagulation is initiated
through the activation of Factor XII. Calcium is added which is essential for the
binding of coagulation factors to the phospholipid. Factor X and Prothrombin
activations are accelerated by phospholipid. Thrombin cleaves fibrinogen-producing
fibrin, producing first a visible change in turbidity and then a fibrin clot. Factor XIII
converts fibrin from an insoluble to a soluble form, thereby stabilizing the clot.
COAGULATION INHIBITOR STUDIES
The coagulation process can be retarded by several mechanisms and influencing

factors. The two main categories are factor deficiencies and inhibitors. Inhibitors are
factors, which have a direct effect on the coagulation process by inhibiting the
factors involved in the process. The following process describes the steps to be
followed when an inhibitor needs to be identified. While reading through the process
it will become clear that you would need to distinguish between an inhibitor and a
factor deficiency. This is done by performing correction studies. If the coagulation
time corrects by adding normal plasma, then there is a factor deficiency, but if the
coagulation time does not correct or corrects only very little, then you can suspect the
presence of an inhibitor.
By performing an APTT and PT you will immediately see which cascade is prolonged
and which factors are influenced. If the PT is prolonged, it will be factors VII, II, V, X
and/or fibrinogen. If the APTT is prolonged, it wil be factors XII, XI, IX, and/or VIII.
If both the PT and APTT is prolonged it can be a multiple factor deficiency or involve
only the factors of the common pathway. It is not very likely that you will encounter a
factor deficiency within the extrinsic pathway, since inhibitors such as Warfarin works
directly onto this pathway.
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D-DIMER
Principle
Latex particles coupled with a highly specified D-dimer monoclonal antibody are
mixed with the plasma test sample. Agglutination of the particles will occur if the
sample contains the target antigens (XL-FDP) at a concentration of approximately 0.25
mg/l or greater. The particles will remain un-agglutinated if XL-FDP antigens are absent
or at a concentration below 0.25 mg/l.
Specimen type
Plasma prepared from whole blood anti-coagulated with sodium citrate. Samples may
be stored at room temperature for 4 hours and plasma at -20°C for 2months.
Spin sample for 15 minutes at 3000 rpm to obtain platelet poor plasma.
FIBRIN DEGRADATION PRODUCTS TEST METHOD
Principle
In the presence of the corresponding antigens, the latex particles coated with
monoclonal anti-FDP antibodies agglutinate to form macroscopic clumps. The FDP
plasma kit allows the detection and the semi-quantitation of FDP.
XDP AND D-DIMERS TEST METHOD
Principle
Under the action of thrombin, fibrinogen is cleaved to give rise to fibrin monomers.
These monomers tend to associate themselves to form intermediate polymers, which
are subsequently stabilized by thrombin-activated F XIII (i.e. F XIIIa) with the
introduction of covalent cross-links in the region of the D-domain to produce the
insoluble fibrin clot.
The presence of the fibrin clot triggers the fibrinolytic system. Plasmin is formed at the
site of the fibrin clot. As a potent lysing enzyme, plasmin attacks the fibrin clot as well
as fibrinogen. However, unlike plasmin action on fibrinogen which produces
fibrinogen degradation products (FDP), which on the fibrin clot leads exclusively to the
generation of derivatives of cross-linked fibrin containing D-dimer. See figure 5.2:
FIBRINOGEN TEST METHOD
Principle
Fibrinogen (Factor I) is produced in the liver and has a half-life of 80–90 hours.
Fibrinogen is consumed during coagulation and is therefore absent in serum.
It is not Vitamin K dependant and tends to participate and precipitate together with
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Factors V, VIII and XIII.
Thrombin hydrolyses fibrinogen, releasing fibrinopeptides A and B to form fibrin

monomers. Fibrinogen may be increased in pregnancy, inflammatory disease and in
women using oral contraceptives. Fibrinogen can rapidly loose its activity in stored
plasma.
The principle of the fibrinogen test is that the clotting time of the plasma after the
addition of thrombin in excess is directly proportional to the concentration of
fibrinogen. The thrombin time can also be used to detect fibrinogen deficiency or
dysfunction.
THROMBIN TIME
Thrombin time is measured by adding thrombin to plasma and measuring the time it
takes to clot. Thrombin time is abnormal in cases of fibrinogen deficiency or when
conversion of fibrinogen to fibrin is inhibited by heparin or fibrin degradation products
(FDP). Heparin will prolong the thrombin time. This must be taken in consideration
when interpreting the results. Heparin can be neutralised by the addition of protamine
sulphate. Normal values ranges from 18 to 25 seconds.
Principle
Thrombin is added to plasma and the clotting time measured. The thrombin time is
affected by the concentration and reaction of fibrinogen, and by the presence of
inhibitory substances, including fibrinogen/fibrin degradation products and heparin.
The clotting time and the appearance of the clot are equally informative.
A prolonged thrombin time is indicative of low fibrinogen and of interference in the

thrombin- fibrinogen reaction, e.g. heparin, abnormal fibrinogen, very high FDP's,
intravascular coagulation (DIC), heparin therapy, qualitative and quantitative
fibrinogen abnormalities and increased fibrinolysis.
BLEEDING TIME TEST METHOD
The bleeding time test is done to detect abnormal platelet function. The test measure
platelet plug formation in vivo. In the Ivy template method, after the application of 40
mm Hg pressure to the upper arm with a blood pressure cuff, two 1-mm deep, 1-cm
long incisions are made in the flexor surface of the forearm skin. Bleeding normally
stops within 3-9 minutes. Prolongation is common in cases of platelet counts less than
75 x 10⁹/ℓ and platelet function disorders.
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Principle
The Bleeding Time Test is done to measure both vascular, VWF and platelet factors
associated with haemostasis.
This test primarily detects platelet function disorders and VWD.
PLATELET AGGREGATION STUDIES
Principle
The study of platelet aggregation response to a number of stimuli is an essential part of
the investigation of a patient with suspected platelet dysfunction. Necessary
equipment for platelet aggregometry includes an instrument known as a platelet
aggregometer and a chart recorder.
Platelet-rich plasma is placed in a siliconized tube, warmed at 37°C in the heating

