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1
 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.
No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical,
including photocopying, recording, or any information storage and retrieval system, without permission in writing
from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies
and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency,
can be found at our website: www.elsevier.com/permissions.
This book and the individual contributions contained in it are protected under copyright by the Publisher (other
than as may be noted herein).
Notices
Knowledge and best practice in this field are constantly changing. As new research and experience broaden our
understanding, changes in research methods, professional practices or medical treatment may become necessary.
Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any
information, methods, compounds or experiments described herein. In using such information or methods they should be
mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility.
With respect to any drug or pharmaceutical products identified, readers are advised to check the most current
information provided (i) on procedures featured or (ii) by the manufacturer of each product to be administered, to
verify the recommended dose or formula, the method and duration of administration and contraindications. It is the
responsibility of practitioners, relying on their own experience and knowledge of their patients, to make diagnoses, to
determine dosages and the best treatment for each individual patient and to take all appropriate safety precautions.
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability
for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from
any use or operation of any methods, products, instructions, or ideas contained in the material herein.
ISBN: 978-0-7020-6696-2
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 Preface
Sir John V. Dacie, MD, FRCPath, FRS (1912–2005).
This 12th edition celebrates the 66th year of Practical
Haematology, a notable achievement. The first edition by
JV (later Professor Sir John) Dacie was published in 1950.
This work, and subsequent editions with Mitchell Lewis as
co-­author, were based on the haematology course for the
University of London Diploma of Clinical Pathology and
subsequently the MSc in Haematology at the then Royal
Postgraduate Medical School.
In the last 66 years the techniques and instrumentation
available to the laboratory haematologist have expanded
at a rate once undreamed of. What has not changed is that
laboratory haematology continues to provide the b ­ edrock
that supports the equally astonishing developments in
clinical haematology. Haematology as a discipline ­remains
strongest when it is an integrated discipline with a very
close relationship between the laboratory and the ­clinical
service. Reflecting this ideal state, the authors of t­his
­edition include laboratory scientists and clinical and labo-
ratory haematologists.
vi
S. Mitchell Lewis, BSc, MD, DCP(London), FRCPath, FIBMS (b. 1924).
The 12th edition, like its predecessors, incorporates
the latest advances in laboratory haematology while
­continuing to describe traditional techniques that remain
applicable, particularly, but not only, in under-resourced
laboratories in low- and middle-income countries.
It is with sadness that we record the death of one of the
authors, Ms Carol Briggs, BSc FIBMS, during the prepara-
tion of this edition.
We are honoured to have taken over the editorship of
Practical Haematology from our distinguished ­predecessors,
Sir John Dacie and Dr Mitchell Lewis. We hope that our
efforts have done them justice.
Barbara J. Bain
Michael A. Laffan
Imelda Bates
 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
Hammersmith Hospital
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
Hammersmith Hospital
London, UK

