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Dacie and Lewis
Practical Haematology
Dacie and Lewis
Practical TWELFTH
EDITION
Haematology
BARBARA J. BAIN, MB BS, FRACP, FRCPath
Professor of Diagnostic Haematology, Imperial College
Faculty of Medicine, St. Mary’s Hospital, London, UK
IMELDA BATES, MB BS, MD, MA, FRCPath

Professor of Tropical Haematology, Liverpool School of Tropical
Medicine, Liverpool, UK
MICHAEL A. LAFFAN, DM, FRCP, FRCPath

Professor of Haemostasis and Thrombosis, Honorary Consultant
Haematologist, Imperial College Faculty of Medicine,
Hammersmith Hospital, London, UK
Editor Emeritus S. MITCHELL LEWIS, BSc, MD,

FRCPath, DCP, FIBMS
Emeritus Reader in Haematology, Imperial College Faculty of Medicine,
Hammersmith Hospital, London, UK
Search full text online at ExpertConsult.com

© 2017, Elsevier Limited. All rights reserved.
First edition 1950 Seventh edition 1991
Second edition 1956 Eighth edition 1995
Third edition 1963 Ninth edition 2001
Fourth edition 1968 Tenth edition 2006
Fifth edition 1975 Eleventh edition 2012
Sixth edition 1984 Twelfth edition 2017
Preface figure of John V. Dacie, with permission from Lewis M; Sir John Dacie MD, FRS, FRCP, FRCPath, Br J Haematol.
2005 Sep;130(6):834-44.
The right of Barbara J. Bain, Imelda Bates, Michael A. Laffan and S. Mitchell Lewis, to be identified as authors of this
work has been asserted by them in accordance with the Copyright, Designs and Patents Act 1988.
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This book and the individual contributions contained in it are protected under copyright by the Publisher (other
than as may be noted herein).
Notices
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Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any
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With respect to any drug or pharmaceutical products identified, readers are advised to check the most current
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Preface
Sir John V. Dacie, MD, FRCPath, FRS (1912–2005). S. Mitchell Lewis, BSc, MD, DCP(London), FRCPath, FIBMS (b. 1924).
This 12th edition celebrates the 66th year of Practical The 12th edition, like its predecessors, incorporates
Haematology, a notable achievement. The first edition by the latest advances in laboratory haematology while
JV (later Professor Sir John) Dacie was published in 1950. continuing to describe traditional techniques that remain
This work, and subsequent editions with Mitchell Lewis as applicable, particularly, but not only, in under-resourced
co-author, were based on the haematology course for the laboratories in low- and middle-income countries.
University of London Diploma of Clinical Pathology and It is with sadness that we record the death of one of the
subsequently the MSc in Haematology at the then Royal authors, Ms Carol Briggs, BSc FIBMS, during the prepara-
Postgraduate Medical School. tion of this edition.
In the last 66 years the techniques and instrumentation We are honoured to have taken over the editorship of
available to the laboratory haematologist have expanded Practical Haematology from our distinguished predecessors,
at a rate once undreamed of. What has not changed is that Sir John Dacie and Dr Mitchell Lewis. We hope that our
laboratory haematology continues to provide the b edrock efforts have done them justice.
that supports the equally astonishing developments in
clinical haematology. Haematology as a discipline remains Barbara J. Bain
strongest when it is an integrated discipline with a very Michael A. Laffan
close relationship between the laboratory and the clinical
service. Reflecting this ideal state, the authors of this Imelda Bates
edition include laboratory scientists and clinical and labo-
ratory haematologists.
vi
Contributors
The editor would like to acknowledge and offer grateful thanks for the input of all previous editions’ contributors, with-
out whom this new edition would not have been possible.
Barbara J. Bain, MB BS, FRACP, FRCPath Barbara De la Salle, MSc

Professor of Diagnostic Haematology Director, UK NEQAS Haematology
Centre for Haematology, Imperial College UK NEQAS Haematology and Transfusion
Faculty of Medicine West Hertfordshire Hospitals NHS Trust
St. Mary’s Hospital Watford, UK
London, UK
Letizia Foroni, MD, PhD, FRCPath
Imelda Bates, MB BS, MD, MA, FRCPath Principal Teaching Fellow
Professor of Tropical Haematology Centre for Haematology
Liverpool School of Tropical Medicine Imperial College Faculty of Medicine
Liverpool, UK Hammersmith Hospital
London, UK
Anne E. Bradshaw, BSc, FIBMS, DMLM
Head of Operations and Regulatory Affairs
Gareth Gerrard, BSc, PGCert, MSc, PhD
John Goldman Centre for Cellular Therapy
Pathology Core Facility Manager
Hammersmith Hospital
UCL Cancer Institute
London, UK
London, UK
Carol Briggs,* BSc, FIBMS
Previously Head of Haematology Evaluation Unit Dominic J. Harrington, MSc, PhD
Department of Haematology Evaluations Consultant Clinical Scientist and Reader in
University College London Hospital Diagnostic Haematology
London, UK King’s College London
Department of Haemostasis, Thrombosis
John Burthem, PhD, FRCP, FRCPath and Nutristasis (Viapath)
Clinical Senior Lecturer and Honorary Consultant Guy’s and St. Thomas’ Hospital
Haematologist London, UK
Department of Clinical Haematology
Manchester Royal Infirmary Sandra Hing, BSc
Manchester, UK Principal Molecular Geneticist
Cellular and Molecular Pathology
Carol Cantwell, CSci, FIBMS, DMS Imperial College Healthcare NHS Trust
Previously Transfusion Laboratory Manager London, UK
St Mary’s Hospital
Imperial College NHS Trust Michael A. Laffan, DM, FRCP, FRCPath
London, UK Professor of Haemostasis and Thrombosis and
Honorary Consultant Haematologist
Jane Y. Carter, MB BS, FRCPC Centre for Haematology, Imperial College
Technical Director, Clinical and Diagnostics Faculty of Medicine
Amref Health Africa Headquarters Hammersmith Hospital
Nairobi, Kenya London, UK
*Deceased
vii
viii Contributors
Mark Layton, FRCP, FRCPH Kuldip S. Nijran, BSc, MSc, DMS, PhD,
Consultant Haematologist MIPEM, CSci
Imperial College Healthcare NHS Trust Head of Nuclear Medicine Physics
Hammersmith and St. Mary's Hospitals Radiological Sciences Unit
London, UK Imperial College Healthcare NHS Trust
S. Mitchell Lewis, BSc, MD, FRCPath, DCP, FIBMS London, UK
Emeritus Reader in Haematology
Centre for Haematology, Imperial College Andrew Osei-Bimpong, MSc, CSci, FIBMS, MIHM
Faculty of Medicine Laboratory Manager
Hammersmith Hospital Blood Sciences
London, UK Hammersmith Hospital
Imperial College Healthcare NHS Trust
Richard A. Manning, BSc, CSci, FIBMS London, UK
Chief Biomedical Scientist
Specialist Coagulation David J. Perry, MD PhD FRCPEdin FRCPLond
Imperial College Healthcare NHS Trust FRCPath FAcadMEd
Hammersmith Hospital Consultant Haematologist and Associate Lecturer
London, UK Cambridge Haemophilia & Thrombophilia Centre
Cambridge University Hospital NHS Foundation Trust
Alison M. May, PhD Addenbrooke’s Hospital
Previously Senior Research Fellow Cambridge UK
Department of Haematology
Cardiff University School of Medicine Fiona A.M. Regan, MB BS, FRCP, FRCPath
Cardiff, UK Consultant Haematologist
NHS Blood and Transplant (North London)
Christopher McNamara, FRACP, FRCPA FRCPath and Imperial College Healthcare NHS Trust
Consultant Haematologist Hammersmith Hospital
Department of Haematology London, UK
University College Hospital
London, UK Alistair G. Reid, BSc, PhD, FRCPath
Consultant Clinical Scientist
Clare Milkins, BSc CSci FIBMS Cellular and Molecular Pathology
Manager, UK NEQAS Blood Transfusion Laboratory Practice Imperial College Healthcare NHS Trust
UK NEQAS Haematology and Transfusion London, UK
West Hertfordshire Hospitals NHS Trust
Watford, UK Stephen J. Richards, PhD FRCPath
Consultant Clinical Scientist
Ricardo Morilla, MSc, FRMS Haematological Malignancy Diagnostic Service
Head of Immunophenotyping Leeds Cancer Centre
Haemato-Oncology Section St James's University Hospital
Royal Marsden Hospital NHS Foundation Trust Leeds, UK
Sutton, Surrey, UK
Lynn D. Robertson, MSc
Alison M. Morilla, BSc Laboratory Manager
Senior Clinical Scientist Core and Special Haematology
Haemato-Oncology Section Imperial College Healthcare NHS Trust
Royal Marsden Hospital NHS Foundation Trust Hammersmith Hospital
Sutton, Surrey, UK London, UK
Elisabet Nadal-Melsió, MD David Roper, MSc, CSci, FIBMS