block of the instrument and stirred by means of a small bar magnet or mechanical
stirrer. Light transmission through the plasma is monitored continuously on a chart
recorder. The addition of an aggregating agent results in the formation of increasingly
larger platelet aggregates with a corresponding decrease in optical density. The
change in optical density is recorded as a tracing by the chart recorder.
Platelet aggregation responses
Arachidonic
"Strong"
Disorder Collagen Adrenalin Ristocetin acid (Na
ADP
salt)
Thrombosthaemia Abnormal Abnormal Abnormal Normal † Abnormal
Bernard-Soulier
Normal Normal Normal Abnormal Normal
syndrome
Von Willebrand
Normal Normal Normal Abnormal* Normal
disease
Cyclooxygenase or
Thromboxane
Normal Abnormal Abnormal Normal Abnormal
synthetase
deficiency
Storage pool
Normal Abnormal Abnormal Normal Normal
disease
Page 315 of 336

"Aspirin defect" Normal Abnormal Abnormal Normal † Abnormal
* The finding of a normal aggregation response to ristocetin does not exclude the
diagnosis of Von Willebrand's disease. In the presence of other suggestive evidence,
plasma should be assayed for factor VIII:WF
† NOTE: Ristocetin 15 mg/ml.
In 40% of VWD Type IIb you may get some aggregation at this dilution, but it will be
abnormal aggregation. This is why the 0.5 mg/1 dilution becomes important to
differentiate this sub type. (Treatment is different)
In normal individuals no aggregation should be observed at the 0.5 mg/1
concentration. Only a straight line will be observed. If a patient is Type IIb, you will
have aggregation, but you need to do the ristocetin co-factor and VW antigen
before you could make a diagnosis.
In normal individuals, normal aggregation will be obtained using the 15 mg/ml
concentration.
LUPUS ANTI COAGULANT
Lupus anticoagulant testing is frequently requested in routine coagulation laboratories.

4.5 ml blood (9 volumes) is collected in 0.5 ml of 32% tri-sodium citrate (1 volume).
These tubes are available commercially (light blue capped Vacutainer). For accurate
results, the volume of blood must be within 10% of the stated volume
Centrifuge the blood sample at 3 000rpm for 15minutes, remove the plasma, avoiding
the plasma-buffy-coat interface, and transfer to a plastic tube
This plasma is then centrifuged again at 3 000rpm for 15 minutes and the plasma again
removed avoiding the interface to obtain platelet poor plasma.
Great care must be taken with the centrifuging as the presence of platelets in the
plasma could mask the effect of the lupus anticoagulant.
Tubes should remain capped throughout centrifugation to minimise pH changes and
to prevent aerosol affect or evaporation.
Haemolysed or jaundiced plasma should be stated on the test report.
A platelet count is done on each sample, to verify that the count is < 10 x 109/l.
Read the patient history form carefully. Medication is important, especially Warfarin or
Heparin.
Principle of the methods:

Lupus anticoagulants are a fascinating paradox occurring frequently in the
coagulation laboratory. Although often prolonging clotting tests to a greater degree
than in the haemophilias, they are rarely associated with bleeding but frequently
occur along with thrombotic problems, recurrent miscarriages and phospholipid-
binding antibodies.
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Criteria for a positive lupus anticoagulant:
 Prolongation of a phospholipid-dependant clotting test such as the APTT-LA,

Kaolin Clotting Time (KCT) or diluted Russel’s Viper Venom Test (dRVVT)
 Clotting time of mixture of test and normal plasma should be longer than the
clotting time of normal plasma.
 There should be a relative correction of the defect by the addition of lysed
platelets or phospholipids.
 The absence of any other specific coagulation factor defect must be ruled out.
There must be no specific factor-inhibitor or factor deficiency. The only factor
inhibitor that can occur with LA is a Factor II inhibitor (prothrombin deficiency).
 A confirmatory test such as the Platelet Neutralisation Procedure must be
positive.
The lupus anticoagulant is an antibody which exerts an inhibitory effect on