Collection and
Handling of Blood
CHAPTER OUTLINE
Biohazard precautions, 1
Procurement of venous blood, 1
Equipment, 1
Specimen containers, 1
Phlebotomy procedure, 2
Postphlebotomy procedure, 3
Capillary blood, 3
Collection of capillary blood, 3
Blood film preparation, 3
Differences between capillary and venous blood, 3
Following an informed decision to analyse a blood sam-
ple, a specimen must be safely and correctly procured. It
is essential to be aware that variation in this pre-analytical
phase of the testing process can lead to errors in the ana-
lytical phase (see Box 1-1).
Venous blood is used for most examinations. Capillary
blood samples may be satisfactory for some purposes but
in general the use of capillary blood should be restricted to
children and to some point-of-care screening tests.
BIOHAZARD PRECAUTIONS
Laboratory policies must be in place to ensure that staff
who collect blood samples and transfer them to the labora-
tory minimise the risk of infection from various pathogens
during all aspects of specimen handling (see Chapter 24).
Additional precautions should be taken when handling
high-risk specimens (e.g. those from patients suspected of
having a viral haemorrhagic fever).1 In this circumstance,
the collection policy should stipulate the use of personal
protective equipment, such as disposable gloves, body
apron and protective eyewear. Care must be taken to pre-
vent injuries, especially when handling and disposing of
needles and lancets. Recommendations for standardising
blood collection have been published.2,3
1
Christopher McNamara
Sample homogeneity, 4
Serum, 4
Cold agglutinins, 4
Anticoagulants, 4
Ethylenediaminetetra-acetic acid (EDTA), 4
Trisodium citrate, 4
Heparin, 5
Effects of storage on the blood count, 5
Effects of storage on blood cell morphology, 5
PROCUREMENT OF VENOUS
BLOOD
Equipment
It is important to assemble a tray or prepare a workspace
that has all the requirements for blood collection (Box 1-2).
The selection of needle diameter is a compromise between
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
(about 15 mm). It may be helpful to collect the blood by
means of a winged needle (often referred to as a ‘butterfly’)
connected to a length of plastic tubing that can be attached
to the nozzle of the syringe or to a needle for entering the
cap of an evacuated container (see Specimen Containers).
Specimen containers
Containers for testing whole blood are available
­commercially with dipotassium, tripotassium or disodium
*The International Organisation for Standardisation has established a
standard (ISO 7864), which relates the following diameters for the
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
related to specimen collection
PRE-COLLECTION
• Urination within 30 min; food or water intake within
2h
• Smoking
• Physical activity (including fast walking) within 20 min
• Stress
• Drugs or dietary supplement administration within 8 h
DURING COLLECTION
• Different times (diurnal variance)
• Posture: lying, standing or sitting
• Haemoconcentration from prolonged tourniquet
pressure
• Excessive negative pressure when drawing blood into
syringe
• Incorrect type of tube
• Capillary versus venous blood
HANDLING OF SPECIMEN
• Insufficient or excess anticoagulant
• Inadequate mixing of blood with anticoagulant
• Error in patient and/or specimen identification
• Inadequate specimen storage conditions
• Delay in transit to laboratory
BOX 1-2 Items to be included in a
phlebotomy tray
• Syringes and needles
• Tourniquet
• Specimen containers (tubes or evacuated tube system) –
plain and with various anticoagulants
• Request form
• 70% isopropanol swabs or 0.5% chlorhexidine
• Sterile gauze swabs
• Adhesive dressings
• Self-sealing plastic bags with a separate compartment
for the request form
• Rack to hold specimens upright during process of filling
(except when an evacuated tube system is used)
• Puncture-resistant disposal container
ethylenediaminetetra-acetic acid (EDTA) anticoagulant,
and often have a mark to indicate the correct amount of
blood to be added.4 Containers are also available contain-
ing trisodium citrate, heparin or acid–citrate–dextrose, as
well as containers with no additive which are used when
serum is required. Design requirements and other specifi-
cations for specimen collection containers have been de-
scribed in a number of national and international standards
(e.g., that of the International Council for Standardisation
in Haematology5) and the European standard (EN 14820).
There is no universal agreement regarding the colours used
for identifying containers with different additives so phle-
botomists should familiarise themselves with the colours
used by their local suppliers.
Evacuated tube systems in common use consist of a
glass or plastic tube under a defined vacuum, a needle
and a needle holder, which secures the needle to the tube.
The main advantage is that the cap can be pierced so that
it is not necessary to remove it either to fill the tube or
subsequently to withdraw samples for analysis, thus min-
imising the risk of aerosol discharge of the contents. An
evacuated system is useful when multiple samples in dif-
ferent anticoagulants are required. The vacuum controls
the amount of blood that enters the tube, ensuring an ad-
equate volume for testing with the correct proportion of
any anticoagulant.
Phlebotomy procedure
Staff undertaking this procedure should be adequately
trained. The phlebotomist must check that the patient’s
identity corresponds to the details on the request form
and also ensure that the phlebotomy tray contains all the
required specimen containers and other equipment neces-
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
that the skin be cleaned with 70% alcohol (e.g., isopro-
panol) and allowed to dry spontaneously before being
punctured. The tourniquet should be released as soon as
the vein is punctured and blood begins to flow into the sy-
ringe or evacuated tube – delay in releasing the tourniquet
leads to fluid shift and haemoconcentration as a result of ve-
nous blood stagnation.6 After the vein has been successfully
punctured, the piston of the syringe should be withdrawn
slowly with no attempt being made to withdraw blood faster
than the vein is filling. Anticoagulated specimens must be
mixed by inverting the container several times. The risk of
unwanted haemolysis of the specimen can be minimised
by using minimal tourniquet time, withdrawing blood
carefully, using an appropriately sized needle, delivering the
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
the anticoagulant some coagulation may occur, rendering
the sample unsuitable. After collection, containers must be
firmly capped to minimise the risk of leakage.
If blood collection fails, it is important to remain calm,
communicate with the patient and consider the possible
causes. These include poor technique (e.g., passing the
needle through the vein, or poor selection of veins), scar-
ring of tissues and haematoma formation.
After obtaining the necessary specimens, remove the
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
worn by the patient. If this type of system is used both
the specimen label and the request form should be bar-
coded with identical data, unless the sample is to be used
for blood transfusion tests, in which case the label should
be handwritten (see Chapter 22).
Specimens should be sent in individual plastic bags
separated from the request forms to prevent contamina-
tion of the forms in the event of leakage. Samples and
form should remain together until the request is registered
in the laboratory reception area.
CAPILLARY BLOOD
Collection of capillary blood
Skin puncture is carried out with a needle or lancet. In
adults and older children, blood can be obtained from a
finger; the recommended site is the distal digit of the third
or fourth finger on its palmar surface, lateral to the nail
bed. In infants, satisfactory samples can be obtained by
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-
fants, especially newborns, to avoid the risk of injury and
possible infection to the underlying tarsal bones.
The area selected for capillary puncture should be
cleaned with an antiseptic and allowed to dry. The skin is
punctured to a depth of 2–3 mm with a sterile, disposable
lancet. After wiping away the first drop of blood with dry
sterile gauze the finger (or heel in infants) is squeezed gen-
tly to encourage a free flow of blood for collection. Free
FIGURE 1-1 Skin puncture in infants. Puncture must be restricted
to the outer medial and lateral portions of the plantar surface of the
foot indicated by the shaded area.
flow of blood is essential and only very gentle squeezing
is permissible; ideally, large drops of blood should exude
slowly but spontaneously.
After use, lancets should be placed in a puncture-­resistant
container for subsequent waste disposal. They must never
be re-used on another individual.
BLOOD FILM PREPARATION
Ideally, blood films should be made immediately after the
blood has been collected. However, in practice, blood
samples are usually sent to the laboratory after a variable
delay. Automated methods for making films are available
and often employed in large centres. When films are not
made on site they should be made in the laboratory soon
after arrival as blood film morphology will deteriorate
with any delay beyond a few hours.
DIFFERENCES BETWEEN
CAPILLARY AND VENOUS
BLOOD
Venous blood and capillary blood are not equivalent.
Blood from a skin puncture is a mixture of blood from
arterioles, veins and capillaries and it contains some in-
terstitial and intracellular fluid.7 The packed cell volume/
haematocrit (PCV/Hct), red blood cell count (RBC) and
 4 Practical Haematology
haemoglobin concentration (Hb) of capillary blood may
be slightly higher than those of venous blood. The to-
tal leucocyte and neutrophil counts may also be higher.
Conversely, the platelet count appears to be higher in ve-
nous than in capillary blood; this may be due to adhesion
of platelets to the site of the skin puncture. All of these
differences are minimised when a free flow of blood has
been obtained after skin puncture.
SAMPLE HOMOGENEITY
To ensure even dispersal of the blood cells it is essential
that specimens are mixed effectively in the laboratory, im-
mediately before being tested. The specimen tube can be
placed on a mechanical rotating mixer for 2 min or the
tube can be inverted 8–10 times by hand. If the specimen
has been stored at 4 °C, it will be viscous and the blood
should be allowed to warm to room temperature before
being mixed.
SERUM
The difference between plasma and serum is that the latter
lacks fibrinogen and some coagulation factors. Blood col-
lected in order to obtain serum should be delivered into
sterile tubes with caps or into commercially available plain
(no anticoagulant) evacuated collection tubes and allowed
to clot undisturbed for about 1 h at room temperature be-
fore centrifugation.† Some containers have silica particles
and an inert polymer gel, which floats between the serum
and the red cells when centrifuged, facilitating separation
of serum and making the specimen suitable for use on
some automated analysers. This eliminates the need to de-
cant the serum and preserves the integrity of the specimen.
The tubes, whether with or without a serum separa-
tor, are then centrifuged for 5 minutes at 3000 revolutions
per minute (rpm). Some tests require a further centri­
fugation to remove any remaining particulate material.
Supernatant serum may be transferred to tubes for further
tests or stored. For most tests, serum should be kept at
4 °C until used, but if testing is delayed, serum can be
stored at −20 °C for up to 3 months and at −40 °C or be-
low for long-term storage. Validation of storage conditions
and sample viability should be undertaken for all tests
performed. Frozen specimens should be thawed in a wa-
ter bath or in a 37 °C incubator, and then inverted several
times to ensure homogeneity before being used for a test.
COLD AGGLUTININS
If cold agglutinins are suspected the blood must be kept
at 37 °C from the point of collection until the sample has
been processed. If cold agglutinins are suspected, it is
best to bring the patient to a suitable location close to the
†
Room temperature is usually considered 18–25 °C.
laboratory and to collect blood into a previously warmed
syringe and then to deliver the blood into containers that
have been kept warm at 37 °C. When filled, the ­containers
should be promptly replaced in the 37 °C water bath. In
this way, it is possible to assess the effect of any putative
antibodies acting, in vivo, at body temperature. When this
is not feasible, specimens can be tightly capped and placed
in a thermos at 37 °C.
ANTICOAGULANTS
Ethylenediaminetetra-acetic acid
(EDTA)
EDTA and sodium citrate remove calcium, which is essen-
tial for coagulation. Calcium is either precipitated as in-
soluble oxalate (crystals of which may be seen in oxalated
blood) or bound in a non-ionised form. Heparin binds
to antithrombin, thus inhibiting the interaction of several
clotting factors.
EDTA anticoagulation is used for blood counts; sodium
citrate is used for coagulation testing and for the erythro-
cyte sedimentation rate. For better long-term preservation
of red cells for certain tests and for transfusion purposes,
citrate is used in combination with dextrose in the form
of acid–citrate–dextrose (ACD) or citrate–phosphate–­
dextrose (CPD).
An excess of EDTA affects both red cells and leuco-
cytes, causing shrinkage and degenerative changes. EDTA
in excess of 2 mg/ml of blood may result in a significant
decrease in PCV assessed by centrifugation and an increase
in mean cell haemoglobin concentration (MCHC).8 The
platelets may also be affected; an excess of EDTA causes
them to swell and then disintegrate, causing an artificially
high platelet count, as the fragments are large enough to
be counted as platelets. Care must therefore be taken to
ensure that the correct amount of blood is added, and that
by repeated inversions of the container the anticoagulant
is thoroughly mixed with the blood specimen. EDTA is re-
sponsible for the activity of a naturally occurring antiplatelet
autoantibody, which sometimes causes platelet aggregation
of platelet adherence to neutrophils in blood films. All pa-
tients with apparent thrombocytopenia therefore require
a blood film to identify this in vitro phenomenon. Repeat
estimation of the platelet count in an alternative antico-
agulant will resolve this problem, as the aforementioned
antibody is inactive in the absence of EDTA.9
Trisodium citrate
For coagulation studies, 9 volumes of blood are added to
1 volume of 109 mmol/l sodium citrate solution (32 g/l of
Na3C6H5O7•2H2O‡).10 This ratio of anticoagulant to blood
is critical as osmotic effects and changes in free calcium
‡
38 g/l of 2Na3C6H5O7•11H2O.

