2.
9
C H A P T E R
Haematology
of the Mouse
Anne Provencher Bolliger
Charles River Laboratories, Sherbrooke, QC, Canada
Nancy Everds
Amgen Inc., Seattle, Washington, USA
Introduction
This chapter is intended to provide useful and
practical advice to those involved in assaying
and interpreting haematological results from
mice. The interpretation of haematological
changes is similar whether the changes are due
to an infectious disease, a toxin or a mutation.
Identification of underlying causes is dependent
on knowing which haematological tests should be
used and how to interpret the test results.
A complete review of haematological malig-
nancies of the mouse is beyond the scope of this
chapter. However, the investigator who is studying
murine haematology must be aware of the
common haematologic malignancies of mice, and
is referred to a review by Frith et al. [1]. Also avail-
able are review articles focusing on the haematol-
ogy of certain genetically modified mice and
mouse models of haematological diseases [2–4].
The Laboratory Mouse
Ó 2012 Elsevier Ltd. All rights reserved.
ISBN 978-0-12-382008-2
HAEMATOLOGY
331
ANATOMY
AND
Much of the information in this chapter is not
published, but has been gleaned from practical
NORMATIVE BIOLOGY
experience and from discussions with colleagues.
When applicable, exact references have been
specifically cited.
Terminology
Haematology is the study of the physiology and
pathology of the cellular elements of blood.
The three major cellular components of blood
are red blood cells (erythrocytes), white blood
cells (leukocytes) and platelets (thrombocytes).
Basic haematological concepts are similar across
most mammalian species, and can be found in
several human and veterinary textbooks. The
reader may consult any of the excellent haema-
tology and clinical pathology reference books
listed at the end of this chapter for guidance
about general haematological principles.
DOI: 10.1016/B978-0-12-382008-2.00014-3
 Clinical terms (e.g. anaemia, leukopenia,
lymphocytosis, pancytopenia) have specific
medical connotations. These terms generally
refer to conditions in which parameters fall
outside of an appropriate reference range for
a widely inclusive population (i.e. male humans,
female dogs, dairy cows). In research, changes in
haematological parameters for mice are usually
compared to a control group or a very narrowly
defined reference interval. Therefore, the use
of these clinical terms is generally not appro-
priate in mouse research, and should not be
used. Changes can be referred to as ‘increases’
or ‘decreases’ in the affected parameter. For
HAEMATOLOGY
example, instead of stating that an experimental
treatment caused anaemia, one can state that
mean haemoglobin concentration decreased by
X g/dl and/or to X% of the control group
mean. For more details about evaluation and
interpretation of haematological changes in the
context of experimental studies, the reader is
332 referred to specific chapters of some haematol-
ogy textbooks [5].
NORMATIVE BIOLOGY
Blood collection and
handling
Blood collection techniques
Several references describe methods for blood
collection from mice [6–8]. For haematological
AND
testing, it is important to collect blood quickly
with a minimum of tissue trauma. Thus, methods
ANATOMY
that collect blood directly from a vessel or plexus
are preferred to those that may cause more
tissue trauma. The following sites are most
commonly used for blood collection for haema-
tology in mice: orbital sinus, tail vein or artery,
sublingual vein, heart, aorta and vena cava.
Depending on the site used, blood collection
must be a terminal procedure (heart, aorta,
vena cava) or may be a survivable procedure
(orbital sinus, tail, sublingual vein). Historically,
blood collection from the retro-orbital plexus
was used commonly as a survivable procedure.
Today its use as a survivable procedure is
discouraged because collection of blood from
this site is associated with greater tissue injury,
and thus may impact animal welfare.
Blood collected from a mouse should be
immediately placed in a tube containing an anti-
coagulant. The preferred anticoagulant for
routine haematological testing is EDTA (ethyle-
nediamine tetra-acetic acid). If the blood is
collected for preparation of smears only, blood
without anticoagulant can be used, provided
that smears are made immediately (seconds)
after blood collection. Heparin should not be
used as an anticoagulant because it tends to
cause clumping of platelets, especially in mice,
and negatively impacts the tinctorial quality of
Romanowsky staining. When a capillary tube is
used it is important to use plain rather than
heparin-coated capillary tubes for blood
collection.
Preparation of blood smears
Blood smear preparation is a skill that is easily
learned and is covered in the standard haema-
tology texts referenced at the end of this
chapter. A blood smear can be prepared with
a minimum of blood (<50 ml), and thus can be
a survivable procedure in mice. Blood smears
should either be stained within a few hours of
preparation or fixed, once thoroughly dry,
with methanol.
Basics of
haematological
evaluation
Evaluation methods
The scope of haematological evaluation may
vary greatly, depending on the needs of the
investigator and the capabilities of the clinical
pathology laboratory. The methods used will
determine the type and number of parameters
measured and the accuracy of the results. The
haematological evaluation can include some
or all of the following tests; these are listed
below in the order of most simple and least
expensive to those that are most complicated
and require significant investment in
instrumentation.
Blood smear evaluation
The blood smear is a landmark of any haematolog-
ical evaluation and is examined microscopically.
Regardless of the methods used to determine
blood cell counts, blood smear evaluation remains
the same. First, the density of white and red blood
cells and platelets is estimated and compared to the
counts obtained. A differential white blood cell
count is performed. The blood smear is reviewed
for morphologic changes in any of the cell popula-
tions (red blood cells, white blood cells and plate-
lets). For this examination, only a microscope is
required. The validity of results is highly depen-
dent on the skill and experience level of the exam-
iner. If automated differentials are conducted (see
below), blood smears are still prepared, but are
often examined only if deemed necessary after
review of the automated results.
Spun haematocrit, haemocytometer cell
counts and blood smear examination
A microcapillary tube is centrifuged to determine
the spun haematocrit (also called packed cell volume).
White and red blood cells and platelets are
counted microscopically, using a haemacytometer.
Haemoglobin concentration may be measured,
and some or all red blood cell indices are
calculated. The blood smear is examined micro-
scopically, and a differential white blood cell
count is performed. Absolute white blood cell
differential counts are calculated. The blood
smear is reviewed for morphologic changes. This
method requires a minimum of equipment
(microcapillary haematocrit centrifuge, hemacy-
tometer, microscope,  method of determining
haemoglobin concentration). The results are
moderately accurate.
Instrument cell counts and differential with
blood smear review
Whole blood is analysed on a haematology cell
counter using either impedance or optical tech-
nology. The cell counter determines red and
white blood cell counts, platelet counts and hae-
moglobin concentration, and measures or calcu-
lates red blood cell indices. The blood smear is
examined microscopically, and a differential
white blood cell count is performed. Absolute
white blood cell differential counts are
calculated. The blood smear may be reviewed
for morphological changes. This method
requires a dedicated haematology analyser and
trained personnel. The results are very accurate
if the instrument has been shown to be valid
for the determination of mouse haematological
tests, and if the operators have sufficient training
to operate the instrument.
Instrument cell counts and optional blood
smear examination
Whole blood is analysed on a flow cytometer-
based haematology instrument. The instrument
HAEMATOLOGY
determines red and white blood cell counts,
platelet counts and haemoglobin concentration,
and measures or calculates red blood cell
indices. The instrument also estimates the
differential white blood cell count populations.
The blood smear is examined microscopically
to confirm the automated differential, and is
also reviewed for morphological changes. This 333
method requires a sophisticated haematology
analyser and well-trained personnel. The
ANATOMY
results are very accurate if the instrument has
been shown to be valid for the determination
of mouse haematological tests, and if the oper-
ators have sufficient training to operate the
AND
instrument. Today the Siemens Advia 120 and
the Sysmex XT-2000 IV are the most
NORMATIVE BIOLOGY
commonly used flow cytometry-based haema-
tology analysers with software that can deter-
mine mouse white blood cell differential
counts (Figure 2.9.1).
Almost all cell counters and flow cytometry-
based analysers developed for haematology
were originally designed to determine haemato-
logical parameters for humans. These instru-
ments have specialized software to discriminate
and count the different cellular constituents of
blood. Some haematology analysers have been
adapted for animal species by using species-
specific software or settings. Analysers adapted
for animal species vary markedly in their ability
to accurately count and differentiate animal
blood cells. This is especially true with blood
from rodents. Therefore, for accurate results it
is essential to use a laboratory with instrumenta-
tion validated for the analysis of mouse blood,
and whose employees are skilled at mouse blood
evaluation.
HAEMATOLOGY
Figure 2.9.1 Example of analysis of mouse blood on a Siemens Advia 120 Automated Hematology Analyzer.
334 The instrument uses a flow cytometry-based analyzer to measure individual white blood cell types (automated
differential). Notice the scatterplots used to classify the various cell types.
NORMATIVE BIOLOGY
Complete blood count
The report of results from a standard haemato-
logical evaluation is called a complete blood count
(CBC). The CBC generally includes most or all
of the parameters listed below. As discussed
briefly above, these parameters are determined
using one or all of the following methods: auto-
AND
mated haematology analysers, haematology ana-
lysers plus microscopic examination, or by
ANATOMY
a combination of manual and automated
methods. In most countries haematology results
are expressed in SI units, as described by Lapo-
sata [9].
Red blood cell mass parameters
The functional red blood cell mass is measured
by three parameters: red blood cell count (RBC),
haematocrit (HCT) and haemoglobin concentra-
tion (HGB).
RED BLOOD CELL COUNT (RBC)
The RBC is the number of red blood cells in
a given volume of whole blood. It is usually deter-
mined using an automated counter. In this
method red blood cells are counted while they
flow through an aperture in single file, using
either impedance or optical technology. For
manual haemocytometer counts, a commercially
available diluent system (UnopetteÓ, Becton-
Dickinsen, Test 5850) is used to dilute blood prior
to counting red blood cells. A qualitative estima-
tion (density) of the RBC can be determined by
microscopic evaluation of well-prepared blood
smears. Mouse RBCs are higher than in most
other species, because of the small size of their
red blood cells. Counts range from approxi-
mately 7–11  1012/L [10, 11]. Mouse red blood cells
have smaller mean cell volumes than other
species, but since their red cell counts are higher,
mouse haematocrits are similar to those of other
species.
The primary function of red blood cells is to
carry oxygen from the lungs to tissues, and to
carry carbon dioxide back to the lungs. In the
mouse red blood cells are normally produced in
bone marrow and in the spleen, even in adults.
This is in contrast to other species in which
splenic haematopoiesis does not occur in healthy
adults. In the mouse the amount of extramedul-
lary haematopoiesis in the spleen and liver