Consultant Haematologist Previously Principal Biomedical Scientist;
Cellular and Molecular Pathology Diagnostic Haematology
Imperial College Healthcare NHS Trust Imperial College Healthcare NHS Trust
Hammersmith Hospital Hammersmith Hospital
London, UK London, UK
Contributors ix
Megan Rowley, FRCP, FRCPath Barbara J. Wild, PhD, FIBMS

Consultant in Haematology and Transfusion Medicine Haemoglobinopathy Specialist Consultant
St. Mary’s Hospital UK NEQAS Haematology and Transfusion
Imperial College Healthcare NHS Trust West Hertfordshire Hospitals NHS Trust
London, UK Watford, UK
Jecko Thachil, MRCP, FRCPath Nay Win, MB BS, FRCP, FRCPath, CTM(Edin)
Consultant Haematologist Consultant Haematologist
Haematology Department Red Cell Immunohaematology
Manchester Royal Infirmary NHS Blood and Transplant (Tooting)
Manchester, UK London, UK
Sarmad Toma, MBChB, MSc Mark Worwood, PhD, FRCPath, FMedSci

Clinical Scientist Emeritus Professor
Cellular and Molecular Pathology Cardiff University School of Medicine
Imperial College Healthcare NHS Trust Cardiff, UK
London, UK
1
Collection and
Handling of Blood
Christopher McNamara
CHAPTER OUTLINE
Biohazard precautions, 1 Sample homogeneity, 4
Procurement of venous blood, 1 Serum, 4
Equipment, 1 Cold agglutinins, 4
Specimen containers, 1 Anticoagulants, 4
Phlebotomy procedure, 2 Ethylenediaminetetra-acetic acid (EDTA), 4
Postphlebotomy procedure, 3 Trisodium citrate, 4
Capillary blood, 3 Heparin, 5
Collection of capillary blood, 3 Effects of storage on the blood count, 5
Blood film preparation, 3 Effects of storage on blood cell morphology, 5
Differences between capillary and venous blood, 3
Following an informed decision to analyse a blood sam- PROCUREMENT OF VENOUS

ple, a specimen must be safely and correctly procured. It
is essential to be aware that variation in this pre-analytical BLOOD
phase of the testing process can lead to errors in the ana-
lytical phase (see Box 1-1).
Equipment
Venous blood is used for most examinations. Capillary It is important to assemble a tray or prepare a workspace
blood samples may be satisfactory for some purposes but that has all the requirements for blood collection (Box 1-2).
in general the use of capillary blood should be restricted to The selection of needle diameter is a compromise between
children and to some point-of-care screening tests. achieving adequate flow with minimal turbulence and
minimising patient discomfort. A 19-gauge (19G) or 21G*
needle is suitable for most adults. A 23G needle is often se-
lected for children. The shaft of the needle should be short
BIOHAZARD PRECAUTIONS (about 15 mm). It may be helpful to collect the blood by
Laboratory policies must be in place to ensure that staff means of a winged needle (often referred to as a ‘butterfly’)
who collect blood samples and transfer them to the labora- connected to a length of plastic tubing that can be attached
tory minimise the risk of infection from various pathogens to the nozzle of the syringe or to a needle for entering the
during all aspects of specimen handling (see Chapter 24). cap of an evacuated container (see Specimen Containers).
Additional precautions should be taken when handling
high-risk specimens (e.g. those from patients suspected of Specimen containers
having a viral haemorrhagic fever).1 In this circumstance,
the collection policy should stipulate the use of personal Containers for testing whole blood are available
protective equipment, such as disposable gloves, body commercially with dipotassium, tripotassium or disodium
apron and protective eyewear. Care must be taken to pre-
vent injuries, especially when handling and disposing of *The International Organisation for Standardisation has established a
needles and lancets. Recommendations for standardising standard (ISO 7864), which relates the following diameters for the
blood collection have been published.2,3 different gauges: 19G = 1.1 mm; 21G = 0.8 mm; 23G = 0.6 mm.
1
2 Practical Haematology
BOX 1-1 Causes of misleading results There is no universal agreement regarding the colours used
related to specimen collection for identifying containers with different additives so phle-
botomists should familiarise themselves with the colours
PRE-COLLECTION used by their local suppliers.
• Urination within 30 min; food or water intake within Evacuated tube systems in common use consist of a
2h glass or plastic tube under a defined vacuum, a needle
• Smoking and a needle holder, which secures the needle to the tube.
• Physical activity (including fast walking) within 20 min The main advantage is that the cap can be pierced so that
• Stress it is not necessary to remove it either to fill the tube or
• Drugs or dietary supplement administration within 8 h
subsequently to withdraw samples for analysis, thus min-
DURING COLLECTION imising the risk of aerosol discharge of the contents. An
• Different times (diurnal variance)
evacuated system is useful when multiple samples in dif-
• Posture: lying, standing or sitting ferent anticoagulants are required. The vacuum controls
• Haemoconcentration from prolonged tourniquet the amount of blood that enters the tube, ensuring an ad-
pressure equate volume for testing with the correct proportion of
• Excessive negative pressure when drawing blood into any anticoagulant.
syringe
• Incorrect type of tube
• Capillary versus venous blood Phlebotomy procedure
Staff undertaking this procedure should be adequately
HANDLING OF SPECIMEN trained. The phlebotomist must check that the patient’s
• Insufficient or excess anticoagulant identity corresponds to the details on the request form
• Inadequate mixing of blood with anticoagulant and also ensure that the phlebotomy tray contains all the
• Error in patient and/or specimen identification
• Inadequate specimen storage conditions
required specimen containers and other equipment neces-
• Delay in transit to laboratory sary for the procedure.
A tourniquet should be applied just above the intended
venepuncture site. Blood is best withdrawn from an ante-
cubital vein or other visible veins of the forearm by means
of either an evacuated tube or a syringe. It is recommended
BOX 1-2 Items to be included in a that the skin be cleaned with 70% alcohol (e.g., isopro-
phlebotomy tray panol) and allowed to dry spontaneously before being
punctured. The tourniquet should be released as soon as
• Syringes and needles
the vein is punctured and blood begins to flow into the sy-
• Tourniquet
• Specimen containers (tubes or evacuated tube system) – ringe or evacuated tube – delay in releasing the tourniquet
plain and with various anticoagulants leads to fluid shift and haemoconcentration as a result of ve-
• Request form nous blood stagnation.6 After the vein has been successfully
• 70% isopropanol swabs or 0.5% chlorhexidine punctured, the piston of the syringe should be withdrawn
• Sterile gauze swabs slowly with no attempt being made to withdraw blood faster
• Adhesive dressings than the vein is filling. Anticoagulated specimens must be
• Self-sealing plastic bags with a separate compartment mixed by inverting the container several times. The risk of
for the request form unwanted haemolysis of the specimen can be minimised
• Rack to hold specimens upright during process of filling by using minimal tourniquet time, withdrawing blood
(except when an evacuated tube system is used)
carefully, using an appropriately sized needle, delivering the
• Puncture-resistant disposal container
blood slowly into the receptacle and avoiding unnecessary
agitation when mixing with the anticoagulant. Note that if
blood is drawn too slowly or is inadequately mixed with
ethylenediaminetetra-acetic acid (EDTA) anticoagulant, the anticoagulant some coagulation may occur, rendering
and often have a mark to indicate the correct amount of the sample unsuitable. After collection, containers must be
blood to be added.4 Containers are also available contain- firmly capped to minimise the risk of leakage.
ing trisodium citrate, heparin or acid–citrate–dextrose, as If blood collection fails, it is important to remain calm,
well as containers with no additive which are used when communicate with the patient and consider the possible
serum is required. Design requirements and other specifi- causes. These include poor technique (e.g., passing the
cations for specimen collection containers have been de- needle through the vein, or poor selection of veins), scar-
scribed in a number of national and international standards ring of tissues and haematoma formation.
(e.g., that of the International Council for Standardisation After obtaining the necessary specimens, remove the
in Haematology5) and the European standard (EN 14820). needle and press a sterile swab over the puncture site.
1 Collection and Handling of Blood 3
Gentle pressure should be applied to the swab with the