ion concentration affect coagulation test results. This ratio
of citrate to blood may need to be adjusted when samples
with a high haematocrit require coagulation studies (see
Chapter 18).
Heparin
Lithium or sodium salt of heparin at a concentration of
10–20 iu/ml of blood is a commonly used anticoagulant
for chemistry, gas analysis and emergency tests. It does
not alter the size of the red cells and it is recommended
when it is important to reduce to a minimum the chance
of lysis occurring after blood has been withdrawn. When
red cells are required for testing, as in the investigation of
certain types of haemolytic anaemia, the sample can be
defibrinated (see previous editions for details) although
heparinised blood is now more often used for such tests.
Heparin is not suitable for blood counts and films as it
often induces platelet and leucocyte clumping11 and gives
a faint blue colouration to the background when films are
stained by Romanowsky dyes (especially, but not only,
in the presence of abnormal proteins). Heparin inhibits
enzyme activity and it should not be used when a poly-
merase chain reaction with restriction enzymes is to be
performed.12
EFFECTS OF STORAGE ON THE
BLOOD COUNT
Various changes take place in anticoagulated blood when
it is stored at room temperature, and these changes occur
more rapidly at higher ambient temperatures. These occur
regardless of the anticoagulant. The RBC, white blood cell
count (WBC), platelet count and red cell indices are usu-
ally stable for up to 8 h after blood collection, although
as the red cells start to swell the PCV/Hct and mean cell
volume (MCV) start to increase, osmotic fragility increases
and the erythrocyte sedimentation rate decreases. When
the blood is kept at 4 °C the effects on the blood count
are not usually significant for up to 24 h. Thus, for many
purposes blood can safely be allowed to stand overnight
in the refrigerator if precautions against freezing are taken.
Nevertheless, it is best to count leucocytes and especially
platelets within 2 h and it should be noted that the de-
crease in the leucocyte count and a progressive decrease
in the absolute lymphocyte count may become marked
within a few hours, especially if there is an excessive
amount of EDTA (>4.5 mg/ml).13 Storage beyond 24 h at
4 °C results in erroneous data for automated white cell dif-
ferential counts. One study using an aperture impedance
analyser on blood left at room temperature showed WBC
and neutrophil counts to be stable for 2–3 days but other
leucocyte counts were stable for only a few hours.14
Reticulocyte counts are unchanged when the blood is
kept in either EDTA or ACD anticoagulant for 24 h at 4 °C,
1 Collection and Handling of Blood 5
but at room temperature the count begins to fall within
6 h. Nucleated red cells disappear in the blood specimen
within 1–2 days at room temperature.
Haemoglobin concentration remains unchanged for
days. However, within 2–3 days, and especially at high
ambient temperatures, the red cells begin to lyse, resulting
in a decrease in the RBC and PCV/Hct, with an increase in
the calculated MCH and MCHC.
Coagulation test stability is critical for diagnosis and
treatment of coagulopathies; it is recommended that tests
be carried out within 2 h when the blood or plasma is
stored at 22–24 °C, within 4 h when stored at 4 °C, within
2 weeks when stored at −20 °C, and within 6 months
when stored at −70 °C.3
For a serum or plasma test, blood should be centri-
fuged within 5 h of collection. For vitamin B12 and folate
assays, the serum or plasma should be kept at 4 °C or
at −20 °C if storage for more than 2–3 weeks is required.
For long-term storage, specimens should be divided into
several aliquots to avoid repeated freezing and thawing.
Inappropriate handling of blood specimens during
transfer to the laboratory (e.g. excess shaking or being
exposed to temperature extremes) may cause haemoly-
sis, partial coagulation and cell disintegration. Shipping
of specimens requires special packaging and should reach
certain minimum specifications.14
EFFECTS OF STORAGE ON
BLOOD CELL MORPHOLOGY
Changes in blood cell morphology of stored samples ­occur
within a few hours of blood collection. Irrespective of anti­
coagulant, films made from blood that has been standing
for <1 h at room temperature are not easily distinguished
from films made immediately after collection of the blood.
By 3 h, changes may be discernible and by 12–18 h these
become striking. Some but not all neutrophils are affected;
their nuclei may stain more homogeneously than in fresh
blood, the nuclear lobes may become separated and the
cyto­plasmic margin may appear ragged or less well-­defined;
small vacuoles appear in the cytoplasm (Fig. 1-2, A, B).
Some or many of the large monocytes develop marked
changes; small vacuoles appear in the cytoplasm and the
nucleus undergoes irregular lobulation, which may almost
amount to disintegration (Fig. 1-2, C). Lymphocytes un-
dergo similar changes: a few vacuoles may be seen in the
cytoplasm, nuclei stain more homogeneously than usual
and in some the nucleus undergoes budding, giving rise
to nuclei with two or three lobes (Fig. 1-2, D–F). Normal
red cells are little affected by standing for up to 6 h at room
temperature. Longer periods lead to progressive crenation
(Fig. 1-2, B, E, F). With an excess of EDTA, a marked de-
gree of red cell crenation occurs within a few hours. All
the aforementioned changes are retarded but not abolished
in blood stored at 4 °C. Their occurrence underlines the
 6 Practical Haematology