Figure 2.9.2 Peripheral blood erythrocytes from
a Charles River CD-1 mouse (1003 oil objective;
Wright–Giemsa stain). Note the presence of poly-
chromasia (light purple/blue staining cells), and var-
iably sized cells (anisocytosis).
increases greatly when there is increased demand
for red blood cells.
Compared to other mammalian species, the
mouse red blood cell has a fairly short lifespan,
estimated at 30–52 days [10, 12, 13]. The red blood
cell lifespans of other common species are much
longer (rats 45–50 days, dogs 110 days, humans
120 days). Because the lifespan of murine red cells
is so short, there is a higher percentage of circu-
lating immature red blood cells at any given
time. Immature red blood cells are larger than
mature erythrocytes and stain with a blue tint
with Romanowsky-stained blood smears and are
called polychromatophils. Therefore, polychromasia
(purple/blue staining cells) and anisocytosis (varia-
bly sized red blood cells) occur to a greater extent
in normal healthy mice compared to humans and
many other animals. In addition, Howell–Jolly
bodies (small, dark blue intracellular particles
with Romanowsky-stained blood smears, which
are remnants of nuclear DNA) are also more
common in circulating red blood cells in mice
(Figure 2.9.2).
HAEMOGLOBIN (HGB)
The haemoglobin concentration (HGB) is the
measurement of total haemoglobin per volume
of whole blood. It is determined spectrophoto-
metrically after lysis of red blood cells. All forms
of haemoglobin, whether functional or not, are
included in the measurement of haemoglobin
concentration. Mouse haemoglobin ranges from
130 to 180 g/L.
HAEMATOCRIT (HCT)
The HCT is a measurement of the volume of red
blood cells as a percentage of whole blood. For
automated procedures, the haematocrit is the
product of the RBC and the mean cell volume
(see below). For manual determinations, the hae-
matocrit is measured after centrifugation of
a microcapillary tube filled with whole blood.
The percentage of blood composed of red blood
cells is the haematocrit (sometimes called packed
cell volume). Manual or ‘spun’ haematocrits tend
HAEMATOLOGY
to be a few percentage points higher than calcu-
lated haematocrits, because trapped plasma is
included in the apparent red blood cell volume.
Haematocrit is expressed as a number without
units between 0.00 and 1.00. Haematocrit values
for mice are generally between 0.40 and 0.50,
but may range up to 0.60 depending on sampling
site and fasting status. 335
Red blood cell indices and other red blood
ANATOMY
cell parameters
MEAN CELL VOLUME (MCV)
The MCV is the average size of red blood cells.
AND
When the RBC is determined by a haematology
instrument, the MCV is measured. When cell
NORMATIVE BIOLOGY
counts are determined by a haemocytometer,
MCV is calculated. For instrument-generated
cell counts, red blood cell volume is measured
during the cell count described above. A histo-
gram is generated from the RBC and size
(Figure 2.9.3) and the MCV is determined from
this histogram. If cells are counted by a haemocy-
tometer, the MCV is determined by dividing the
haematocrit by the RBC. The MCV of mice is
approximately 40–55 fl [10, 11, 14].
MEAN CELL HAEMOGLOBIN (MCH)
The MCH is the average amount of haemoglobin
found in each individual red blood cell. It is
determined by dividing the HBG by the RBC. In
general, MCH is the least useful haematology
parameter, because it is insensitive to change
and provides little additional information than
other red blood cell parameters. MCH for mice
ranges from 13 to 17 pg. MCH is sometimes
expressed in fmol.
 dye causes clumping and staining of residual
Mean cell volume
nucleic acid present in immature cells. The
stained cells (reticulocytes) are counted as
a percentage of total red blood cells. The absolute
reticulocyte count is determined by multiplying
the total RBC by the percentage of reticulocytes.
The number of circulating reticulocytes is higher
in mice (200–500  109/L) than in most other
species, due to the short lifespan of the mouse
red blood cell. Units for reticulocyte count vary
among laboratories, but generally are reported
in the same units as red blood cells or in the
same units as platelets (cells  109/L).
Figure 2.9.3 Red cell histogram. Red cell size is
HAEMATOLOGY

