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Dacie and Lewis Practical Haematology

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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
International ISBN: 978-0-7020-6930-7
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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
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
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
4 Practical Haematology

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.
1 Collection and Handling of Blood 5

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
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
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.
1 Collection and Handling of Blood 7

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.
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 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.
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