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
FIGURE 1-3 Morphological features of apoptosis.
importance of making films as soon as possible after the
blood has been collected.
These artefactual changes must be distinguished from
apoptosis, which can be seen in high-grade haemato-
logical neoplasms. Apoptosis is characterised, morpho-
logically (Fig. 1-3), by cell shrinkage, a homogeneously
glassy appearance of the nucleus, cytoplasmic conden-
sation around the nuclear membrane and indentations
in the nucleus, followed by its fragmentation. Apoptotic
neutrophils with a single apoptotic body may be confused
with nucleated red cells if the cytoplasmic features are not
appreciated.
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.
3. CLSI. Procedures for the collection of diagnostic blood specimens by
venipuncture. Approved standard. 6th ed. Wayne, PA: CLSI; 2007.
Document H3-A6.
4. NCCLS. Tubes and additives for venous blood specimen collection.
Approved standard. 5th ed. Wayne, PA: NCCLS; 2003.
5. Tatsumi N, van Assendelft OW, Naka K. ICSH recommendation for
blood specimen collection for hematological analysis. Lab Hematol
2002;8:1–6.
6. Saleem S, Mani V, Chadwick M, et al. A prospective study of causes
of haemolysis during venepuncture: tourniquet time should be kept
to a minimum. Ann Clin Biochem 2009;46:244–6.
7. Yang Z-W, Yang S-H, Chen L, et al. Comparison of blood counts in
venous, finger tip and arterial blood and their measurement varia-
tion. Clin Lab Haematol 2001;23:155–9.
8. Zini G. Stability of complete blood count parameters with storage:
toward defined specifications for different diagnostic applications.
Int J Lab Hematol 2014;36:111–3.