plotted on the x-axis, and measurement frequency is Platelet parameters

plotted on the y-axis. The mean value for red cell size
is the mean cell volume (MCV); the frequency is the PLATELET COUNT (PLT)
red cell count, and the spread of red cell histogram is
reflected in the red cell distribution width (RDW). Platelets are essential for primary haemostasis
and form a temporary haemostatic plug prior
to activation of the clotting cascade. The PLT is
MEAN CELL HAEMOGLOBIN CONCENTRATION the number of platelets in a given volume of
(MCHC) whole blood. It is usually determined using an
336
automated counter, using either impedance or
The MCHC is the HBG divided by the HCT, and
optical technology. Platelets are counted as they
thus is the average concentration of haemoglobin
NORMATIVE BIOLOGY

flow through an aperture in single file, similar

in all red blood cells. MCHC is much more relevant
to red blood cells. Platelets can also be counted
than the MCH, discussed above, because it is more
microscopically with a haemocytometer. A quali-
sensitive to changes affecting red blood cells.
tative estimation of PLT can be determined by
Mouse MCHC generally ranges between 270 and
microscopic evaluation of well-prepared blood
330 g/L but can vary between strains of mice.
smears. Counting platelets in mouse blood is
RED BLOOD CELL DISTRIBUTION WIDTH (RDW) problematic because mouse platelets tend to
The RDW is the coefficient of variation of the form clumps (Figure 2.9.4). The presence of
clumped platelets can interfere with the accuracy
AND

red blood cell volume, and is calculated from

the red blood cell histogram described above of both platelet and white blood cell counts, and
ANATOMY

and in Figure 2.9.3. It is therefore a quantitative

indicator of the variation in red blood cell
size (anisocytosis). Mouse RDW generally falls
between 11 and 15%.

RETICULOCYTE COUNT (RETIC)

Reticulocytes are immature red blood cells con-
taining residual RNA. Reticulocytes can be
counted by instrumentation or by haemocytome-
ter. Automated reticulocyte counts are performed
similarly to automated RBCs. Before counting, red
blood cells are stained with a dye that stains for
nucleic acid (such as acridine orange) to differen-
tiate reticulocytes from mature red blood cells.
For manual reticulocyte counts, whole blood is
mixed with a supravital dye such as new methy- Figure 2.9.4 Platelet clump, peripheral blood smear
lene blue and blood smears are prepared. The (1003 oil objective; Wright–Giemsa stain).
also invalidates PLT. Depending on the analyser,
clumped platelets can either be counted as white
blood cells or specifically as eosinophils. There-
fore, regardless of the method used to count
platelets, the smear must be evaluated for platelet
clumps before the PLT value is accepted.
Mice have the highest circulating platelet
count of any laboratory animal species (approxi-
mately 900–2000  109/L) [11, 14]. Mouse platelet
half-life is approximately 4–5 days [11, 15, 16].
Platelets are variable in morphology, but range
from 1 to 4 mm in diameter and 4 to 7 fl in
volume. The primary growth factor controlling
platelet production is thrombopoietin, but eryth-
smears by a trained observer. The WBC of
mice ranges from 2 to 10  109/L.
DIFFERENTIAL WHITE BLOOD CELL COUNT (DIFF)
The DIFF enumerates the various individual
white blood cell types found in peripheral blood.
The predominant circulating leukocytes in mice
are lymphocytes, followed by neutrophils, mono-
cytes, eosinophils and lastly basophils (Figures
2.9.5–2.9.7).
The DIFF can be performed on an automated
instrument or by microscopy. Automated analy-
sers with species-specific software are flow
cytometers dedicated to differentiating periph-