phospholipids, due to a neutralisation of the phospholipids used in some clotting tests
such as the activated partial thromboplastin time. The principle of the APTT-LA test is
based on the measurement of a plasma recalcification time in the presence of
cephalin and an activator. The presence of a lupus anticoagulant will prolong the
clotting time. The reagent has been sensitised which specifically enhances the
prolongation of the clotting time due to lupus anticoagulant in plasma.
Diluted Russel’s Viper Venom (dRVVT) has emerged as the second most frequently
used test for confirmation of LA. The dRVVT evaluates the final common pathway of
coagulation (RVV directly activates X to Xa). The dRVVT may yield false positive results
in the case of factor VIII or IX inhibitors or deficiencies.
The Kaolin Clotting Time (KCT) is essentially an activated partial thromboplastin time
(APTT) test without any added phospholipid. The latter is believed to be the main
target of antiphospholipid antibodies which are often associated with lupus
anticoagulants and which therefore prolong the test.
The Platelet Neutralisation Procedure (PNP) is done with washed frozen-thawed
platelets prepared in the laboratory. The PNP is based on the ability of an excess of
phospholipids (present in platelets) to bypass the inhibition through the inhibitor and
therefore significantly correct in vitro coagulation abnormalities.
RUSSELL'S VIPER VENOM TEST
Principle
Russel’s viper venom directly activates factor X, “by-passing” factor VII of the extrinsic
coagulation pathway and the contact and antihaemophilic factors of the intrinsic
pathway. Therefore the DRVVT is more specific for LA than APTT’s as it is neither
affected by contact factor abnormalities nor by factor VII deficiencies or antibodies.
Additional phospholipid is present in DRVV Confirm Reagent to neutralise LA.
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Russell’s viper venom directly activates factor X to factor Xa in the presence of
phospholipid and calcium, and completes the final conversion of fibrinogen to fibrin
leading to detectable clot formation in the plasma. This direct activation bypasses the
contact and intrinsic factors in the coagulation cascade thereby excluding
interference from deficiencies factors VIII, IX, XI and XII and their respective inhibitors.
This test may also be performed on samples with a normal APTT as the dilution and
type of phospholipid contained in the reagent increases the tests sensitivity and
specificity to LA.
The DVV confirm is formulated similarly to DVV except that it contains a higher
phospholipid concentration. This higher concentration allows the reagent to correct
the prolonged result of the DVV test in the manner of the modified platelets
neutralisation procedure if LA is present. The use of this confirmatory reagent provides
the means for the diagnosis of LA’s interfering antibodies.
Page 318 of 336

HAEMATO-IMMUNOLOGY
ABO BLOOD GROUPING PROCEDURE
Principle
There are 4 main groups i.e. A, B, AB, O, based on the presence of 2 antigens A &
B. The patient’s red cells and serum are both grouped and the 2 results compared.
Anti-A, Anti-B, and Anti-AB (group O) sera are required for the cell grouping test. A
and B cells are required for the serum grouping tests.
Serum of group O persons normally contains Anti-A and Anti-B; that of group A
contains Anti-B; that of group B persons contain Anti-A and that of group AB persons
contain neither Anti-A or Anti-B. Anti-A and Anti-B are always, to some extent,
naturally occurring and IgM in type.
The test used with test reagents is based on the principle of direct haemagglutination.
Normal human erythrocytes will clump or agglutinate when mixed with anti-A, or Anti-B
or Anti-A and B if they possess A and / or B antigens.
RH SYSTEM
This system is coded by allelic genes at three closely linked loci; alternative antigens
Cc, Ee together with D or no D (termed “d”) exist. Thus a person may inherit CDe from
the mother and cde from the father to have a genotype CDe/cde. There is a
shortened nomenclature for these linked sets of genes shown in table 5.4.
The Rh system – genotypes:
Caucasian
CDE nomenclature Short symbol Rh D status
frequency (%)
Cde/cde Rr 15 Negative
CDe/cde R1r 32 Positive
CDe/Cde R1R1 17 Positive
cDE/cde R2r 13 Positive
CDe/cDE R1R2 14 Positive
cDE/cDE R2R2 4 Positive
Positive (almost
Other genotypes 5
all)
Rh antibodies rarely occur naturally; most are immune, i.e. they result from previous
transfusion or pregnancy. Anti-D is responsible for most of the clinical problems
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associated with the system and a simple subdivision of subjects into Rh D positive and
Rh negative using anti-D is sufficient for routine clinical purposes. Anti-C, anti-c, anti-E
and anti-e are occasionally seen and may cause both transfusion reactions and
haemolytic disease of the newborn. Anti-d does not exist.
DIRECT COOMBS TEST
Principle
Polyspecific antihuman globulin (AHG) reagents are used which contain an antibody
against human IgG and the C3d component of human complement and may also
react with IgA and IgM molecules. The most important function of the AHG reagent is
to detect the presence of IgG. A positive Direct Antiglobulin generally indicates that
the red cells are coated in vivo with immunoglobulin and/or complement.
The Ortho Biovue system utilizes column agglutination technology comprised of glass
beads contained in a column, which, upon centrifugation of the cassette trap
agglutinated red blood cells and allow non-agglutinated red blood cells to travel to
the bottom of the column.
Specimen
Fresh EDTA or Citrate blood or clotted specimen without gel
INDIRECT COOMBS TEST
Principle
Polyspecific antihuman globulin (AHG) reagents are used which contain antibody to
human IgG and the C3d component of human complement and may also react with
lgA and IgM molecules. The most important function of the AHG reagent is to detect
the presence of IgG. A positive Direct Antiglobulin generally indicates that the red cells
are coated in vivo with immunoglobulin and/or complement.
The Ortho Biovue system utilizes column agglutination technology comprised of glass
beads contained in a column, which upon centrifugation of the cassette trap
agglutinated red blood cells and allow non-agglutinated red blood cells to travel to
the bottom of the column.
Specimen
1 x 5ml fresh EDTA specimen
1 x 5ml clotted tube without gel
Page 320 of 336