9. Schuff-Werner P, Steiner M, Fenger S, et al. Effective estimation
of correct platelet counts in pseudothrombocytopenia using an
alternative anticoagulant based on magnesium salt. Br J Haematol
2013;162:684–92.
10. Ingram GIC, Hills M. The prothrombin time test; effect of varying
citrate concentration. Thromb Haemost 1976;36:230–6.
11. Salzman EW, Rosenberg RD. Effect of heparin and heparin fractions
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.
1 Collection and Handling of Blood 7
13. Hadley GG, Weiss SP. Further notes on use of salts of ethylene di-
aminetetraacetic acid (EDTA) as anticoagulants. Am J Clin Pathol
1955;25:1090–3.
14. Gulati GL, Hyland LJ, Kocher W, et al. Changes in automated com-
plete blood cell count and differential leucocyte count results in-
duced by storage of blood at room temperature. Arch Pathol Lab Med
2002;126:336–42.
 2
Reference Ranges and
Normal Values
Imelda Bates
CHAPTER OUTLINE
Reference ranges, 8
Statistical procedures, 9
Confidence limits, 9
Normal reference values, 10
Physiological variations in the blood count, 13
Red cell components, 13
A number of factors affect haematological values in appar-
ently healthy individuals. As described in Chapter 1, these
include the technique and timing of blood collection, the
transport and storage of specimens, the posture of the sub-
ject when the sample is taken, the prior physical activity
and the degree of ambulation (e.g. whether the subject is
confined to bed or not). Variation in the analytical methods
used may also affect the measurements. These can all be
standardised.
More problematic are the inherent variables as a result
of gender, age, occupation, body build, genetic background
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-
tain that the ‘normal’ subjects used for constructing normal
ranges are completely healthy and do not have nutritional
deficiencies, mild chronic infections, parasitic infestations
or the effects of smoking.
Haematological values for the normal and abnormal will
overlap and a value within the recognised normal range may
be definitely pathological in a particular subject. For these
reasons the concept of ‘normal values’ and ‘normal ranges’
has been replaced by reference values and the reference range,
which is defined by reference limits and obtained from meas-
urements on the reference population for a particular test.
Unless a reference range is derived in this manner, the term
should not be used. The reference range is also termed the
reference interval.1,2 Ideally, each laboratory should establish
8
Leucocyte count, 15
Platelet count, 16
Other blood constituents, 16
Effects of smoking on haematological normal
reference values, 16
a databank of reference values that takes account of the
variables mentioned earlier and the test method, so that an
individual’s result can be expressed and interpreted relative
to a comparable apparently normal population, insofar as
normal can be defined.
New haematological parameters such as the number
of immature cells or the number of red cell fragments
are often initially developed for research purposes but
can be used for clinical decision making once internal
quality control and external quality assessment processes
are in place.3
REFERENCE RANGES
A reference range for a specified population can be es-
tablished from measurements on a relatively small num-
ber of subjects (discussed later) if they are assumed to be
representative of the population as a whole.2 The condi-
tions for obtaining samples from the individuals and the
analytical procedures must be standardised, whereas data
should be analysed separately for different variables relat-
ing to individuals – recumbent or ambulant, smokers or
nonsmokers and so on. One approach is that specimens
are collected at about the same time of day, preferably in
the morning before breakfast; the last meal should have
been eaten no later than 9 p.m. on the previous evening,
and at that time alcohol should have been restricted to
one bottle of beer or an equivalent amount of another

alcoholic drink.4 An alternative approach is that, unless
a test is usually done on a fasting patient, specimens are
collected throughout the day on subjects who are not
fasting or resting, as this will produce a reference range
that is more relevant to results from patients. It is some-
times appropriate that the reference population is defined
as having normal results for specific laboratory tests. For
example, if determining a reference range for blood count
components it may be necessary, in some populations,
to exclude iron deficiency, β thalassaemia heterozygosity
and, when relevant, α thalassaemia.
STATISTICAL PROCEDURES
In biological measurements, it is usually assumed that the
data will fit a specified type of pattern, either symmet-
ric (Gaussian) or asymmetric with a skewed distribution
(non-Gaussian). With a Gaussian distribution, the arith-
metic mean (x) can be obtained by dividing the sum of all
measurements by the number of observations. The mode is
the value that occurs most frequently and the median (m)
is the point at which there are an equal number of obser-
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.
If the data fit a Gaussian distribution, when plotted as
a frequency histogram the pattern shown in Figure 2-1 is
obtained. Taking the mode and the calculated SD as ref-
erence points, a Gaussian curve is superimposed on the
histogram. From this curve, practical reference limits can
be determined even if the original histogram included
outlying results from some subjects not belonging to the
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.
2 Reference Ranges and Normal Values 9
normal population. Limits representing the 95% reference
range are calculated from the arithmetic mean ±2SD (or
more accurately ±1.96SD).
When there is a log normal (skewed) distribution of
measurements, the range to −2SD may even extend to
zero (Fig. 2-2, A). To avoid this anomaly, the data should
be plotted on semilogarithmic graph paper to obtain a
normal distribution histogram (Fig. 2-2, B). To calculate
the mean and SD the data should be converted to their
logarithms. The log–mean value is obtained by adding the
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
antilogs to express the data in the arithmetic scale. This
process is now generally carried out using an appropriate
statistical computer program.
When it is not possible to make an assumption about
the type of distribution, a nonparametric procedure may
be used instead to obtain the median and SD. To obtain
an approximation of the SD, the range that comprises the
middle 50% spread (i.e. between 25 and 75% of results) is
read and divided by 1.35. This represents 1SD.
Confidence limits
In any of the methods of analysis, a reasonably reliable
estimate can be obtained with 40 values, although a larger
number (≥120) is preferable (Fig. 2-3).5 When a large set
of reference values is unattainable and precise estimation
is impossible, a smaller number of values may still serve as
a useful clinical guide. Confidence limits define the relia-
bility (e.g. 95% or 99%) of the established reference values
 10 Practical Haematology