ropoietin and iron status also affect platelet
production. Under conditions of increased
demand for platelets, platelet production is
increased, and large platelets are sometimes
observed on the peripheral blood smear.
MEAN PLATELET VOLUME (MPV)
The MPV is an estimation of the average size of
platelets, and is analogous to the MCV of red
blood cells. The MPV is only available on auto-
HAEMATOLOGY
eral white blood cells, and use nuclear and cyto-
plasmic characteristics of white blood cells for
classification (see Figure 2.9.1). The DIFF can
also be determined microscopically by exam-
ining and categorizing 100 white blood cells on
a peripheral smear. The percentage of each cell
type is multiplied by the total WBC to arrive at
absolute differential counts for the various cell 337

ANATOMY
mated cell counters. As platelets are counted,
their size is measured. A histogram is generated
from the platelet counts and platelet sizes. The
MPV is determined from this histogram. Units

AND
for MPV are femtolitres (fl).

NORMATIVE BIOLOGY
White blood cell parameters
WHITE BLOOD CELL COUNT (WBC)
White blood cells participate in immune and Figure 2.9.5 Lymphocytes, peripheral blood smear
(1003 oil objective; Wright–Giemsa stain). Note the
inflammatory processes. The WBC may be
high nuclear/cytoplasmic ratio, eccentric nucleus,
determined quantitatively (automated analyser smudged chromatin and blue cytoplasm.
counts or manual haemocytometer counts) or
qualitatively (determination of density with
blood smear review). Before quantitative white
blood cell determinations, red blood cells are
lysed using a hypotonic solution. The resulting
preparation contains only white blood cells and
platelets. Automated counters measure white
blood cells as they flow in single file past
a detector. For manual haemocytometer counts,
a commercially available diluent system
(UnopetteÓ, Becton-Dickinsen, Test 5855) is
used to dilute blood and lyse red blood cells Figure 2.9.6 Neutrophil, peripheral blood smear
(1003 oil objective; Wright–Giemsa stain). The cyto-
prior to counting white blood cells. A qualitative plasm is clear or stains faintly. The nuclei of neutro-
estimation of the WBC can be determined by phils show multilobulation and the chromatin is
microscopic evaluation of well-prepared blood variably clear or condensed.

Figure 2.9.7 Monocyte, peripheral blood smear
(1003 oil objective; Wright–Giemsa stain). The
monocyte is the largest peripheral blood cell of mice.
Note the grey-blue vacuolated cytoplasm, lobulated
nucleus and clear cytoplasmic vacuoles.
HAEMATOLOGY
types. Interpretation of leukocyte changes should
be based on absolute numbers (number of a given
cell type per unit volume of blood) rather than
relative numbers (percentage of a given cell
type). Units for absolute DIFF are the same as
for total WBC (cells  109/L).
338 The main categories of white blood cells
counted during a differential count are lympho-
NORMATIVE BIOLOGY
cytes, neutrophils, monocytes, eosinophils and
basophils. Additional cell types may be observed;
these might be subcategories of the five major
cell types, or perhaps other cells not normally
observed in peripheral blood. Interpretation of
white blood cell changes is covered in a later
section of this chapter.
1. Lymphocytes are responsible for immune
surveillance, production of antibodies,
AND
production of cytokines and antigenic
memory. Unlike other circulating leukocytes,
ANATOMY
lymphocytes are capable of division. Mouse
lymphocytes are generally similar in appear-
ance to those of other species. Both small and
large lymphocytes can be observed on
peripheral smears. Lymphocytes are approxi-
mately 7–12 mm in diameter, and have round to
oval dark blue nuclei with pale blue scant
cytoplasm on Romanowsky-stained blood
smears. In the mouse, lymphocytes make up
70–80% of the differential count [11].
2. Neutrophils are circulating phagocytes and
modulators of the immune response. In the
mouse, neutrophils account for 20–30% of
DIFF [11]. The morphologic appearance of the
mouse neutrophil is similar in most respects to
those of other species. Mouse neutrophils are
10–25 mm in diameter [10] and have pale gran-
ules. Mouse neutrophils sometimes exhibit
circular doughnut-shaped nuclei; similar
morphology is sometimes observed in rat
neutrophils as well.
3. Monocytes and eosinophils are minor cell types.
Generally there are more monocytes than
eosinophils in peripheral blood. Basophils are
very rarely observed in the peripheral blood
of mice. Some authors have questioned the
presence of basophils in mouse blood,
however, microscopic and ultrastructural
characteristics of murine basophils have been
described in the literature [17, 18].
Morphological evaluation
of the blood smear
The blood smear is evaluated microscopically for
alterations in appearance (morphology) of white
blood cells, red blood cells and platelets. At the
same time, any other unusual findings are noted.
Examples of appropriate comments would be
observations of variation in size, shape, and color-
ation of cells; cellular organization (agglutination,
rouleaux, platelet clumps); parasites and other
relevant findings.
Bone marrow evaluation
Bone marrow smears are analysed to determine
the underlying pathophysiology of peripheral
blood changes. Both qualitative and quantitative
evaluations of bone marrow cells can be useful
in determining the cause of peripheral blood
changes. Bone marrow smears can be qualita-
tively evaluated for relative proportions, matu-
rity of precursor cells, storage pool and other
changes. Quantitative evaluations of various cell
types (differential) are generally not necessary,
but may be done on occasion if extremely precise
data is required. For a complete review and best-
practice approach to bone marrow evaluation in
laboratory animals, readers are referred to
a best-practice publication [19]. This review article
covers approaches for indications when to eval-
uate bone marrow in laboratory animals, with
techniques available including histological, cyto-
logical or flow cytometric methods.
 Variables affecting
haematology results
Numerous factors can interfere with accurate
analysis of haematology parameters. Potential
interfering factors for analysis of mouse blood
are listed in Table 2.9.1. Some of these interfering
factors only occur with mouse samples, while
others can occur with samples from other species.
The results of haematology tests are not only
affected by pathologic processes, but are also
Figure 2.9.8 Bone marrow smear (1003 oil objective;
affected by the status of the mouse at the time