arm slightly elevated for a minute before checking that
bleeding has completely ceased. Finally, the puncture site
should be covered with a small adhesive dressing.
Obtaining blood from an indwelling line or catheter is
an important potential source of error. It is common prac-
tice to flush indwelling lines with heparin, so they must
be flushed free from heparin and the first 5 ml of blood
must be discarded before any blood is collected for lab-
oratory testing. If intravenous fluids are being transfused
into an arm, blood should generally not be collected from
that arm; however, if this is essential, the specimen should
be obtained from below the intravenous infusion with the
tourniquet being placed below the site of infusion.
Postphlebotomy procedure
It is essential that every specimen is labelled with adequate
patient identification immediately after the samples have
been obtained and at the patient’s bedside. The information
should include, as a minimum, surname and forename or
initials, hospital number or other unique identifying num-
ber, date of birth and date and time of specimen collection.
Many centres have adopted automated patient identifi-
cation using a bar code printed on a wrist or ankle band FIGURE 1-1 Skin puncture in infants. Puncture must be restricted
worn by the patient. If this type of system is used both to the outer medial and lateral portions of the plantar surface of the
the specimen label and the request form should be bar- foot indicated by the shaded area.
coded with identical data, unless the sample is to be used
for blood transfusion tests, in which case the label should flow of blood is essential and only very gentle squeezing
be handwritten (see Chapter 22). is permissible; ideally, large drops of blood should exude
Specimens should be sent in individual plastic bags slowly but spontaneously.
separated from the request forms to prevent contamina- After use, lancets should be placed in a puncture-resistant
tion of the forms in the event of leakage. Samples and container for subsequent waste disposal. They must never
form should remain together until the request is registered be re-used on another individual.
in the laboratory reception area.
BLOOD FILM PREPARATION
CAPILLARY BLOOD Ideally, blood films should be made immediately after the
Collection of capillary blood blood has been collected. However, in practice, blood
samples are usually sent to the laboratory after a variable
Skin puncture is carried out with a needle or lancet. In delay. Automated methods for making films are available
adults and older children, blood can be obtained from a and often employed in large centres. When films are not
finger; the recommended site is the distal digit of the third made on site they should be made in the laboratory soon
or fourth finger on its palmar surface, lateral to the nail after arrival as blood film morphology will deteriorate
bed. In infants, satisfactory samples can be obtained by with any delay beyond a few hours.
a deep puncture of the plantar surface of the heel in the
area shown in Figure 1-1. The central plantar area and the
posterior curvature should not be punctured in small in- DIFFERENCES BETWEEN
fants, especially newborns, to avoid the risk of injury and CAPILLARY AND VENOUS
possible infection to the underlying tarsal bones. BLOOD
The area selected for capillary puncture should be
cleaned with an antiseptic and allowed to dry. The skin is Venous blood and capillary blood are not equivalent.
punctured to a depth of 2–3 mm with a sterile, disposable Blood from a skin puncture is a mixture of blood from
lancet. After wiping away the first drop of blood with dry arterioles, veins and capillaries and it contains some in-
sterile gauze the finger (or heel in infants) is squeezed gen- terstitial and intracellular fluid.7 The packed cell volume/
tly to encourage a free flow of blood for collection. Free haematocrit (PCV/Hct), red blood cell count (RBC) and
haemoglobin concentration (Hb) of capillary blood may laboratory and to collect blood into a previously warmed
be slightly higher than those of venous blood. The to- syringe and then to deliver the blood into containers that
tal leucocyte and neutrophil counts may also be higher. have been kept warm at 37 °C. When filled, the containers
Conversely, the platelet count appears to be higher in ve- should be promptly replaced in the 37 °C water bath. In
nous than in capillary blood; this may be due to adhesion this way, it is possible to assess the effect of any putative
of platelets to the site of the skin puncture. All of these antibodies acting, in vivo, at body temperature. When this
differences are minimised when a free flow of blood has is not feasible, specimens can be tightly capped and placed
been obtained after skin puncture. in a thermos at 37 °C.
SAMPLE HOMOGENEITY ANTICOAGULANTS

To ensure even dispersal of the blood cells it is essential Ethylenediaminetetra-acetic acid
that specimens are mixed effectively in the laboratory, im-
mediately before being tested. The specimen tube can be
(EDTA)
placed on a mechanical rotating mixer for 2 min or the EDTA and sodium citrate remove calcium, which is essen-
tube can be inverted 8–10 times by hand. If the specimen tial for coagulation. Calcium is either precipitated as in-
has been stored at 4 °C, it will be viscous and the blood soluble oxalate (crystals of which may be seen in oxalated
should be allowed to warm to room temperature before blood) or bound in a non-ionised form. Heparin binds
being mixed. to antithrombin, thus inhibiting the interaction of several
clotting factors.
EDTA anticoagulation is used for blood counts; sodium
SERUM citrate is used for coagulation testing and for the erythro-
The difference between plasma and serum is that the latter cyte sedimentation rate. For better long-term preservation
lacks fibrinogen and some coagulation factors. Blood col- of red cells for certain tests and for transfusion purposes,
lected in order to obtain serum should be delivered into citrate is used in combination with dextrose in the form
sterile tubes with caps or into commercially available plain of acid–citrate–dextrose (ACD) or citrate–phosphate–
(no anticoagulant) evacuated collection tubes and allowed dextrose (CPD).
to clot undisturbed for about 1 h at room temperature be- An excess of EDTA affects both red cells and leuco-
fore centrifugation.† Some containers have silica particles cytes, causing shrinkage and degenerative changes. EDTA
and an inert polymer gel, which floats between the serum in excess of 2 mg/ml of blood may result in a significant
and the red cells when centrifuged, facilitating separation decrease in PCV assessed by centrifugation and an increase
of serum and making the specimen suitable for use on in mean cell haemoglobin concentration (MCHC).8 The
some automated analysers. This eliminates the need to de- platelets may also be affected; an excess of EDTA causes
cant the serum and preserves the integrity of the specimen. them to swell and then disintegrate, causing an artificially
The tubes, whether with or without a serum separa- high platelet count, as the fragments are large enough to
tor, are then centrifuged for 5 minutes at 3000 revolutions be counted as platelets. Care must therefore be taken to
per minute (rpm). Some tests require a further centri ensure that the correct amount of blood is added, and that
fugation to remove any remaining particulate material. by repeated inversions of the container the anticoagulant
Supernatant serum may be transferred to tubes for further is thoroughly mixed with the blood specimen. EDTA is re-
tests or stored. For most tests, serum should be kept at sponsible for the activity of a naturally occurring antiplatelet
4 °C until used, but if testing is delayed, serum can be autoantibody, which sometimes causes platelet aggregation
stored at −20 °C for up to 3 months and at −40 °C or be- of platelet adherence to neutrophils in blood films. All pa-
low for long-term storage. Validation of storage conditions tients with apparent thrombocytopenia therefore require
and sample viability should be undertaken for all tests a blood film to identify this in vitro phenomenon. Repeat
performed. Frozen specimens should be thawed in a wa- estimation of the platelet count in an alternative antico-
ter bath or in a 37 °C incubator, and then inverted several agulant will resolve this problem, as the aforementioned
times to ensure homogeneity before being used for a test. antibody is inactive in the absence of EDTA.9
COLD AGGLUTININS Trisodium citrate