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
when it is on the borderline between normal and abnor-
mal. Calculation of confidence limits is described on page
566. Another important measurement is the coefficient of
variation (CV) of the test because a wide CV is likely to
influence its clinical utility (see p. 566).
NORMAL REFERENCE VALUES
The data given in Tables 2-1, 2-2 and 2-3 provide general
guidance to normal reference values that are applicable to
most healthy adults and children in high-income coun-
tries. However, slightly different ranges may be found
in individual laboratories where different analysers and
methods are used. The reference interval, which comprises
a range of ±2SD from the mean, indicates the lim-
its that should cover 95% of normal subjects; 99% of
normal subjects will be included in a range of ±3SD.
Age and gender differences have been taken into ac-
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
expected under standardised conditions. Because mod-
ern analysers provide a high level of technical precision,
even small differences in successive measurements may
be significant. It is thus important to establish and un-
derstand the limits of physiological variation for various
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.
Another random document with
no related content on Scribd:
These slit-footed, or fissiped creatures, are divided into several
species, and there is considerable difference in their way of treading;
some, as the cats, walk on their toes. Come here, kitty, let us see
your feet. Kitty can draw her claws close into the “velvet,” or fur, of
her paw so that you would not know she had sharp nails. As she
does this she curls her foot up into a cushion, and walks on the ends
of her toes. Look at her foot when she stretches it; how well you can
see the slit-foot arrangement and the clawed toes. Kitty is a mild and
domestic representative of the great, feline race, at the head of
which stands the lion. Ponto, on the other hand, is a civilized and
educated specimen of the canine race, which numbers among its
members the wolf.
While kitty draws in her claws and walks on her toes, the bears are
what is called plantigrade carnivora, for they walk on the flat soles of
their feet. These carnivora are all flesh-eaters, but when educated
and refined, like Ponto and Kitty, they will eat any kind of food.
Among the carnivora we enumerate lions, tigers, bears, cougars,
hyenas, panthers, wolves, jackals, leopards, cats, dogs, badgers,
and many more. You see they embrace all the fiercest and most
dangerous wild beasts, as well as our domestic friends, the cat and
the dog. If you will open Kitty’s mouth, or Ponto’s, you will see what
style of teeth these carnivora have; they are made for tearing flesh,
and are accompanied by sharp claws for holding fast the living prey.
All the carnivora are singularly strong, agile, and tenacious of life.
The carnivora are so varied and interesting, that we leave them to be
studied elsewhere, and indeed so we must serve the final order of
mammals—the primates.[94]
These primates are divided into two sub-orders, the quadrumana, or
four-handed animals, and the human race. Many scientists, instead
of the order primates, with these two sub-orders, give us the order
quadrumana, or monkeys, and the order bimana, or man, and that is
the better division.
The quadrumana, or ape families, contain a large number of curious
and interesting animals, all natives of tropical regions. They are hairy
creatures, with three kinds of teeth, and are adapted for eating
nearly all kinds of food. They are well known to us in menageries,
and are frequently seen on our streets, fancifully dressed, and in
company with a man and an organ, surrounded also by a group of
admiring children.
“How many children are there in your family, Pierre?” we one day
asked a bright little Italian boy.
“Four, and the monkey, signora.”
“But the monkey, Pierre, does not count with the children.”
“O signora! for care and trouble he counts more than the children!
He must have the warmest corner and the best food. At night, for
warmth, he sleeps in my father’s arms. He gets cold ten times as
often as we do; and if he gets a cold he must have the doctor, for he
is likely to die. If he dies, away go all our saved dollars for more
monkey! Gracious! O signora! do you think our father would let our
monkey run round in the streets and look out for his own dinner as
Marie and I do? The thing is impossible!”
THE END.

FOOTNOTES:
[92] The animal called a buffalo in America is not a buffalo, but a
bison. The buffalo of India and Italy is the Bubalus, the buffalo of
the Western plains is the bison. The large, wild bison of Germany
is called the Aurochs. All these animals belong to the family
Bovidæ.
[93] If we examine the toes of the cat and dog, we shall see that
the skin extends web-like partly up the toe bones.
[94] It has been impossible in so small a book to discuss with any
minuteness the subjects noticed in the closing chapters. It is
hoped that the hints given will incite pupils to study and observe
the domestic animals of which we see so much and know so little.
 SEA-SIDE AND WAY-SIDE.
BOOK I.

Illustration from No. 1.—Mr. and Mrs. Crab.