Wright–Giemsa stain). The majority of the cells in this
field are late granulocytic precursors with doughnut-
shaped nuclei, admixed with a few erythroid precursors.
HAEMATOLOGY
of blood collection, collection techniques and
handling of blood prior to and during analysis.
Therefore, it is essential to keep variables as
consistent as possible so that results are compa-
rable across experiments.
Haematological parameters vary with the
status of the animal being tested. Fasting status,
hydration status, time of collection, prior experi- 339
mental manipulations, prior and concurrent
anaesthesia, sex and a myriad of other factors

ANATOMY
will influence the results of haematology tests.
In general, mice are not fasted prior to haematol-
ogy sample collection, because fasted mice tend
to drink less water and may become dehydrated.

AND
Age has a very significant effect on haemato-
logical parameters. For example, the erythroid

Figure 2.9.9 Bone marrow smear (1003 oil objective;
Wright–Giemsa stain). The majority of the cells in this
field are erythroid precursors (dark nuclei, high
nuclear/cytoplasmic ratio, blue cytoplasm), admixed
with a few late-stage granulocytic precursors.
NORMATIVE BIOLOGY
parameters for newborn mice are very different
from those of mice aged 6 weeks or more.
Newborn mice have much higher reticulocyte
counts and lower red blood cell mass parameters.
During the first few weeks of life, red blood cell

TABLE 2.9.1: Factors affecting results of haematology tests

Animal physiology Collection process Analysis

Age Study design Sample quality (e.g. haemolysis,

lipaemia, clots)
Sex Movement of cages Sample storage and handling
Strain Prior handling or dosing Instrumentation
Transport Anaesthesia requirement Order of analysis
Time of collection Order of collection Platelet clumps
Fasting/fed status Site of collection
Concurrent illness Anticoagulant used
Other experimental Anticoagulant/blood ratio
procedures
 mass actually decreases, but then rebounds
steadily and is near adult levels at weaning.
The order of blood collection can affect hae-
matology results. For this reason, it is important to
collect blood from controls alternately with
treated (experimentally manipulated) mice.
Collection site can also play a role: in general,
blood collected from central arteries or heart
tends to have lower white and RBCs than blood
collected from distal sites such as ocular plexus
or tail [20]. Underfilling of tubes results in excess
anticoagulant for the amount of blood, and can
result in artefacts in haematological parameters.
It is important to fill blood tubes to the volume
HAEMATOLOGY
recommended by the manufacturer.
Once collected it is important that all samples
are stored appropriately (generally room temper-
ature or refrigerated), and are analysed at
approximately the same length of time after
collection. A recent publication showed that
mouse blood samples stored at 4  C for 24 h or
more had changes in some haematological
340
parameters compared to those analysed within
1 h after collection [21].
NORMATIVE BIOLOGY
Analytical variables that need to be avoided
include operator error, bias due to order of anal-
ysis, instrument malfunction, outdated reagents
or control material, dilution errors, calculation
errors and calibration errors. For more informa-
tion about general quality control and quality
assurance in a veterinary clinical laboratory, the
reader is referred to published guidelines from
the American Society of Veterinary Clinical
AND
Pathology [22].
ANATOMY
Pathophysiology
and interpretation
of results
Red blood cells
The most important effects on red blood cells are
those that result in changes in red blood cell mass.
These effects can be divided into those
that increase red blood cell mass, and those that
decrease red blood cell mass. Red blood cell
mass changes are summarized in Table 2.9.2.
Increased red blood cell mass
Increases in red blood cell mass can be classified
as either relative or absolute changes. Relative
increases in red blood cell mass are the result of
dehydration, which can occur very rapidly in
mice. Dehydration may be associated with other
signs of poor health, including inactivity,
hunched appearance, decreased appetite and
poor skin turgor.
Absolute increases in red blood cell mass
result from increased red blood cell production,
and may occur whenever there is increased
production of growth factors or cytokines that
stimulate erythropoiesis. The primary cytokine
driving erythropoiesis is erythropoietin (EPO),
but other cytokines, especially thrombopoietin,
can also affect red blood cell production. EPO-
like drugs may also have similar effects.
Active erythropoiesis, resulting in increased
red blood cell mass, can be secondary and appro-
priate (occurring in response to a disorder with
decreased oxygen delivery to tissues) or may be
primary and inappropriate (occurring in the
absence of any need for increased oxygen
delivery). Causes of appropriately increased red
blood cell mass include cardiovascular disorders,
pulmonary disorders or abnormal oxygen-
carrying capacity of haemoglobin. Causes of inap-
propriately increased red blood cell mass include
conditions resulting in excess erythropoietin
(autonomous production) or excess stimulation
of the erythropoietin receptor (activating muta-
tions). Polycythaemia, a myeloproliferative disea-
se, also results in increased red blood cell mass.
In mice increased red blood cell production,
regardless of the cause, is associated with extra-
medullary erythropoiesis in the spleen and liver,
hypercellularity of bone marrow, and increased
reticulocytes in the peripheral blood.
Decreased red blood cell mass
Decreases in circulating red blood cell mass are
indicated by decreases in RBCs, haemoglobin
concentration and haematocrit. Decreased red
blood cell mass can be either relative (expansion
of plasma volume) or absolute.
Relative decreases in red blood cell mass are
rare, but may occur in pregnant dams and
neonates, or as a result of other perturbations in
 TABLE 2.9.2: Alterations in red blood cell mass parameters in mice