If cold agglutinins are suspected the blood must be kept For coagulation studies, 9 volumes of blood are added to
at 37 °C from the point of collection until the sample has 1 volume of 109 mmol/l sodium citrate solution (32 g/l of
been processed. If cold agglutinins are suspected, it is Na3C6H5O7•2H2O‡).10 This ratio of anticoagulant to blood
best to bring the patient to a suitable location close to the is critical as osmotic effects and changes in free calcium
†
Room temperature is usually considered 18–25 °C. ‡
38 g/l of 2Na3C6H5O7•11H2O.
ion concentration affect coagulation test results. This ratio but at room temperature the count begins to fall within
of citrate to blood may need to be adjusted when samples 6 h. Nucleated red cells disappear in the blood specimen
with a high haematocrit require coagulation studies (see within 1–2 days at room temperature.
Chapter 18). Haemoglobin concentration remains unchanged for
days. However, within 2–3 days, and especially at high
ambient temperatures, the red cells begin to lyse, resulting
Heparin in a decrease in the RBC and PCV/Hct, with an increase in
Lithium or sodium salt of heparin at a concentration of the calculated MCH and MCHC.
10–20 iu/ml of blood is a commonly used anticoagulant Coagulation test stability is critical for diagnosis and
for chemistry, gas analysis and emergency tests. It does treatment of coagulopathies; it is recommended that tests
not alter the size of the red cells and it is recommended be carried out within 2 h when the blood or plasma is
when it is important to reduce to a minimum the chance stored at 22–24 °C, within 4 h when stored at 4 °C, within
of lysis occurring after blood has been withdrawn. When 2 weeks when stored at −20 °C, and within 6 months
red cells are required for testing, as in the investigation of when stored at −70 °C.3
certain types of haemolytic anaemia, the sample can be For a serum or plasma test, blood should be centri-
defibrinated (see previous editions for details) although fuged within 5 h of collection. For vitamin B12 and folate
heparinised blood is now more often used for such tests. assays, the serum or plasma should be kept at 4 °C or
Heparin is not suitable for blood counts and films as it at −20 °C if storage for more than 2–3 weeks is required.
often induces platelet and leucocyte clumping11 and gives For long-term storage, specimens should be divided into
a faint blue colouration to the background when films are several aliquots to avoid repeated freezing and thawing.
stained by Romanowsky dyes (especially, but not only, Inappropriate handling of blood specimens during
in the presence of abnormal proteins). Heparin inhibits transfer to the laboratory (e.g. excess shaking or being
enzyme activity and it should not be used when a poly- exposed to temperature extremes) may cause haemoly-
merase chain reaction with restriction enzymes is to be sis, partial coagulation and cell disintegration. Shipping
performed.12 of specimens requires special packaging and should reach
certain minimum specifications.14
EFFECTS OF STORAGE ON THE