CONTENTS.

Lesson.
I. Mr. and Mrs. Crab.
II. Mr. Crab and his House.
III. More about Mr. Crab.
IV. Mr. and Mrs. Crab get New Coats.
V. What the Crab does.
VI. Mr. Crab and his Friends.
VII. Some Other Crabs.
VIII. The Hermit Crab.
IX. The Crab’s Enemies.
X. The Uses of Crabs.
XI. Mrs. Wasp and her Home.
XII. What Mrs. Wasp can do.
 XIII. A Look at Mrs. Wasp.
XIV. Mrs. Wasp’s Year.
XV. Mrs. Wasp at Home.
XVI. Review.
XVII. The Bee and the Man.
XVIII. How the Bee is Made.
XIX. The Bee at Home.
XX. The Bee Babies.
XXI. The Bee War.
XXII. The Bee’s Work.
XXIII. The Wise Bees.
XXIV. Earth Bees.
XXV. Other Bees.
XXVI. More about Bees.
XXVII. The Spider and his Dress.
XXVIII. The Spider at Home.
XXIX. The Little Nest.
XXX. The Spider and his Food.
XXXI. Very Queer Spiders.
XXXII. Review.
XXXIII. Out of Harm’s Way.
XXXIV. Shell-Fish.
XXXV. The Story of Mr. Conch.
XXXVI. Sea-Babies.
XXXVII. More about Sea-Babies.
XXXVIII. About Mr. Drill.
 XXXIX. The Story of a War.
XL. How Shell-Fish Feed.
XLI. Review.
Book I. 95 pages. 32 illustrations. 30 cts.

BOOK II.

Illustration from No. 2.—The Parasol Ants.

CONTENTS.

Lesson.
I. A Look at an Ant.
II. The Life of an Ant.
III. The Ant’s Home.
IV. The Ants at Home.
V. The Ants on a Trip.
VI. The Farmer Ants.
 VII. Ants and their Trades.
VIII. The Slave Ants.
IX. Wonder Ants.
X. The Ways of Ants.
XI. Mr. Worm and his Family.
XII. The Earth-worm at Home.
XIII. Mr. Worm at Work.
XIV. Mr. Worm’s Cottage by the Sea.
XV. Mr. Worm at Home.
XVI. A Look at a House-Fly
XVII. How to Look at a Fly.
XVIII. Mrs. Fly and her Foes.
XIX. Of what Use are Flies.
XX. A Swarm of Flies.
XXI. Some Queer Flies.
XXII. In Armor Clad.
XXIII. When Mr. Beetle was Young.
XXIV. How to Learn about Beetles.
XXV. The Rose Beetle.
XXVI. Princes and Giants.
XXVII. The Little Sexton.
XXXVIII. The Story of the Stag Beetle.
XXIX. Mr. Beetle Seeks for a Home.
XXX. The Little Water-Men.
XXXI. Whirligig Beetles.
XXXII. What a Fisherman Told.
 XXXIII. Mr. Barnacle and his Son.
XXXIV. A Fishing Party.
XXXV. A Last Look at Mr. Barnacle.
XXXVI. Flowers of the Sea.
XXXVII. The Life of a Jelly-Fish.
XXXVIII. Sea-Stars.
XXXIX. A Sea-Change.
XL. The Star-Fish with an Overcoat.
XLI. The Flying Flowers.
XLII. Under the Water.
XLIII. A Happy Change.
XLIV. The Dragon-Fly and his Cousins.
XLV. The Wings of the Dragon.
Review Lessons.
Book II. 184 pages. 39 illustrations. 40 cts.

BOOK III.
 Illustration from No. 3.—Beaks.

CONTENTS.

Lesson.
I. The Great Mother.
II. The Earth’s Eldest Child.
III. A Look at a Plant.
IV. A Year in a Plant’s Life.
V. The Growth of Plants.
VI. The Food of Plants.
VII. Seeds and Leaves.
VIII. The Color of Plants.
IX. The Motion of Plants.
X. Plants and their Partners.
XI. Air, Water, and Sand Plants.
 XXII. Plants that eat Animals.
XIII. Weather Prophet Plants.
XIV. Plant Clocks.
XV. The School Cabinet.
XVI. The Old Man of the Meadow.
XVII. The Life of the Old Man.
XVIII. The Robber Cousin.
XIX. The Merry Cousins.
XX. Queer Cricket.
XIX. Other Hoppers.
XXII. A Real Live Fairy.
XXIII. The Child of the Day.
XXIV. Life Among Snow and Roses.
XXV. Joseph’s Coat.
XXVI. Cousin Moth.
XXVII. The Child of the Night.
XXVIII. The Bird.
XXIX. Beaks and Claws.
XXX. Trees, Ground, and Water Birds.
XXXI. On the Wing.
XXXII. Nest Building.
XXXIII. The Bird at Home.
XXXIV. Birds of Song.
XXXV. The Other Partner.
XXXVI. A Brigade of Birds.
XXXVII. The Birds in the Woods.
XXXVIII. The Birds in the House.
XXXIX. The Lost Birds.
XL. The Fin Family
XLI. Outside and Inside.
XLII. Where they live.
XLIII. How they Behave.
XLIV. Fry and School.
XLV. Scales and Teeth.
XLVI. Big and Little Brothers.
Book III. 300 pages. 29 illustrations. 50 cts.

BOOK IV.
Illustration from No. 4.—The Reign of the Pine.

CONTENTS.