KLEIHAUER TEST FOR FOETAL HAEMOGLOBIN IN RED CELLS
Principle
The identification of red cells containing HbF depends on the fact that they resist acid
elution to a greater extent than do normal adult red cells. Therefore after elution in an
acid medium, cells containing HbF appear as darkly stained cells, whereas cells
containing normal adult haemoglobin appear as pale staining ghost cells. Cells,
which stain to an intermediate degree, may be reticulocytes.
Rh haemolytic disease of the newborn is almost always the result of the destruction of
an infant’s D-positive red cells by IgG anti-D antibody produced by its D-negative
mother. Large volumes of foetal blood (0.5 ml or more) are usually found after delivery,
and they are found more commonly in woman who have had traumatic and
manipulative procedures during labour and delivery such as manual removal of the
placenta or caesarean section – it is these women who are particularly likely to form
Rh antibodies. It has been shown that the injection of anti-Rh antibody into an
unsensitised Rh negative woman immediately after the delivery (within 24 hours) of an
Rh positive infant can prevent the possible formation of Rh antibody. To identify such
mothers, a blood sample should be taken within 2 hours of delivery and examined with
the Kleihauer test for foetal cells.
The size of the Transplacental haemorrhage (TPH), in ml of foetal cells, can be

calculated from the following formula:
_______________2400________________________
TPH in ml = proportion of adult: foetal red cells in maternal blood
(The red cell volume of the mother is assumed to be 1800ml, the ratio of the MCV of
foetal cells to that of adult red cells is assumed to be 1.20 and 90% of the foetal red
cells are assumed to be stained)
This method can also be used to distinguish HPFH (Hereditary persistence of foetal
haemoglobin) from other conditions where HbF arises (e.g. β thalassaemia). In HPFH,
all cells containing foetal haemoglobin have the same amount of foetal haemoglobin
in each cell and the stain is distributed evenly in the cells.
In other conditions where HbF is present in the red cells, the amount of HbF present in
individual’s cells may vary.
DONATH-LANDSTEINER
Principle
The Donath Landsteiner antibody (D-L antibody) of Paroxysmal cold haemoglobinuria
differs from the high titre cold antibodies in that it is an IgG antibody and is relatively
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far more lytic to normal cells, so much so that the lysis titre may be the same as or
greater than the agglutination titre. Almost maximal lysis develops in unacidified
serum. It is an IgG antibody which is activated by cold temperatures e.g. hands and
feet exposed to cold in winter, but is functionally active at body temperature (37 ˚C)
and cause haemolysis at body temperature.
OSMOTIC FRAGILITY
Principle
The osmotic fragility test determines the resistance of erythrocytes to haemolysis in
hypotonic saline solutions and is used mainly in the differential diagnosis of haemolytic
anaemias (and others) in which the physical properties of the RBC's are changed. The
ability of red cells to take up water is determined by their volume to surface area ratio
and functional state of the cell membrane.
A normal red cell withstands hypotonic solutions because its biconcave shape allows
the cell to increase its volume by about 70 %. Once this limit is reached, lysis occurs
(about 50 % of normal erythrocytes will haemolyse at saline concentrations of 0.40 -
0.44 % NaCl.)
Increased osmotic fragility will be seen in cells where the volume to surface area ratio
is increased (i.e. cells which have lost membrane or cells which are relatively "full" of
water before testing) and also in cells where membrane function is abnormal so that
hydrophilic (water carrying) substances like Na⁺ will enter the cells in increased
amounts. An increased osmotic fragility is seen in most acquired haemolytic
anaemias with spherocytosis including warm AIHA, HDN due to ABO incompatibility,
malaria and most notably in hereditary spherocytosis (other inherited HA's are usually
normal). Incubation of blood for 24 h @ 37°C may reveal abnormalities not detected
at room temperature (as in hereditary elliptocytosis.)
Decreased osmotic fragility occurs in cells whose volume to surface area is decreased
for some reason - either "empty" cells lacking Hb, cells with "extra" membranes as in the
thin leptocytes of liver disease, hypochromic cells and target cells of iron deficiency,
thalassaemia and sickle cells. Reticulocytes and normoblasts also demonstrate
decreased osmotic fragility and it is an expected finding in post-splenectomy and
during pregnancy.
Specimen
2 x 5ml Heparin, 1x 5ml EDTA for patient
2 x 5ml Heparin, 1x 5ml EDTA for control
1x 5ml Heparin of patient and control to be stored in the waterbath at 37˚C overnight
for post incubation fragility
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If only 1 Heparin specimen of patient and/or control is received, divide specimen into
equal amounts, by using a syringe. Do not open the specimen cap. Do not squirt the
blood through the needle into the second tube (to prevent haemolysis).
Put the EDTA specimen of patient through the instrument for a blood count, make a
slide, check for spherocytes, do a reticulocyte count.
Put the EDTA specimen of control through the instrument to verify that the count is
normal. If not normal, then another control specimen must be drawn.
Quality control
Normal control - 2 x 5 ml Heparin, treated the same as the patient. Graph of control
should be within range for the test to be acceptable.
Procedure
For unicubated fragility, make up 1% saline (1g NaCl made up with 100 ml of distilled
water).
Mark two sets of plastic screw cap tubes. One set for the patient, 1-15 for example P1,
P2 etc. and one set for the control, 1-15 example C1, C2 etc.
Using the 1% saline and distilled water make up the following saline concentrations.
These dilutions must be very accurate.
NB. The saline must be made up freshly before the test is done.
Page 323 of 336