BLOOD COUNT EFFECTS OF STORAGE ON
Various changes take place in anticoagulated blood when
BLOOD CELL MORPHOLOGY
it is stored at room temperature, and these changes occur Changes in blood cell morphology of stored samples occur
more rapidly at higher ambient temperatures. These occur within a few hours of blood collection. Irrespective of anti
regardless of the anticoagulant. The RBC, white blood cell coagulant, films made from blood that has been standing
count (WBC), platelet count and red cell indices are usu- for <1 h at room temperature are not easily distinguished
ally stable for up to 8 h after blood collection, although from films made immediately after collection of the blood.
as the red cells start to swell the PCV/Hct and mean cell By 3 h, changes may be discernible and by 12–18 h these
volume (MCV) start to increase, osmotic fragility increases become striking. Some but not all neutrophils are affected;
and the erythrocyte sedimentation rate decreases. When their nuclei may stain more homogeneously than in fresh
the blood is kept at 4 °C the effects on the blood count blood, the nuclear lobes may become separated and the
are not usually significant for up to 24 h. Thus, for many cytoplasmic margin may appear ragged or less well-defined;
purposes blood can safely be allowed to stand overnight small vacuoles appear in the cytoplasm (Fig. 1-2, A, B).
in the refrigerator if precautions against freezing are taken. Some or many of the large monocytes develop marked
Nevertheless, it is best to count leucocytes and especially changes; small vacuoles appear in the cytoplasm and the
platelets within 2 h and it should be noted that the de- nucleus undergoes irregular lobulation, which may almost
crease in the leucocyte count and a progressive decrease amount to disintegration (Fig. 1-2, C). Lymphocytes un-
in the absolute lymphocyte count may become marked dergo similar changes: a few vacuoles may be seen in the
within a few hours, especially if there is an excessive cytoplasm, nuclei stain more homogeneously than usual
amount of EDTA (>4.5 mg/ml).13 Storage beyond 24 h at and in some the nucleus undergoes budding, giving rise
4 °C results in erroneous data for automated white cell dif- to nuclei with two or three lobes (Fig. 1-2, D–F). Normal
ferential counts. One study using an aperture impedance red cells are little affected by standing for up to 6 h at room
analyser on blood left at room temperature showed WBC temperature. Longer periods lead to progressive crenation
and neutrophil counts to be stable for 2–3 days but other (Fig. 1-2, B, E, F). With an excess of EDTA, a marked de-
leucocyte counts were stable for only a few hours.14 gree of red cell crenation occurs within a few hours. All
Reticulocyte counts are unchanged when the blood is the aforementioned changes are retarded but not abolished
kept in either EDTA or ACD anticoagulant for 24 h at 4 °C, in blood stored at 4 °C. Their occurrence underlines the
A C E
B D F
FIGURE 1-2 Effect of storage on blood cell morphology. Photomicrographs from films made from ethylenediaminetetra-acetic acid (EDTA)
blood after 24 h at 20 °C. (A, B) Polymorphonuclear neutrophils; (C, D) monocytes; (E, F) lymphocytes. Red cell crenation is prominent in
all images.
ACKNOWLEDGEMENT
The author wishes to acknowledge the contribution of
previous authors of this chapter – the late Corrine Jury,
Yutaka Nagai and the late Noriyuki Tatsumi – and of
Gareth Ellis, who reviewed the content of this chapter.
REFERENCES
1. Advisory Committee on Dangerous Pathogens. Management of
Hazard Group 4 viral haemorrhagic fevers and similar human infectious
diseases of high consequence. Health and Safety Executive; November
2014. Available at www.gov.uk/government/uploads/system/
uploads/attachment_data/file/377143/VHF_guidance_document_
updated_19112014.pdf [accessed May 2016].
2. Tatsumi N, Miwa S, Lewis SM, International Council for
Standardization in Haematology/ International Society of
Hematology. Specimen collection, storage, and transportation to the
laboratory for hematological tests. Int J Hematol 2002;75:261–8.
FIGURE 1-3 Morphological features of apoptosis. 3. CLSI. Procedures for the collection of diagnostic blood specimens by
venipuncture. Approved standard. 6th ed. Wayne, PA: CLSI; 2007.
Document H3-A6.
importance of making films as soon as possible after the 4. NCCLS. Tubes and additives for venous blood specimen collection.
blood has been collected. Approved standard. 5th ed. Wayne, PA: NCCLS; 2003.
These artefactual changes must be distinguished from 5. Tatsumi N, van Assendelft OW, Naka K. ICSH recommendation for
apoptosis, which can be seen in high-grade haemato- blood specimen collection for hematological analysis. Lab Hematol
logical neoplasms. Apoptosis is characterised, morpho- 2002;8:1–6.
6. Saleem S, Mani V, Chadwick M, et al. A prospective study of causes
logically (Fig. 1-3), by cell shrinkage, a homogeneously of haemolysis during venepuncture: tourniquet time should be kept
glassy appearance of the nucleus, cytoplasmic conden- to a minimum. Ann Clin Biochem 2009;46:244–6.
sation around the nuclear membrane and indentations 7. Yang Z-W, Yang S-H, Chen L, et al. Comparison of blood counts in
in the nucleus, followed by its fragmentation. Apoptotic venous, finger tip and arterial blood and their measurement varia-
tion. Clin Lab Haematol 2001;23:155–9.
neutrophils with a single apoptotic body may be confused 8. Zini G. Stability of complete blood count parameters with storage:
with nucleated red cells if the cytoplasmic features are not toward defined specifications for different diagnostic applications.
appreciated. Int J Lab Hematol 2014;36:111–3.
9. Schuff-Werner P, Steiner M, Fenger S, et al. Effective estimation 13. Hadley GG, Weiss SP. Further notes on use of salts of ethylene di-
of correct platelet counts in pseudothrombocytopenia using an aminetetraacetic acid (EDTA) as anticoagulants. Am J Clin Pathol
alternative anticoagulant based on magnesium salt. Br J Haematol 1955;25:1090–3.
2013;162:684–92. 14. Gulati GL, Hyland LJ, Kocher W, et al. Changes in automated com-
10. Ingram GIC, Hills M. The prothrombin time test; effect of varying plete blood cell count and differential leucocyte count results in-
citrate concentration. Thromb Haemost 1976;36:230–6. duced by storage of blood at room temperature. Arch Pathol Lab Med
11. Salzman EW, Rosenberg RD. Effect of heparin and heparin fractions 2002;126:336–42.
on platelet aggregation. J Clin Invest 1980;65:64–73.
12. Yokota M, Tatsumi N, Nathalang O, et al. Effects of heparin on
polymerase chain reaction for blood white cells. J Clin Lab Anal
1999;13:133–40.
2
Reference Ranges and
Normal Values
Imelda Bates
CHAPTER OUTLINE
Reference ranges, 8 Leucocyte count, 15
Statistical procedures, 9 Platelet count, 16
Confidence limits, 9 Other blood constituents, 16
Normal reference values, 10 Effects of smoking on haematological normal
Physiological variations in the blood count, 13 reference values, 16
Red cell components, 13
A number of factors affect haematological values in appar- a databank of reference values that takes account of the
ently healthy individuals. As described in Chapter 1, these variables mentioned earlier and the test method, so that an
include the technique and timing of blood collection, the individual’s result can be expressed and interpreted relative
transport and storage of specimens, the posture of the sub- to a comparable apparently normal population, insofar as
ject when the sample is taken, the prior physical activity normal can be defined.
and the degree of ambulation (e.g. whether the subject is New haematological parameters such as the number
confined to bed or not). Variation in the analytical methods of immature cells or the number of red cell fragments
used may also affect the measurements. These can all be are often initially developed for research purposes but
standardised. can be used for clinical decision making once internal
More problematic are the inherent variables as a result quality control and external quality assessment processes
of gender, age, occupation, body build, genetic background are in place.3
and adaptation to diet and to environment (especially alti-
tude). These factors must be recognised when establishing
physiologically normal values. It is also difficult to be cer-
REFERENCE RANGES
tain that the ‘normal’ subjects used for constructing normal A reference range for a specified population can be es-
ranges are completely healthy and do not have nutritional tablished from measurements on a relatively small num-
deficiencies, mild chronic infections, parasitic infestations ber of subjects (discussed later) if they are assumed to be
or the effects of smoking. representative of the population as a whole.2 The condi-
Haematological values for the normal and abnormal will tions for obtaining samples from the individuals and the
overlap and a value within the recognised normal range may analytical procedures must be standardised, whereas data
be definitely pathological in a particular subject. For these should be analysed separately for different variables relat-
reasons the concept of ‘normal values’ and ‘normal ranges’ ing to individuals – recumbent or ambulant, smokers or
has been replaced by reference values and the reference range, nonsmokers and so on. One approach is that specimens
which is defined by reference limits and obtained from meas- are collected at about the same time of day, preferably in
urements on the reference population for a particular test. the morning before breakfast; the last meal should have
Unless a reference range is derived in this manner, the term been eaten no later than 9 p.m. on the previous evening,
should not be used. The reference range is also termed the and at that time alcohol should have been restricted to
reference interval.1,2 Ideally, each laboratory should establish one bottle of beer or an equivalent amount of another
8
2 Reference Ranges and Normal Values 9
alcoholic drink.4 An alternative approach is that, unless normal population. Limits representing the 95% reference
a test is usually done on a fasting patient, specimens are range are calculated from the arithmetic mean ±2SD (or
collected throughout the day on subjects who are not more accurately ±1.96SD).
fasting or resting, as this will produce a reference range When there is a log normal (skewed) distribution of
that is more relevant to results from patients. It is some- measurements, the range to −2SD may even extend to
times appropriate that the reference population is defined zero (Fig. 2-2, A). To avoid this anomaly, the data should
as having normal results for specific laboratory tests. For be plotted on semilogarithmic graph paper to obtain a
example, if determining a reference range for blood count normal distribution histogram (Fig. 2-2, B). To calculate
components it may be necessary, in some populations, the mean and SD the data should be converted to their
to exclude iron deficiency, β thalassaemia heterozygosity logarithms. The log–mean value is obtained by adding the
and, when relevant, α thalassaemia. logs of all the measurements and dividing by the number
of observations. The log SD is calculated by the formula
on page 566 and the results are then converted to their
STATISTICAL PROCEDURES antilogs to express the data in the arithmetic scale. This
In biological measurements, it is usually assumed that the process is now generally carried out using an appropriate
data will fit a specified type of pattern, either symmet- statistical computer program.
ric (Gaussian) or asymmetric with a skewed distribution When it is not possible to make an assumption about
(non-Gaussian). With a Gaussian distribution, the arith- the type of distribution, a nonparametric procedure may
metic mean (x) can be obtained by dividing the sum of all be used instead to obtain the median and SD. To obtain
measurements by the number of observations. The mode is an approximation of the SD, the range that comprises the
the value that occurs most frequently and the median (m) middle 50% spread (i.e. between 25 and 75% of results) is
is the point at which there are an equal number of obser- read and divided by 1.35. This represents 1SD.
vations above and below it. In a true Gaussian distribution
they should all be the same. The standard deviation (SD)
can be calculated as described on page 565.
Confidence limits
If the data fit a Gaussian distribution, when plotted as In any of the methods of analysis, a reasonably reliable
a frequency histogram the pattern shown in Figure 2-1 is estimate can be obtained with 40 values, although a larger
obtained. Taking the mode and the calculated SD as ref- number (≥120) is preferable (Fig. 2-3).5 When a large set
erence points, a Gaussian curve is superimposed on the of reference values is unattainable and precise estimation
histogram. From this curve, practical reference limits can is impossible, a smaller number of values may still serve as
be determined even if the original histogram included a useful clinical guide. Confidence limits define the relia-
outlying results from some subjects not belonging to the bility (e.g. 95% or 99%) of the established reference values
Number of subjects
FIGURE 2-1 Example of establishing a reference range. Histogram of data of Hb measurements in a population, with Gaussian curve su-
perimposed. The ordinate shows the number that occurred at each reference point. The mean was 140 g/l; the reference ranges at 1SD, 2SD
and 3SD are indicated.
FIGURE 2-2 Example of conversion to a log normal distribution. Data of serum cobalamin (vitamin B12) measurements in a population. (A)
Arithmetic scale: mean 340 pg/ml; 2SD range calculated as 10–665. (B) Geometric scale: mean 308 pg/ml; 2SD range calculated as 120–780.
Number of Hb measurements from a group
of normal women
FIGURE 2-3 Effect of sample size on reference values. A smoothed distribution graph was obtained for Hb measurements from a group
of normal women; the ordinate shows the frequency distribution. The 95% reference range is defined by the lower and higher reference
limits, which are 115 and 165 g/l, respectively. The confidence levels for these values are shown for three sample sizes of 20, 40 and 165,
respectively.
when assessing the significance of a test result, especially methods are used. The reference interval, which comprises
when it is on the borderline between normal and abnor- a range of ±2SD from the mean, indicates the lim-
mal. Calculation of confidence limits is described on page its that should cover 95% of normal subjects; 99% of
566. Another important measurement is the coefficient of normal subjects will be included in a range of ±3SD.
variation (CV) of the test because a wide CV is likely to Age and gender differences have been taken into ac-
influence its clinical utility (see p. 566). count for some values. Even so, the wide ranges that
are shown for some tests reflect the influence of various
factors, as described below. Narrower ranges would be
NORMAL REFERENCE VALUES expected under standardised conditions. Because mod-
The data given in Tables 2-1, 2-2 and 2-3 provide general ern analysers provide a high level of technical precision,
guidance to normal reference values that are applicable to even small differences in successive measurements may
most healthy adults and children in high-income coun- be significant. It is thus important to establish and un-
tries. However, slightly different ranges may be found derstand the limits of physiological variation for various
in individual laboratories where different analysers and tests. The blood count data and other test results can
TA BL E 2- 1
HAEMATOLOGICAL VALUES FOR NORMAL ADULTS (PREDOMINANTLY FROM EUROPE AND