Causes Characteristics

Increased red cell mass Increased haemoglobin, haematocrit, RBC

Relative increased red cell mass
Dehydration
Absolute increased red cell mass
Increased erythropoietin activity
Myeloproliferative disease
Recognized by clinical signs
May see increased total protein or albumin
Increased extramedullary erythropoiesis
Increased reticulocytes

Decreased red cell mass Decreased haemoglobin, haematocrit, RBC

Relative decrease in red cell mass

HAEMATOLOGY
Pregnant dams Plasma volume expansion
Neonates Rare occurrence

Absolute decreases in red cell mass

Red cell loss (haemorrhage)

Internal or external (overt, Recognized by clinical signs
gastrointestinal, genitourinary)
341
Red cell destruction

ANATOMY
Toxins, immune-mediated Y reticulocytes
Cell membrane alterations [ MCV
Biochemical alterations [ RDW
Vascular injury/turbulence Y MCHC

AND
[ extramedullary erythropoiesis
[ splenic weights

NORMATIVE BIOLOGY
Decreased red cell production
Decreased erythropoietin Y reticulocytes
Renal disease Y MCV
Endocrine disease Y RDW
Chronic inflammatory disease No change or [ MCHC
Bone marrow toxicity
Abnormal maturation
Abnormal haem or nucleic acid
synthesis
Haematopoietic neoplasia

plasma volume homeostasis. Apparent decreases
in red blood cell mass can be artefactual when
blood cells are insufficiently resuspended in
plasma due to poor mixing of the blood with anti-
coagulant prior to analysis.
Absolute decreases in red blood cell mass are
much more common than relative decreases, and
result from loss (haemorrhage), increased
destruction (haemolysis), or decreased
production of red blood cells. Each of these
causes can be recognized by particular changes
in haematological parameters.
Haemorrhage
Loss of red blood cells is called haemorrhage, and
can be either overt or occult. Overt haemorrhage
can result from disorders of haemostasis,
 ulcerated masses, surgical procedures or other
trauma. Overt haemorrhage is a clinical diag-
nosis, and is best identified by observing the phys-
ical condition of the mouse. Occult haemorrhage
is generally due to loss of red blood cells into the
alimentary tract or urogenital tract, and can be
detected by tests for occult haemorrhage such
as HemoccultÓ for faeces or dipstick tests for
urine blood. In mice loss of red blood cells is
generally accompanied by marked regeneration
(increased reticulocytes), while very chronic loss
of blood may result in decreased red blood cell
production due to lack of iron, without a concur-
rent increase in reticulocyte count.
HAEMATOLOGY
erythropoiesis. This is in contrast to other species,
in which extramedullary erythropoiesis only
occurs under more severe haematological stress.
In mice, splenic weights can be used as sensitive
and objective measures of red blood cell regener-
ation. The presence of extramedullary erythro-
poiesis should be confirmed histologically.
Decreased production
Any process that has a deleterious effect on red
blood cell precursors in the bone marrow can
cause decreased red blood cell production. The
effects can be grouped into effects on stem cells,
growth factors, or synthesis of haemoglobin or

nucleic acids.
Increased destruction Mice with decreased red blood cell mass due
to decreased red blood cell production will have
Increased destruction of red blood cells, or hae-
inappropriately low reticulocyte counts in
molysis, results in decreased circulating half-life
peripheral blood, with respect to the change in
of red blood cells. In most cases increased
red blood cell mass. The decrease in red blood
destruction of red blood cells occurs by prema-
cell mass due to decreased production is referred
342 ture removal from circulation (extravascular
to as a non-regenerative or poorly regenerative
haemolysis) by the reticuloendothelial system of
process.
the liver and spleen, rather than by rupture of
NORMATIVE BIOLOGY

Reticulocytes, which are larger and have less

red blood cells within the vasculature (intravas-
haemoglobin than mature red blood cells, are
cular haemolysis). Normally, red blood cell life-
normally present in a larger percentage in mice
span in the mouse is 30–52 days. With
than in other species. Therefore, in mice
haemolysis, the lifespan of red blood cells can
decreased reticulocytes seen in decreased red
be decreased markedly. Destruction of red blood
blood cell mass due to decreased production
cells may be due to metabolic, immune-mediated
may result in decreased polychromatophilic cells
or physical causes.
on the blood smear, decreased MCV and
Haemolytic processes result in compensatory
increased MCHC. The morphology of peripheral
increased red blood cell production (regenera-
AND

blood and/or bone marrow may help to elucidate

tion). Regeneration is the result of increased
the mechanism for decreased red blood cell
erythropoietin in response to decreased oxygen
ANATOMY

production. When decreased red blood cell

tension. Erythropoietin recruits stem cells to
differentiate into red blood cell precursors, and
promotes survival of committed red blood cell
precursors, resulting in increased circulating
immature red blood cells in peripheral blood,
and increased absolute reticulocyte count. The
immature red blood cells (which include reticulo-
cytes) are larger than mature red blood cells, and
contain less haemoglobin. Therefore, in regener-
ative haemolytic anaemia, the altered parameters
are associated with cell size (increased MCV,
RDW, macrocytosis) and cell haemoglobin
concentration (decreased MCHC).
In mice even minimal haemolysis results in
compensatory hypercellularity of the bone
marrow and splenic extramedullary
production is suspected, bone marrow histology
and/or cytology should be evaluated to deter-
mine an underlying cause.
Anaemia of chronic disease
The most common cause of decreased red blood
cell mass in humans and animals is anaemia of
chronic disease [23–25]. This condition is usually
secondary to hormonal or inflammatory condi-
tions. It results from decreased red blood cell
production and increased red blood cell destruc-
tion. Although the term ‘chronic’ implies that
this effect only occurs after a long period, it
can actually be observed within days to weeks.
Because of shorter red blood cell half-lives in
 IL-1 TNF differential leukocyte counts. Leukocytes are
summarized in Table 2.9.3.