I. Earth Building.
II. The First Continent.
III. The Age of Crabs and Corals.
IV. The Pines and the Reptiles.
V. The Palm and the Man.
VI. The Starry Heavens.
VII. A Fragment of the Milky Way.
VIII. Plan and Progression.
IX. The King of the Day.
X. The Queen of the Night.
XI. Vanished Fauna.
 XII. A Mountain of Fossils.
XIII. Written in Rocks.
XIV. Footprints in the Sand.
XV. The Winter of the World.
XVI. Fossil Crabs.
XVII. Stone-Fish and Stone-Lilies.
XVIII. Long-Buried Reptiles.
XIX. Birds of other Ages.
XX. The Early Mammals.
XXI. Very Old Families.
XXII. The Marvel in Mail.
XXIII. The Wonderful Builder.
XXIV. An Opossum Hunt.
XXV. A New Fashion of Pappoose.
XXVI. Low Down in the Scale.
XXVII. With a Duck’s Bill.
XXVIII. In Australian Rivers.
XXIX. A Walk Among Wonder Trees.
XXX. Still in the Wonder Grove.
XXXI. A Noisy Family.
XXXII. The Frogs’ Cousin.
XXXIII. Salamanders.
XXXIV. A Citizen of the Marsh Lands.
XXXV. A Stranger from Mexico.
XXXVI. Some Merry Little Friends.
XXXVII. The Ancient Monster.
 XXXVIII. El Lagarto.
XXXIX. Wiser than any Beast of the Field.
XL. Our Common Enemy.
XLI. With a House on His Back.
XLII. A Real Live Mermaid.
XLIII. Great Whales Also.
XLIV. A Seal-Skin Cloak.
XLV. Flying Mammals.
XLVI. Order out of Confusion.
XLVII. Nibblers.
XLVIII. Gnawers.
XLIX. The Thick Skins.
L. The Ruminants.
Book IV. 370 pages. 28 illustrations. 60 cents.
RICKS’ NATURAL HISTORY OBJECT LESSONS.
PART I.—PLANTS AND ANIMALS.

Chapter.
I. Introduction.
II. A Typical Plant.
III. General Classification of Plants.
IV. Minute Structure of Plants.
V. Roots and their Functions.
VI. Stems and their Uses.
VII. Leaves and Buds.
VIII. Flowers, their Parts and Uses.
IX. Fruits and Seeds.
X. The Palm Trees.
XI. Cereals, the Sugar-Cane, etc.
XII. Starches.
XIII. Oils and Fats.
XIV. Gums, Resins, Gum-Resins, etc.
XV. Cotton, Hemp, Flax, Jute.
XVI. Paper.
XVII. Bleaching and Dyeing.
XVIII. Tea, Coffee and Chocolate.
XIX. Spices.
XX. Opium, Quinine and Camphor.
XXI. Indigo, Oak-Galls, etc.
 XXII. Classification of Animals.
XXIII. and XXIV. Classification of Vertebrata.
XXV. Classification of Invertebrata.
XXVI. Coverings of Vertebrate Animals.
XXVII. The Bony Skeleton and its Modifications.
XXVIII. Teeth,—Varieties and Uses.
XXIX. Tongues.
XXX. Tails and their Uses.
XXXI. The Principal Internal Organs of Animals.

PART II.—SPECIMEN LESSONS.

Lesson.
I. Paws and Claws.
II. Cocoa-Nut.
III. Cotton and Wool.
IV. An Egg.
V. Acorn and Hazel-Nut.
VI. Milk.
VII. Onion, Turnip, Carrot.
VIII. Cat and Dog.
IX. Down.
X. A Quill Feather.
XI. Gutta Percha.
XII. Leaves.
XIII., XIV. and XV. Starch.
XVI. The Horse.
 XVII. The Cow and the Sheep.
XVIII. Honey and Wax.
XIX. Ivory.
XX. and XXI. Seeds and Seedlings.
XXII. Olive Oil.
XXIII. Liber.
XXIV. Mammals and Birds.
XXV. Reptiles and Fishes.
XXVI. Mammals.
XXVII. Chewing the Cud.
XXVIII. Horns and their Uses.
XXIX. Parts of a Flower.
XXX. Birds’ Nests.
XXXI. The Hedgehog.
XXXII. Whale Oil.
XXXIII. Leather.
XXXIV. The Mole.
XXXV. Cotton.
XXXVI. Vertebrata and Invertebrata.
XXXVII. The Cockroach.
XXXVIII. The Earthworm.
XXXIX. Spider’s Threads.
XL. Bleaching.
XLI. The Rat and His Relatives.
XLII. Beaks of Birds.
XLIII. and XLIV. Snakes.
 XLV. and XLVI. Fishes.
XLVII. Insects—Form and Structure.
XLVIII. Insects—Benefits and Injuries.
XLIX. Insects,—Metamorphosis.
L. Insects, Legs and Feet.
LI. Insect and Spider.
LII.-LIV. Legs and Feet,—Mammals.
LV.-LVI. Legs and Feet,—Birds.
LVII. Flour.
LVIII. The Frog.
LIX. The Frog,—Life History.
LX. and LXI. Eggs.
LXII. Snails.
LXIII. Snails—Whelk and Periwinkle.
LXIV. Snails.
LXV. The Amoeba and Foraminifera.
LXVI. The Hydra.
LXVII. Sea Anemones and Corals.
LXVIII. Plant Factories.
352 pages. 121 illustrations and seven plates. Cloth $1.50.
 Elementary Science.
Natural History Object Lessons. A Manual for Teachers.
By Geo. Ricks, Inspector of Schools, London School
Board. Cloth. 352 pages. Retail price, 1.50.
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