NORTH AMERICA) EXPRESSED AS A MEAN ± 2SD OR AS A 95% RANGE
Red blood cell count Heparin cofactor II 0.55–1.45 u/ml
Men 5.0 ± 0.5 × 1012/l concentration‡
Women 4.3 ± 0.5 × 1012/l Median red cell fragility (MCF)
Haemoglobin concentration* (g/l NaCl)
Men 150 ± 20 g/l Fresh blood 4.0–4.45 g/l NaCl
Women 135 ± 15 g/l After 24 h at 37 °C 4.65–5.9 g/l NaCl
Packed cell volume (PCV) or Cold agglutinin titre (4 °C) <64
haematocrit (Hct) Blood volume (normalised to
Men 0.45 ± 0.05 l/l ‘ideal weight’)
Women 0.41 ± 0.05 l/l Red cell volume
Mean cell volume (MCV) Men 30 ± 5 ml/kg
Men and women 92 ± 9 fl Women 25 ± 5 ml/kg
Mean cell haemoglobin (MCH) Plasma volume 45 ± 5 ml/kg
Men and women 29.5 ± 2.5 pg Total blood volume 70 ± 10 ml/kg
Mean cell haemoglobin Red cell lifespan 120 ± 30 days
concentration (MCHC) Serum iron
Men and women 330 ± 15 g/l Men and women 10–30 μmol/l
Red cell distribution width (0.6–1.7 mg/l)
(RDW) Total iron-binding capacity 47–70 μmol/l
As coefficient of variation (CV) 12.8% ± 1.2% (2.5–4.0 mg/l)
Red cell diameter (mean Transferrin saturation 16–50%
values) Serum ferritin concentration
Dry films 6.7–7.7 μm Men 15–300 μg/l (median
Red cell density 1092–1100 g/l 100 μg/l)
Reticulocyte count 50–100 × 109/l Women 15–200 μg/l (median
(0.5–2.5%) 40 μg/l)
White blood cell count 4.0–10.0 × 109/l Serum vitamin B12 180–640 ng/l
Differential white cell count concentration
Neutrophils 2.0–7.0 × 109/l (40–80%) Serum folate concentration 3–20 μg/l (6.8–45 nmol/l)
Lymphocytes 1.0–3.0 × 109/l (20–40%) Red cell folate concentration 160–640 μg/l
Monocytes 0.2–1.0 × 109/l (2–10%) (0.36–1.45 μmol/l)
Eosinophils 0.02–0.5 × 109/l (1–6%) Plasma haemoglobin 10–40 mg/l
Basophils 0.02–0.1 × 109/l (<1–2%) concentration
Lymphocyte subsets Serum haptoglobin
(approximations from ranges concentration
in published data) Radial immunodiffusion 0.8–2.7 g/l
CD3 0.6–2.5 × 109/l (60–85%) Haemoglobin binding capacity 0.3–2.0 g/l
CD4 0.4–1.5 × 109/l (30–50%) Haemoglobin A2 2.2–3.5%
CD8 0.2–1.1 × 109/l (10–35%) Haemoglobin F <1.0%
CD4/CD8 ratio 0.7–3.5 Methaemoglobin <2.0%
Platelet count 280 ± 130 × 109/l Erythrocyte sedimentation rate
Bleeding time† (mm in 1 h at 20 ± 3 °C)
Ivy method 2–7 min Men
Template method 2.5–9.5 min 17–50 years ≤10
Thrombin time 15–19 s 51–60 years ≤12
Plasma fibrinogen 1.8–3.6 g/l 61–70 years ≤14
concentration >70 years ≤30
Plasminogen concentration‡ 0.75–1.60 u/ml Women
Antithrombin concentration‡ 0.75–1.25 u/ml 17–50 years ≤12
Protein C concentration‡ 51–60 years ≤19
Functional 0.70–1.40 u/ml 61–70 years ≤20
Antigen 0.61–1.32 u/ml >70 years ≤35
Protein S concentration‡ Plasma viscosity
Total antigen 0.78–1.37 u/ml 25 °C 1.50–1.72 mPa/s
Free antigen 0.68–1.52 u/ml 37 °C 1.16–1.33 mPa/s
Premenopausal women§ 0.55–1.55 u/ml
Functional 0.60–1.35 u/ml
Premenopausal women 0.55–1.35 u/ml
*Haemoglobin concentration may sometimes be reported as g/dl.
†
Bleeding time is no longer recommended for routine assessment of haemostasis but may be useful in suspected collagen disorders.
‡
These ranges are for general guidance only because each laboratory should establish its own normal range.
§
From Dykes AC, Walker ID, McMahon AD et al. Protein S antigen levels in 3788 healthy volunteers. Br J Haematol 2001;113:636–641.
TA BL E 2 - 2
12
HAEMATOLOGICAL VALUES FOR NORMAL INFANTS (AMALGAMATION OF DATA DERIVED FROM VARIOUS SOURCES;
EXPRESSED AS MEAN ± 2SD OR 95% RANGE)*
Practical Haematology
Birth Day 3 Day 7 Day 14 1 Month 2 Months 3–6 Months
Red blood cell count 6.0 ± 1.0 5.3 ± 1.3 5.1 ± 1.2 4.9 ± 1.3 4.2 ± 1.2 3.7 ± 0.6 4.7 ± 0.6
(RBC) (×1012/l)
Haemoglobin 180 ± 40 180 ± 30 175 ± 40 165 ± 40 140 ± 25 112 ± 18 126 ± 15
concentration (g/l)
Haematocrit (Hct) 0.60 ± 0.15 0.56 ± 0.11 0.54 ± 0.12 0.51 ± 0.2 0.43 ± 0.10 0.35 ± 0.07 0.35 ± 0.05
(l/l)
Mean cell volume 110 ± 10 105 ± 13 107 ± 19 105 ± 19 104 ± 12 95 ± 8 76 ± 8
(MCV) (fl)
Mean cell 34 ± 3 34 ± 3 34 ± 3 34 ± 3 33 ± 3 30 ± 3 27 ± 3
haemoglobin
(MCH) (pg)
Mean cell 330 ± 30 330 ± 40 330 ± 50 330 ± 50 330 ± 40 320 ± 35 330 ± 30
haemoglobin
concentration
(MCHC) (g/l)
Reticulocyte count 120–400 50–350 50–100 50–100 20–60 30–50 40–100
(×109/l)
White blood cell 18 ± 8 15 ± 8 14 ± 8 14 ± 8 12 ± 7 10 ± 5 12 ± 6
count (WBC)
(×109/l)
Neutrophils (×109/l) 4–14 3–5 3–6 3–7 3–9 1–5 1–6
Lymphocytes 3–8 2–8 3–9 3–9 3–16 4–10 4–12
(×109/l)
Monocytes (×109/l) 0.5–2.0 0.5–1.0 0.1–1.7 0.1–1.7 0.3–1.0 0.4–1.2 0.2–1.2
Eosinophils (×109/l) 0.1–1.0 0.1–2.0 0.1–0.8 0.1–0.9 0.2–1.0 0.1–1.0 0.1–1.0
Lymphocyte subsets
(×109/l)†
CD3 3.1–5.6 2.4–6.5 2.0–5.3
CD4 2.2–4.3 1.4–5.6 1.5–3.2
CD8 0.9–1.8 0.7–2.5 0.5–1.6
CD4/CD8 ratio 1.1–4.5 1.1–4.4 1.1–4.2
Platelets (×109/l) 100–450 210–500 160–500 170–500 200–500 210–650 200–550
*There have been some reports of WBC and platelet counts being lower in venous blood than in capillary blood samples.
†
Approximations because wide variations have been reported in different studies.
TA BL E 2- 3
HAEMATOLOGICAL VALUES FOR NORMAL CHILDREN (AMALGAMATION OF DATA DERIVED FROM