IL-6 Hepcidin
IFN-β
Lymphocytes
IFN-γ
Transferrin
In response to inflammation, mice usually have
increased lymphocyte counts; this response is in
contrast to that of most other laboratory species,
EPO RBC
but similar to that of rats. Increased circulating
Fe release
production precursors
from macrophages lymphocytes are also observed secondary to cate-
cholamines (excitement), altered trafficking or
Figure 2.9.10 Pathophysiology of anaemia of chronic
lymphoid malignancy. Lymphocyte increases
disease. Adapted from Means and Krantz, 1992, Ganz,
2006, Nairz and Weiss, 2006. due to catecholamines are generally transitory
(minutes to hours), whereas inflammatory-

mice, processes affecting red cell mass have
more rapid effects in mice than in other species.
Figure 2.9.10 outlines the pathophysiology of
anaemia of chronic disease.
Leukocytes
Interpretation of changes in total WBC should be
made on the basis of changes in the absolute
HAEMATOLOGY
related increases are more persistent (days to
weeks).
Decreased lymphocytes can be due to stress
[26], altered trafficking or genetic manipulation
resulting in immunomodulation. A decrease in
lymphocytes due to stress occurs secondary to
the effects of increased corticosteroids. This
stress response in lymphocytes occurs within 343
hours of the stressful event, and can persist for

ANATOMY
TABLE 2.9.3: Most common peripheral blood leukocyte patterns in mice

AND
Short-term (acute) Decreased neutrophils (may not be observed) during severe
inflammation inflammation
Immature neutrophils (less common in mice than larger animals)