Tube Concentration saline Volume saline in ml Volume distilled water
in ml
1. 0.24% 1.2 ml 3.8 ml
2. 0.28% 1.4 ml 3.6 ml
3. 0.32% 1.6 ml 3.4 ml
4. 0.36% 1.8 ml 3.2 ml
5. 0.40% 2.0 ml 3.0 ml
6. 0.44% 2.2 ml 2.8 ml
7. 0.48% 2.4 ml 2.6 ml
8. 0.52% 2.6 ml 2.4 ml
9. 0.56% 2.8 ml 2.2 ml
10. 0.60% 3.0 ml 2.0 ml
11. 0.64% 3.2 ml 1.8 ml
12. 0.68% 3.4 ml 1.6 ml
13. 0.72% 3.6 ml 1.4 ml
14. 0.76% 3.8 ml 1.2 ml
15. 0.80% 4.0 ml 1.0 ml
After adding saline, each successive tube should show a volume gradient, i.e. each
successive tube should have 0.2 ml more saline than the previous tube. After adding
distilled water, each tube should show the same volume of 5 ml.
 Mix each tube well after making up saline and distilled water dilutions. Take
care not to spill any of the dilution, as the volume of each tube must stay
constant at 5 ml in order to make a standard comparison. If the tubes are left
to stand for any length of time, they must be mixed again before continuing.
 Label two 15 ml tubes, one for patient, and one for control. Add 1 ml of patient
blood to patient tube and 1 ml control blood to control tube. Roll the tubes for
5 minutes on the bench in order to aerate the blood.
 Gently add 50 μl patient's aerated blood (to standardize method) to all patient
dilutions and 50 μl control aerated blood to control dilutions starting from the
lowest saline concentration i.e. from tube 15-1. Wipe the pipette tip after each
dilution is made. Put the caps back on the tubes.
 Mix gently by inverting and allow standing undisturbed at room temperature for
15 minutes.
 Mix gently and allow standing undisturbed at room temperature for a further 15
minutes.
 Spin down in centrifuge at 4000 rpm for 15 minutes.
 Use the patient blank supernatant, tube 15, to zero the spectrophotometer.
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 Read the optical density of all the patient's tubes supernatants at 540 nm,
starting with tube 14 and ending with tube 1 and enter onto the patients result
sheet.
 Repeat for the control, using control blank, tube 15 to zero the
spectrophotometer and enter onto the results sheet.
 If either the patient or control tube 15 has visible haemolysis, the tube cannot be
used as a blank, in such a case use 5 ml of normal saline (0.85 %) and 50 μl of
the relevant aerated cells. (Spin down first, before reading)
 For the incubated fragility repeat the entire method 24 hours after receiving the
specimen in the laboratory. Specimen must have been incubated at 37°C in
the waterbath.
 Calculate the % haemolysis of each dilution in the following way.
 Tube 1 = 100% haemolysis. If the optical density of tube 1 = 62, then an optical
of 40 will give % haemolysis as follows:
 Graphically plot the % haemolysis on the X axis against the saline concentration
on the Y axis, of both patient and control. Repeat for post incubation.
 Results must be interpreted together with the patient's blood count, blood
smear, reticulocyte count and coombs test.
 Reporting results
 Osmotic fragility result
 Normal
 Increased (if increased when 1st fragility is

done, then no 24 hr fragility must be performed)
 Decreased
 A population of cells show increased red cell fragility. (This occurs when there is a
tail in the result graph)
Notes for interpreting results

The osmotic fragility results are reported as a curve on linear graph paper, relating %
haemolysis to saline concentration for each solution and including both the test
(patient) and normal control curves, and the saline concentration for the patient at
which 50 % haemolysis occurred (MCF).
Read off 50 % haemolysis of patient and control from the graph. If the 50 % haemolysis
is more than 0.43 % then the fragility is increased. If the 50 % haemolysis is less than 0.39
Page 325 of 336

% then the fragility is decreased. If the 50 % haemolysis is between 0.39 % and 0.43 %
then the fragility is normal.
If the patient graph gives an increased tail at the bottom, but the 50 % haemolysis is
normal, then the patient has a population of cells with an increased fragility.
NB: The control graph must be normal.
Limitations of procedure
1 % Saline must be made up accurately.
The saline and distilled water dilutions must be accurate.
All tubes and pipettes must be clean.
The incubated sample may not have the lid removed until the incubation period is
over. If blood is needed from this tube, it must be removed by using a syringe through
the lid and gently (without needle) transferred into a clean tube with a lid.
Heparin specimens must be used.
The control specimen's blood count must be normal.
The test should be repeated if control graph is not acceptable.
Abnormal osmotic fragility results for the patient should correspond (or at least not
conflict) with findings on the blood film. Examples: if the osmotic fragility is increased
then look for spherocytes in the film; if the osmotic fragility is decreased look for
hypochromia, target cells, etc.
The supernatant from the normal control blood 0.80 % NaCl tube must be free from
visible haemolysis. If not, repeat the test with new saline.
The osmotic fragility test is considered to be the most sensitive index of the presence
and extent of spherocytosis, which accompanies much haemolytic anaemia.
SICKLE CELL SOLUBILITY TEST
Principle
Sickle cell tests depend on the decreased solubility of Hb-S in conditions of reduced
oxygen concentration. The mixture of Hb-S in a reducing solution will give a turbid
appearance, whereas haemoglobin with a normal solubility will give a clear solution.
Specimen
Any anticoagulant can be used.
HAM TEST
Paroxysmal nocturnal haemoglobinuria (PNH) red cells are unusually susceptible to lysis
by complement. This can be demonstrated by the acidified serum test (Ham test). In
the acidified serum, complement is activated via the alternative pathway. PNH cells
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undergo lysis because of their greatly increased sensitivity to lysis by complement. PNH
cells are deficient in CD55, red cell acetylcholineesterase, neutrophil alkaline
phosphatase and CD59. CD59 is largely responsible for the complement sensitivity of
PNH red cells. Flow cytometry on CD55 and CD59 has replaced the Ham test as
primary method of diagnosis of PNH.
Principle
The patient’s red cells are exposed to 37˚C and the patient’s own serum suitably
acidified to the optimum pH for lysis (pH 6.5 – 7.0). Red cells can be obtained form
heparin, citrate or EDTA blood. The lysis is measured with a spectrophotometer at
wavelength of 540nm.
Page 327 of 336