VARIOUS SOURCES; EXPRESSED AS MEAN ± 2SD OR 95% RANGE)
1 Year 2–6 Years 6–12 Years
Red cell count (×10 /l)
12
4.5 ± 0.6 4.6 ± 0.6 4.6 ± 0.6
Haemoglobin concentration (g/l) 126 ± 15 125 ± 15 135 ± 20
Haematocrit (Hct) or packed cell volume (PCV) (l/l) 0.34 ± 0.04 0.37 ± 0.03 0.40 ± 0.05
Mean cell volume (MCV) (fl) 78 ± 6 81 ± 6 86 ± 9
Mean cell haemoglobin (MCH) (pg) 27 ± 2 27 ± 3 29 ± 4
Mean cell haemoglobin concentration (MCHC) (g/l) 340 ± 20 340 ± 30 340 ± 30
Reticulocyte count (×109/l) 30–100 30–100 30–100
White cell count (×109/l) 11 ± 5 10 ± 5 9±4
Neutrophils (×109/l) 1–7 1.5–8 2–8
Lymphocytes (×109/l) 3.5–11 6–9 1–5
Monocytes (×109/l) 0.2–1.0 0.2–1.0 0.2–1.0
Eosinophils (×109/l) 0.1–1.0 0.1–1.0 0.1–1.0
Lymphocyte subsets (×109/l)*
CD3 1.5–5.4 1.6–4.2 0.9–2.5
CD4 1.0–3.6 0.9–2.9 0.5–1.5
CD8 0.6–2.2 0.6–2.0 0.4–1.2
CD4/CD8 ratio 1.0–3.0 0.9–2.7 1.0–3.0
Platelets (×109/l) 200–550 200–490 170–450
*Approximations because wide variations have been reported in different studies.
then provide sensitive indications of minor abnormali- difficult to assess. At birth the Hb is higher than at any
ties that may be important in clinical interpretation and period subsequently (Table 2-2). The RBC is high im-
health screening. mediately after birth,8 and values for Hb above 200 g/l,
It should be noted that in Table 2-1 the differential RBC higher than 6.0 × 1012/l and a haematocrit (Hct) over
white cell count is shown as percentages and in absolute 0.65 are encountered frequently when cord clamping is
numbers. Automated analysers provide absolute counts delayed and blood from the placenta and umbilical artery
for each type of leucocyte and, because proportional (per- re-enters the infant’s circulation. There are rapid fluctua-
centage) counting is less likely to indicate correctly their tions in the blood count of newborn babies, infants and
absolute increase or decrease, the International Council for older children. Reference ranges for preterm infants vary
Standardisation in Haematology has recommended that with gestational age. For example, in preterm infants in
the differential leucocyte count should always be given as the United States between 22 and 41 weeks’ gestation,
the absolute number of each cell type per unit volume of the packed cell volume increases from 0.40 to 0.52 l/l, the
blood.6 The neutrophil:lymphocyte ratio obtained from Hb from 140 to 170 g/l and the platelet count from 200
a differential leucocyte count should be regarded only as to 250 × 109/l, whereas the mean cell volume (MCV) and
an approximation. There are variations in the ability of mean cell haemoglobin (MCH) gradually decrease from
different automated blood cell analysers to characterise, 121 to 105 fl and from 40.5 to 35.5 pg, respectively.9
quantify and flag different types of cells. Most analysers After the immediate postnatal period, the Hb falls
show good correlation for neutrophils and eosinophils but fairly steeply to a minimum by about the second month
counts and flags for basophils, blasts and immature granu- (Fig. 2-4). The RBC and Hct also fall, although less steeply,
locytes may not be reliable enough for clinical use.7 and the cells may become microcytic with the develop-
ment of iron deficiency. The changes in the MCH, mean
PHYSIOLOGICAL VARIATIONS IN cell haemoglobin concentration (MCHC) and MCV from
THE BLOOD COUNT the neonate through infancy to early childhood are shown
in Tables 2-2 and 2-3.
Red cell components The Hb and RBC increase gradually through childhood
to reach almost adult levels by puberty. The lower normal
Age and gender limits for Hb (i.e. 2SD below the mean) are usually taken
There is considerable variation in the red blood cell count as 130 g/l for men and 120 g/l for women. The levels in
(RBC) and Hb at different periods of life and there are also women tend to be significantly lower than those in men10
transient fluctuations, the significance of which is often partly due to a hormonal influence on haemopoiesis, and
FIGURE 2-4 Changes in Hb values in the first 2 years after birth. The horizontal lines show the means and the perpendicular lines the
2SD ranges.
possibly subclinical iron deficiency in women. The extent TA B LE 2 -4

to which menstrual blood loss is a significant factor is not
clear because a loss of up to 100 ml of blood with each pe- HAEMOGLOBIN CONCENTRATION VALUES
riod may lead to iron depletion without causing anaemia. IN PREGNANCY
There may also be ethnic differences in Hb (e.g. the Hb is
First trimester 124–135 g/l
5–10 g/l lower in black Americans than in socially com-
Second trimester 110–117 g/l
parable white counterparts). In older adults the threshold Third trimester 106–109 g/l*
Hb level at which mortality increases is lower.11 Mean values postpartum
Day 2 104 g/l
Pregnancy Week 1 107 g/l
In normal pregnancy, there is an increase in erythropoietic Week 3 116 g/l
activity and a simultaneous increase in plasma volume oc- Month 2 119 g/l
curs, which overall results in a progressive decrease in Hb,
*Higher values (120 g/l or higher) may be found when supplementary
Hct and RBC (Table 2-4). There is a slight increase in MCV iron is being given.
during the second trimester. Serum ferritin decreases in
early pregnancy and usually remains low throughout
pregnancy, even when supplementary iron is given.12 The Moderate or severe anaemia should never be attributed to
haematological parameters return to normal about a week ageing per se until underlying disease has been excluded;
after delivery. however, a significant number of elderly subjects with anae-
mia have no identifiable clinical or nutritional causes.
The elderly
In healthy men and women, Hb, RBC, Hct and other red Exercise
cell indices remain remarkably constant until the sixth dec- Optimal athletic performance depends on proper function
ade. Anaemia becomes more common in those older than of many organs, including the blood. Several haemato-
70–75 years13 and is associated with poor clinical outcomes logical parameters can affect or be influenced by physi-
due to reduced cognition, increased frailty and an elevated cal activity, including blood cell counts and coagulation
risk of hospitalisation and of complications during hospi- mechanisms.14 For example, endurance athletes may de-
talisation. In the elderly, the difference in Hb between men velop so-called ‘sports anaemia’, which is thought to be
and women is 10 g/l or less compared with a difference the result of increased plasma volume. Increasing oxygen
of 20 g/l in younger age groups. Serum iron increases as delivery by raising the Hct is a simple acute method to
women age although serum ferritin levels remain higher improve athletic performance. Legal means of raising the
in elderly men than in women. Factors that contribute to Hct include altitude training and use of hypoxic tents.
the lower Hb in the elderly include renal insufficiency, in- Illegal means include blood doping and the administra-
flammation, testosterone deficiency, diminished erythro- tion of erythropoietin (EPO) (see Chapter 6).15 Endurance
poiesis, stem cell proliferative decline and myelodysplasia. athletes may also have decreased levels of serum iron and
ferritin, possibly associated with loss of iron in sweat. Leucocyte count