NORMATIVE BIOLOGY
Chronic inflammation Mildly increased total leukocytes (young adults)
Mildly to markedly increased total leukocytes (old mice)
Increased neutrophils
Increased lymphocytes
Increased monocytes
Excitement (catecholamine/ Proportional increase in both lymphocytes and neutrophils
adrenaline-induced
changes)
Stress (corticosterone- Increased neutrophils  hypersegmentation of neutrophils
induced changes) Decreased lymphocytes
Decreased eosinophils
Decreased monocytes
Bone marrow toxicity May involve only one cell type or may involve all three cell types
If pancellular, cells are usually affected in the following order:
leukocytes, platelets, then red blood cells
Red cell effects:
decreased reticulocytes, red cell distribution width, mean cell volume
increased mean cell haemoglobin concentration
Neoplasia Haematopoietic neoplasia: sometimes leukemic (neoplastic cells in
circulation)
Space-occupying lesions: sometimes leukopenic
Allergy or hypersensitivity Increased eosinophils, possibly increased basophils
 days to weeks. Often, decreased lymphocytes are
incorrectly attributed to stress without correla-
tive findings supporting the diagnosis. Generally,
other effects such as weight loss, clinical signs or
thymic atrophy are present when stress results in
decreases in lymphocytes.
Total peripheral blood lymphocyte counts
are only an estimate of immune system status.
Unexplained effects on lymphocytes may be
further explored using cytochemical or flow
cytometric techniques. In addition, histopa-
thology of other lymphoid tissues, such as lymph
nodes and thymus, may be useful in the under-
standing of peripheral lymphocyte changes.
HAEMATOLOGY
Neutrophils
Increases in circulating neutrophils can be due to
excitement (demargination), increased neutro-
phil production or decreased neutrophil egress
from circulation. Increased production is gener-
344 ally caused by increases in colony stimulating
factors (G-CSF or GM-CSF), as a result of inflam-
mation. Increased production can rarely be
NORMATIVE BIOLOGY
recognized by the presence of immature neutro-
phils in the peripheral blood. These neutrophils
may have the morphological appearance of
band neutrophils, or may show signs consistent
with toxic change (increased granulation, baso-
philic cytoplasm and/or foamy cytoplasm), indi-
cating accelerated neutrophil production.
Decreased egress can be caused by lack of func-
tional adhesion molecules (P-selectin or E-selectin
AND
deficiency) or absence of chemotactic factors to
recruit neutrophils into tissues. Under conditions
ANATOMY
of decreased egress, increased nuclear segmenta-
tion may be observed in a subset of circulating
neutrophils.
Decreased neutrophils can result from
decreased bone marrow production, increased
egress into tissue or destruction of neutrophils.
Because the circulating lifespan of neutrophils
is short (7–14 h) [27], decreased neutrophil count
is often the first peripheral result of bone
marrow toxicity, and usually occurs prior to
platelet or red blood cell decreases. Increased
egress can be due to peracute inflammation. In
the initial stages of inflammation, many circu-
lating neutrophils may be called to the site of
inflammation by inflammatory cytokines. This
may cause a temporary decrease in circulating
neutrophils that is ameliorated when the bone
marrow increases production of neutrophils to
meet the peripheral demand. A decrease in
neutrophils can also result from direct destruc-
tion by immunological or non-immunological
mechanisms.
Monocytes, eosinophils and basophils
Increased circulating monocytes generally
indicate an increased demand for tissue macro-
phages, and thus are an indicator of inflamma-
tion. A decrease in circulating monocytes is
uncommon, but may indicate increased egress
into tissues, or may be a result of increased cortico-
steroid activity [14].
Increased circulating eosinophils result from
allergic or inflammatory conditions. Generally,
increases in circulating eosinophils are accompa-
nied by increased tissue eosinophils as well.
Decreased eosinophils may occur in conjunction
with decreased lymphocytes, as a result of
increased circulating corticosteroids.
Increased basophil counts are very
uncommon in mice. Because of the lack of baso-
phils in most mouse samples, decreases in baso-
phils do not generally occur.
Platelets
Effects on platelets may alter their number, size
or function. The most common reason for an
increase in platelets is a secondary response to
accelerated production of red blood cells
(increased erythropoietin). Other reasons for
increased platelets include iron deficiency and
increased thrombopoietin.
Decreased platelets can result from decreased
production or increased destruction. Decreased
production occurs as a result of injury to mega-
karyocytes, the bone marrow cells responsible
for production of platelets. If decreased produc-
tion is suspected, megakaryocyte number and
morphology may help to elucidate the cause of
decreased platelets. Decreased numbers or
altered morphology of megakaryocytes supports
the hypothesis that decreased platelet production
has occurred. Often, processes that affect bone
marrow production of one cell line can have
ramifications in one of the other two cell lines
produced in the bone marrow. Because platelets
and leukocytes have shorter half-lives than red
blood cells, effects on bone marrow will often
first manifest as peripheral blood abnormalities
on these two cell lines.
Increased destruction/removal of platelets
occurs if there is accelerated activation of plate-
lets or immune recognition of platelets. If platelet
half-life is shortened, megakaryocytes release
large immature platelets containing rRNA. These
‘reticulated platelets’ can be measured using
nucleic acid-binding dyes and flow cytometry.
Alternatively, these large platelets can be
detected by measuring MPV. Bone marrow
generally shows increased numbers of megakar-
yocytes, with or without an increase in younger
megakaryocytes.
Platelet function can also be studied in mice.
Techniques such as aggregometry, bleeding
time assays and flow cytometry [12, 28] can be
used to elucidate functional changes.
Bone marrow
evaluation
Murine haematopoiesis (production of granulo-
cytes, monocytes, platelets and erythrocytes)
primarily occurs in bone marrow at steady state,
although a small amount of haematopoiesis also
occurs in the spleens of healthy mice. Therefore,
alterations in peripheral blood counts may be
investigated by examination of the bone marrow.
Mouse bone marrow includes stromal cells,
macrophages, mast cells, megakaryocytes and
megakaryocyte precursors; erythrocytes and
erythroid precursors; granulocytes, monocytes
and their precursors; and lymphocytes, as well
as non-haematopoietic cells such as osteoblasts
and osteoclasts.
Granulocytes (neutrophils, eosinophils and
basophils) and red blood cells arise from
precursor cells. The precursor cells of both gran-
ulocytes and red blood cells can be divided into
proliferating cells (cells that are capable of
undergoing division) and maturing cells (end-
stage cells in the process of maturation). During
a complete bone marrow differential cell count,
these cells are counted, along with those cells
mentioned above. For a more complete review
of bone marrow smear collection techniques
and evaluation, the readers are referred to Rea-
gan et al. [19].
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Further reading
General haematology texts
Beutler E, Lichtman MA, Collier BS, Kipps TJ,
Siligsohn U. Williams’ Hematology. 6th ed. New
York: McGraw-Hill; 2001.
Foucar K. Bone Marrow Pathology. 2nd ed.
Chicago: American Society of Clinical Patholo-
gists; 2001.
Hoffman R, Benz EJ, Shattil SJ, Furie F,
Cohen HJ, Silberstein LE. Hematology. 3rd ed.
New York: Churchill Livingstone; 1999.
Lee GR. Wintrobe’s Clinical Hematology. 10th ed.
Baltimore, MD: Lippincott Williams & Wilkins;
1999.
McGarry MP, Protheroe CA, Lee JL. Mouse
Hematology; A Laboratory Manual. New York:
Cold Spring Harbor Laboratory Press; 2010.
Animal haematology/clinical
pathology
Everds N. Hematology of the laboratory mouse.
In: Foster HL, et al., editors. The Mouse in
Biomedical Research, vol. 3. Amsterdam: Elsevier;
2006. pp. 133–70.
Hawkey CM, Dennett TB. Color Atlas of
Comparative Veterinary Hematology. Ames, IA:
Iowa State University Press; 1989.
Jain NC. Essentials of Veterinary Hematology.
Philadelphia, PA: Lea & Febiger; 1993.
Latimer KS, Mahaffey EA, Prasse KW,
Duncan J, Proctor NW. Duncan and Prasse’s
Veterinary Laboratory Medicine: Clinical
Pathology. 4th ed. Ames, IA: Iowa State
University Press; 2003.
Reagan WJ, Irizzary-Rovira A, DeNicola DB.
Veterinary Hematology: Atlas of Common
Domestic and Non-Domestic Species. 2nd ed.
Ames, IA: Wiley-Blackwell; 2009.
Smith CA, Andrews CM, Collard JK, Hall DE,
Walker AK. A Color Atlas of Comparative,
Diagnostic, and Experimental Hematology.
London: Mosby-Yearbook; 1994.
Stockham SL, Scott MA. Fundamentals of
Veterinary Clinical Pathology. 2nd ed. Ames, IA:
Blackwell Publishing; 2008.
Thrall MA. Veterinary Hematology and Clinical
Chemistry. Philadelphia, PA: Lippincott Williams
& Wilkins; 2004.
Weiss DJ, Wardrop KJ. Schalm’s Veterinary
Hematology. 6th ed. Ames, IA: Wiley-Blackwell;
2010.
Hematological methods
American Society of Veterinary Clinical
Pathology. Principles of Quality Assurance and
Standards for Veterinary Clinical Pathology.
www.asvcp.org/pubs/pdf/ASVCPQualityControl
Guidelines.pdf; 2009 [accessed October 2011].
Bain BJ. Blood Cells: A Practical Guide.
Philadelphia, PA: JB Lippincott; 1989.
Jones TC, Ward JM, Mohr U, Hunt RD. Hema-
topoietic System. Berlin: Springer-Verlag; 1990.
McGarry MP, Protheroe CA, Lee JL. Mouse
Hematology. A Laboratory Manual. New York:
Cold Spring Harbor Laboratory Press; 2010.
O’Connor BH. A Color Atlas and Instruction
HAEMATOLOGY
Manual of Peripheral Blood Cell Morphology.
Baltimore, MD: Williams & Wilkins; 1984.
Stiene-Martin AE, Lotspeich-Steininger CA,
Koepke JA. Clinical Hematology: Principles,
Procedures, Correlations. 2nd ed. Baltimore, MD:
Lippincott Williams & Wilkins; 1998.
347
ANATOMY
AND
NORMATIVE BIOLOGY