OTHER
BUFFY COAT PREPARATION
Principle
In some cases of leucopenia, leukaemia or carcinoma, the white cells can be
concentrated, stained and examined for the presence of abnormal or immature
forms. It may also be used to demonstrate the lupus erythematosis (LE) phenomenon.
Specimen
3 ml EDTA blood
Procedure
The Wintrobe tube is filled by means of a long-stemmed pipette. Sufficient blood
should be taken up into the pipette to fill the tube in one operation, starting from the
bottom of the tube, raising the pipette as filling occurs.
The filled tube is centrifuged at 1500 rpm for 15 minutes.
Remove plasma with a long-stemmed pipette and discard.
Carefully aspirate with the same pipette the buffy coat layer (layer between the red
cells and the plasma).
Mix inside the pipette.
Place a small drop onto four slides.
If blood on slide is not mixed, mix with the corner of the spreader.
Spread the films in the usual way.
Let dry and stain with Wright's stain.
Procedure notes
If a buffy coat is done on healthy blood then it might be possible to find immature cells
but only in a very small number.
In disease, leaving leukaemias out of consideration, abnormal cells may be seen in the
buffy coat preparations in much larger numbers than in films of whole blood. For
instance, megakaryocytes and immature cells of the granulocyte series are found in
relatively large numbers in disseminated carcinoma. Megaloblasts if found may help
in the diagnosis of a megaloblastic anaemia.
Erythrophagocytosis may be conspicuous in cases of autoimmune haemolytic

anaemia and in systemic lupus erythematosis a few LE-cells may be found.
Page 328 of 336

It is not advisable to attempt an accurate differential count on buffy coat
concentrates as the different leucocytes tend to sediment under influence of gravity
at different rates and form layer upon layer.
ICT MALARIA P. FALCIPARUM P. VIVAX
Principle
ICT Malaria Pf/Pv is a rapid, in-vitro immunodiagnostic test for the detection of
circulating Plasmodium falciparum (P.f) and Plasmodium vivax (P.v) antigens in whole
blood. The test uses two antibodies, which have been immobilized as two separate
lines across a test strip. One antibody (test area 1) is specific for the histidine-rich
protein 2 antigen of P. falciparum (P.fHRP2). The other antibody (test area 2) is specific
for a malarial antigen, which is common to both P. falciparum and P. vivax species.
Whole blood (15µl) is applied to a sample pad impregnated with colloidal gold-
labeled antibodies, which are directed against the two malarial antigens.
When a positive sample is applied, malarial antigens bind to the gold-coupled

antibodies in the pad, and the immune complexes formed migrate along the test strip
where they are captured by the immobilized antibodies. When capture occurs, a pink
line forms in area 1 and/or 2 of the test window. When a negative sample is applied
these lines do not form. A procedural line will appear, in area C of the test window, if
the test has been performed correctly.
Positive control line for ICT
MANUAL METHOD PLATELET COUNT
Principle
In most instances, platelet numbers can be adequately assessed by examination of a
blood film, only low or high numbers of platelets need to be enumerated by counting.
With the increasing use of cytotoxic drugs, knowing the number of platelets is
important for patient management.
The platelet counts are also important in helping to diagnose bleeding disorders.
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The red cells are lysed in the ammonium oxalate, in order to identify the platelets more
easily.
Reagents and Equipment

Neubauer Chamber
The chamber consists of four squares divided into sixteen smaller squares and one
central square divided into twenty five smaller squares, of which each one of the
twenty five is divided into sixteen other squares.
The platelets are counted in squares 5 A, B, C, D and E; altogether eighty small
squares. The depth is 0.1mm (0.02μl in volume).
When counting, it is extremely important to avoid drying of the preparation, as this will
affect the distribution of the cells.
Platelet diluting fluid – 1g Ammonium Oxalate dissolved in 100 ml of distilled water (1%
Ammonium Oxalate)
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The Neubauer Hemacytometer (0.1 mm deep (from cover slip down to chamber)
Results/ Calculations
Depth of the counting Chamber = 0.1mm

Area of square is 1mm x 1mm = 1mm2
Volume of one large square = 1mm x 1mm x 0.1mm = 0.1mm3
Volume of 1/5 of a large square = 1/5 x 0.1mm3 = 0.02mm3
(We count only 5 of the 25 blocks therefore Volume is 1/5 of the large square)
Platelet count = Number of platelets counted X 106/L

Dilution x Depth x Area
= Number of platelets counted X 106/L

1/20 x 1/10 x 1/5
= Number of platelets counted X 20 X 10 X 5 X 106/L
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= Number of platelets counted X 1000 X 106/L
= ______ X 109/L
Example:
Platelets Counted: A = 52
B = 40
C = 53
D = 45
E = 43
Total = 233
The Platelet Count = no. of cells counted X 106 /ℓ