Conversely, in sprinters who require a short burst of very
strenuous muscular activity, there is a transient increase At birth, the total leucocyte count is high; neutrophils pre-
in RBC by 0.5 × 1012/l and in Hb by 15 g/l, largely because dominate, reaching a peak of ≈ 13.0 × 109/l within 6–8 h
of a reduction in plasma volume and to a lesser extent the for neonates of >28 weeks’ gestation and 24 h for those de-
re-entry into the circulation of cells previously sequestered in livered at <28 weeks.9 The count then falls to ≈ 4.0 × 109/l
the spleen. The effects of exercise must be distinguished from over the next few weeks and then stabilises (Tables 2-1,
a form of haemolysis known as ‘runner’s anaemia’ or ‘march 2-2 and 2-3). The lymphocytes decrease during the first
haemoglobinuria’, which occurs as a result of pounding of 3 days of life, often to a low level of ≈ 2.0–2.5 × 109/l, and
the feet on the ground.16 A similar phenomenon has been then rise up to the tenth day; after this time, they are the
reported in djembe drummers from repeated hand trauma. predominant cell (up to about 60%) until the fifth to sev-
enth year, when neutrophils predominate. From that age
Posture onwards, the levels are the same as those of adults. There
There is a small but significant alteration in the plasma vol- are also slight sex differences; the total leucocyte count
ume with an increase in Hb and Hct as the posture changes (WBC) and the neutrophil count may be slightly higher in
from lying to sitting, especially in women;17 conversely, girls than in boys, and in women than in men.21 After the
changing from walking to lying results in a 5–10% de- menopause, the counts fall in women so that they tend to
crease in the Hb and Hct. The difference in position of the become lower than in men of similar age.
arm during venous sampling, whether dependent or held People differ considerably in their leucocyte counts.
at atrial level, can also affect the Hct. Some tend to maintain a relatively constant level over long
These aspects highlight the relevance of using a stand- periods; others have counts that may vary by as much as
ardised method for blood collection, although this is not 100% at different times. In some subjects, there appears
necessarily practicable in routine practice. This is dis- to be a rhythm, occurring in cycles of 14–28 days, and
cussed in Chapter 1 and the differences between venous in women this may be related to the menstrual cycle or
and capillary blood are described on page 3. to the use of oral contraception. There is no clear-cut di-
urnal variation, but minimum counts are found in the
Diurnal and seasonal variation morning with the subject at rest and during the course
Changes in Hb and RBC during the course of the day are of a day there may be differences of 14% for the WBC,
usually slight, about 3%, with negligible changes in the 10% for neutrophils, 14% for lymphocytes and 20% for
MCV and MCH. However, variation of 20% occurs with eosinophils;18 in some cases this may result in a reversed
reticulocyte counts.18 Studies of diurnal variation of serum neutrophil:lymphocyte ratio. Random activity may raise
erythropoietin have shown conflicting results. Pronounced, the count slightly; strenuous exercise causes increases of
but variable, diurnal variations are seen in serum iron and up to 30 × 109/l, partly because of mobilization of mar-
ferritin and in patients taking iron- containing supple- ginated neutrophils and changes in cortisol levels.22 Large
ments.19 It has been suggested that minor seasonal vari- numbers of lymphocytes and monocytes also enter the
ations also occur, but the evidence for this is conflicting. bloodstream during strenuous exercise. However, there
have also been reports of neutropenia and lymphopenia in
Altitude athletes undergoing strenuous exercise.23
The effect of altitude is to reduce the plasma volume, in- Epinephrine (adrenaline) injection causes an increase in
crease the Hb and Hct and raise the number of circulat- the numbers of all types of leucocytes (and platelets), pos-
ing red cells with a lower MCV.20 The magnitude of the sibly reflecting the extent of the reservoir of mature blood
polycythaemia depends on the degree of hypoxaemia. At cells present not only in the bone marrow and spleen but
an altitude of 2000 m, Hb is ≈ 8–10 g/l and Hct is 0.025 also in other tissues and organs of the body. Emotion may
higher than at sea level; at 3000 m, Hb is ≈ 20 g/l and Hct possibly cause an increase in the leucocyte count in a sim-
is 0.060 higher; and at 4000 m, Hb is 35 g/l and Hct is ilar way. A transient lymphocytosis with a reversed neutro-
0.110 higher. Corresponding increases occur at interme- phil:lymphocyte ratio occurs in adults with physical stress
diate and at higher altitudes.20 These increases appear to or trauma. The effect of ingestion of food is uncertain.
be the result of enhanced erythropoiesis secondary to the Cigarette smoking has an effect on the leucocyte count
hypoxic stimulus, and the decrease in plasma volume that (see p. 16).
occurs at high altitudes. A moderate increase in the WBC, of up to 15 × 109/l,
is common during pregnancy, owing to an increase in the
Smoking neutrophil count, with the peak in the second trimester.
Cigarette smoking affects Hb, RBC, Hct and MCV (see The count returns to non-pregnancy levels about a week
p. 16). after delivery.24
In individuals of African ancestry there is a tendency TA B LE 2 -5

for the neutrophil:lymphocyte ratio to be reversed primar-
ily due to a reduction in neutrophil count. This is due EFFECTS OF CIGARETTE SMOKING*
to genetic rather than environmental factors. Significantly
Increased Decreased
lower WBC and neutrophil counts have also been observed
in Africans and Afro-Caribbeans living in Britain as well as Haemoglobin concentration (Hb) Plasma volume
in many African countries. ‘Benign ethnic neutropenia’ oc- Red blood cell count (RBC) Protein S
curs in up to 5% of African Americans and is defined as a Haematocrit (Hct)
neutrophil count <1.5 × 109/l without overt cause or com- Mean cell volume (MCV)
Mean cell haemoglobin (MCH)
plications.25 A Duffy null polymorphism is associated with White blood cell count (WBC)
the difference in WBC and neutrophil counts between Neutrophil count
African Americans and European Americans.26 Elderly Lymphocyte count
people receiving influenza vaccination show a lower total T cells (CD4-positive)
leucocyte count owing to a decrease in lymphocytes.27 Monocyte count
Carboxyhaemoglobin (>2%)
Platelet count (transient)
Platelet count Mean platelet volume
There is a slight diurnal variation in the platelet count Fibrinogen concentration
of about 5%;18 this occurs during the course of a day as β thromboglobulin concentration
von Willebrand factor
well as from day-to-day. Within the wide normal reference Red cell mass
range, there are some ethnic differences, and in healthy Haptoglobin concentration
Afro-Caribbeans and Africans platelet counts may on av- Plasma viscosity
erage be 10–20% lower than those in Europeans living in Whole blood viscosity
the same environment.28 There is also a gender difference; Erythrocyte sedimentation rate (ESR)
thus, in women, the platelet count is about 20% higher
than in men.29 A decrease in the platelet count may occur *Extent of change from normal reference values varies with individuals
and the amount smoked. Some effects may be transient or occur only
in women at about the time of menstruation. In the first during and immediately after smoking.
year after birth the reference range for the platelet count is
higher than the adult reference range. Strenuous exercise
causes a 30–40% increase in platelet count;22 the mecha- level increases by about 1%, and in heavy smokers the
nism is similar to that for leucocytes. carboxyhaemoglobin may constitute ≈ 4–5% of the to-
Further information. Detailed ranges related to age, tal haemoglobin. The WBC increases, largely as a result
gender, ethnic origin and pregnancy status are given in of an increase in the neutrophils; neutrophil function
reference 30. may also be affected. Smoking can cause an increase in
CD4-positive lymphocytes and total lymphocyte count.
Smokers tend to have higher platelet counts than non-
Other blood constituents smokers, but the counts decrease rapidly on cessation of
smoking. Studies of platelet aggregation and adhesiveness
As with the blood count, variations from usual values occur have given equivocal results, but there appears to be a
in relation to gender, age, exercise, stress, diurnal fluctua- consistent increase in platelet turnover with decreased
tion and so on. These are described in the relevant chapters. platelet survival and increased plasma β thromboglob-
ulin. Elevated fibrinogen concentration (with increased
plasma viscosity) and reduced protein S have been re-
EFFECTS OF SMOKING ON ported, but smoking does not seem to have any consist-
HAEMATOLOGICAL NORMAL ent effects on the fibrinolytic system.
REFERENCE VALUES
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Haematology 12TH Edition Barbara J.

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Following an informed decision to analyse a blood sam- PROCUREMENT OF VENOUS

Gentle pressure should be applied to the swab with the

SAMPLE HOMOGENEITY ANTICOAGULANTS

COLD AGGLUTININS Trisodium citrate

EFFECTS OF STORAGE ON THE

HAEMATOLOGICAL VALUES FOR NORMAL ADULTS (PREDOMINANTLY FROM EUROPE AND

HAEMATOLOGICAL VALUES FOR NORMAL CHILDREN (AMALGAMATION OF DATA DERIVED FROM

*Approximations because wide variations have been reported in different studies.

possibly subclinical iron deficiency in women. The extent TA B LE 2 -4

ferritin, possibly associated with loss of iron in sweat. Leucocyte count

In individuals of African ancestry there is a tendency TA B LE 2 -5

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