Volume counted (0.02 μl)
= 233 x 106 /ℓ
1/20 x 1/10 x 1/5
= 233 x 1000 x 106 /L
= 233 X 109/L
Interpretation
The normal range for adults and children is 150 – 400 x 109/ℓ.
The platelet count in individuals is fairly constant, although a decrease can occur,
especially with women during their menstrual cycle.
With the manual method platelet counts can however be doubtful, due to difficulties
in counting small bodies that agglutinate and break up so easily.
An increased platelet count, thrombocytosis, is found in polycythemia vera, idiopathic

thrombocythaemia, chronic myelogenous leukaemia and following a splenectomy
etc.
A decreased platelet count, thrombocytopenia, occurs in thrombocytopenic purpura,

aplastic anaemia, acute leukaemia, Gaucher’s disease, pernicious anaemia and
sometimes following chemotherapy and radiation therapy etc.
DUAL- LABELLED SCHILLING'S TEST
Principle
The vitamin B12 absorption (Schilling) test permits differentiation of causes of vitamin B12
deficiency (pernicious anaemia or intestinal malabsorption).
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R Protein and intrinsic factors are cobalamin-binding proteins in gastric juice. Much
dietary cobalamin binds to R protein, and in this form, it is not absorbable. Pancreatic
proteases cleave cobalamin from R protein, allowing binding by intrinsic factor, which
is absorbed in the ileum. However, in pancreatic insufficiency, the R protein-bound
cobalamin is not released, whereas cobalamin bound to intrinsic factor is absorbed.
This is the basis of the dual-labelled Schilling's test.
The proportion absorbed from orally administered [⁵⁷Co]- or [⁵⁸Co]-labelled vitamin B12
can be measured by determining the radioactivity in faeces, urine or serum or by
externally scanning the liver. The usual procedure is to measure the radioactivity in a
24-h urine sample, which is collected after oral administration of 0.5 μg of radioactive
cobalt-labelled vitamin B12 after an overnight fast.
Procedure
The patient is injected intramuscularly with a "flushing dose" of 1000 μg unlabelled
vitamin B12 to cause urinary excretion of the absorbed vitamin B12. Cobalamin is
administered orally labelled by two different isotopes bound to different cobalamin-
binding proteins. [⁵⁸Co]-cobalamin is bound to hog R protein and [⁵⁷Co]-cobalamin
to human intrinsic factor. In health, the pancreatic proteases cleave [⁵⁸Co]-
cobalamin from the hog R protein so that it binds with intrinsic factor and is absorbed.
The urinary ratio of [⁵⁸Co]-cobalamin to [⁵⁷Co]-cobalamin is about 0.8 or 8% in health,
whereas the ratio decreases with increasing pancreatic insufficiency (usually less than
7% and often 0-3%).
Reduced absorption
A confirmatory test for lack of intrinsic factor (IF) requires ingestion of vitamin B12 and IF
and repeat of the flushing dose 1 or 2 days after the initial vitamin B12 absorption tests.
When gastric secretion of IF is lacking, oral administration of IF simultaneously with
vitamin B12 increases the absorption of vitamin B12 by the intestinal tract, and thus the
urinary excretion increases to 8% or more of the dose administrated. In patients with
malabsorption, oral administration of IF does not increase the percentage of the dose
extracted. However, approximately 25% of patients with pernicious anaemia also
have intestinal malabsorption that is secondary to vitamin B12 deficiency and is
reversible after weeks or months of vitamin B12 therapy. Thus, a low value of vitamin B12
absorption when administered with IF does not rule out pernicious anaemia.
PLASMA VISCOSITY
Principle
The Viscometer II determines viscosity by using a syringe to push a sample through a
capillary at constant pressure. The time taken for a known volume of blood plasma to
flow through the capillary is proportional to its dynamic viscosity.
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The sample is pushed through the capillary by a stepper motor driven syringe. This
motor is directly controlled by a microprocessor that can simultaneously monitor the
pressure in the syringe system. In this way, the sample can be driven through the
capillary at constant pressure by adjusting the motor step rate to compensate for
variations in pressure.
Since erroneous results could potentially be reported if there were air bubbles in the
sample, or it was a short sample, the Viscometer II takes a number of consecutive time
readings for small portions of the sample during the measurement.
Eight time readings are taken, one for each volume unit. These readings span the
entire sampling stroke so that they reflect the approach of the sample to the capillary
and the time taken for a constant pressure to be reached. Not all time readings are
necessarily of use in calculating the result.
All data points are checked against an error percentage band; any point not within
this band is excluded. A new mean is calculated for all points that are within the error
percentage band. This new mean is, therefore, a direct function of the plasma's
viscosity.
Result interpretation for plasma viscosity.
Result Interpretation
Less than 1.50 Abnormal level of one or more plasma protein fractions
1.50-1.72 Normal range
Indicates an abnormally high level of one or more plasma
Over 1.72
protein functions.
Values in this range with slight variation only after 3 weeks
1.75-2.00
suggests a chronic disease
Around 3.00 Suggest myeloma.
Above 3 and up to 20
Suggests macroglobulinaemia.
mPa.s
Specimens must be spun down properly for clear plasma.
Specimens containing clots are not acceptable.
Specimen for a viscosity should not be older than a week.
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REFERENCES
HAEMOGLOBIN ELECTROPHORESIS
Hb electrophoresis at pH 8.4 – 8.6 using cellulose acetate membrane is simple, reliable

and rapid.
At an alkaline pH haemoglobin is negatively charged and will therefore move to the
anode (positive electrode) when subject to an electrical current. Structural variants
that have a change in the charge on the surface of the molecule will separate from
Hb A. The haemoglobin pattern is visualized by staining the film.
Alkaline agarose electrophoresis effectively resolves normal haemoglobins (Hb A and

Hb F) and the major variants (Hb S and Hb C), but cannot distinguish the minor variants
G and D, E and O, from Hb S and Hb C respectively.
Electrophoresis at acid pH (6.0) not only resolves normal and major haemoglobins, but
also allows HbD and G to be distinguished from HbS; and HbE, HbO and HbC to be
resolved from one another.
Alkaline gel Acid gel
_ + _ +
O
A2 S F A F A S C
C D A2
E G D
O E
G
High performance liquid chromatography (HPLC) is being used increasingly to

differentiate between the haemoglobins. The advantages are automation (thus less
staff time), small samples are sufficient, quantification of normal and variant
haemoglobins is available on every sample and a provisional identification of a larger
proportion of variant haemoglobins can be made.
Page 335 of 336

Bain, B.J., 1995. Blood cells a practical guide. 2nd ed. London: Gower Medical
Publishing
Hoffbrand, A.V., Lewis, S.M., Tuddenham, E.G.D., 1999. Postgraduate Haematology. 4th
ed. Oxford: Butterworth-Heineman
Hoffbrand, A.V., Moss, P.A.H., Pettit, J.E., 2006. Essential Haematology. 5th ed. Oxford:
Blackwell Publishing Ltd
Howard, M.R., Hamilton, P.J., 2002. Haematology an illustrated colour text. 2nd ed.
Harcourt Publishers Ltd
Lewis, S.M., Bain, B.J., Bates, I., 2006. Dacie and Lewis Practical Haematology. 10th ed.
Philadelphia: Churchill Livingstone Elsevier
[Available online from:] http://www.uq.edu.au/vdu/HDUAcuteLeukaemia.htm
[Available online from:]

http://www2.kumc.edu/coa/Education/AMED900/HypoproliferativeAnemia.htm#HYP
OPROLIFERATIVE
[Available online from:]

http://www.merckmanuals.com/professional/hematology_and_oncology/anemias_c
aused_by_deficient_erythropoiesis/hypoproliferative_anemias.html
[Available online from:] http://www.practical-

haemostasis.com/Platelets/platelet_function_testing_pfa100.html
Pictures are referenced directly underneath the picture.
Page 336 of 336

Ampath

Uploaded by

Ampath

Uploaded by

2014

Developed: June 2012

Written By: Alvine Janse van Rensburg and Shanta Padiachy

Learning Content description Page number

Although it is designed as a reference for student Medical Technicians,

Purpose of the Learning Unit

Learning Unit 1 provides you with the underpinning knowledge,

Size: Slightly larger than a red cell

Size: Same size as myeloblast

G-CFU, granulocyte colony-forming unit; GM-CFU, granulocyte/macrophage colony-

http://www.nature.com/nri/journal/v3/n4/images/nri1056-f1.gif 27 October 2010

 The bone marrow is the site of blood formation

Tabulate the differences between heparin, sodium citrate and EDTA

Where do all blood cells originate from?

What stain will be used to show progress of erythropoiesis?

What percentage of blasts is normal in peripheral blood?

Sketch the following cells and label the contents properly.

Purpose of the Learning Unit

Learning Unit 2 provides you with the underpinning knowledge,

Before we understand the automated cell counter and it principles, we have

And the following calculated parameters:

WBC count A WBC count is a measurement of the number of white blood

Hb Haemoglobin is a protein in red blood cells that carries oxygen.

Hct Measures the percentage per volume of whole

MCHC The MCHC is the average concentration of Hb in a given volume

MCV It is an estimate of the average size (volume) of a red blood cell

MPV Is a machine-calculated measurement of the average size of

Why is the test done?

When is a Full blood Count Collected?

All these measurements are now made on automated and semi-automated

Automated blood cell counters aspirate and dilute blood samples to

Counting and sizing are done by either electrical impedance or by light

Note: Flow cytometry (abbreviated: FCM) is a technique for counting and

Impedance (the Coulter principle)

Optical or light scatter

Forward Scatter(FSc) Approximate cell size

Side Scatter(SSc) Cell complexity or granularity

Fluorescent Labelling Used to investigate cell structure and function.

Conductivity is determined using a high-frequency electromagnetic probe

A methodology unique to the Bayer automated haematology series is the use

Method Instrument Impedance Conductivity Light Scatter Cytochemistry

Daily cleaning helps to prevent protein build-up in the tubing.

Haematology analysers require regular maintenance in order for them to work

This may include the following: performing a shutdown and a start up

This may involve extended cleaning, unblocking and removal of protein

Lymph RBC precursor

SIDE SCATTER (COMPLEXITY)

Granulocytes for example reflect more light than smooth surfaced B- or T-

Red cells are distributed according

The black curves on the red cell and platelet

The red curves demonstrate the effect of

Microcytic red cells only affect the right end of

Platelets are distributed according to cell size

The black curves on the red cell

The red curves demonstrate the

Left curve shows interferences

With fluorescence methodology,

The following parameters are calculated on your Haematology analyzer:

 Mean Corpuscular Haemoglobin

These parameters can be calculated using the equations below:

RED CELL INDICES

In order to understand the conversions, here is a refresher:

Measure Units Conversion

We use prefixes to indicate decimal fractions or multiples:

MCH = Hb(g/dl) x 10 UNITS - pg

MCH = 11.5 x 10 (g/l) UNITS - pg

Decreased MCH (hypochromic) Increased MCH

MCHC = Hb (g/dl) x 100 UNITS – g/dl OR

MCHC = Hb (g/dl) UNITS – g/dl

X 100 to compensate for haematocrit in %

MCHC = HB (g/dl) x 100 UNITS – g/dl

= 11.5 x 100 or 11.5

Decreased MCHC Increased MCHC

HCT = RBC (x 1012/l) x MCV (fl) UNITS - %

HCT = RBC x MCV UNITS - %

MCV = HCT (%) x 10