the
beaufort cottage laboratories Guide to
equine clin i c a l
patholo g y
T h e
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Welcome to the Beaufort Cottage Laboratories
Guide to Equine Clinical Pathology.
We hope this Guide will provide practical help to
busy veterinary clinicians, nurses and students in
need of a reference source in the course of their
daily work or studies. It is not intended to be a
textbook, nor do we pretend it is complete.
For further advice on pathology issues raised in this
Guide, please call Beaufort Cottage Laboratories
on +44 (0)1638 663017 (office hours). In case of
emergency, please call Rossdale & Partners on
+44 (0)1638 663150 (24 hours).
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Contents
Introduction
4
Using clinical pathological aids to diagnosis 6
Clinical disease, preventive medicine, laboratory profiles, management aids
8
Sampling requirements
Labelling and request forms, dispatch, post & packaging, sampling equipment
Reference ranges
11
Haematology
12
Erythrocytes 12, Leucocytes 14, Platelets 17
Clinical chemistry
19
Proteins 19, IGG 21, plasma fibrinogen, SAA, AST, 22, CK, LD 24, cardiac troponin 25, SDH,
GLDH, GGT 27, SAP, IAP, bilirubin, bile acids 28, amylase, glucose, oral glucose absorption
test, cholesterol &triglycerides, urea 29, creatinine, urine fractional clearance ratios 30,
calcium, potassium & chloride, calcium, phosphate & magnesium 31, plasma lactate 32
Blood gas analysis
33
Endocrinology
33
Pregnancy tests, progestagens 33, granulosa cell tumour 34, cryptorchidism,
thyroid function, pituitary function, glucose, cortisol, insulin, overnight dexamethasone
suppression test 35, TRH stimulation test, combined DXM suppression/TRH stim. test 36
Urine collection and analysis
37
Parasitology
38
Faeces collection, faecal worm egg counts, faecal lungworm larval counts
Microbiology
39
Bacterialogy, skin scrapings, virology 40
Cytology
42
Fluid samples - peritoneal, pleural 43, synovial, tracheal washes, bronchoalveolar lavage 44,
cerebrospinal fluid, bone marrow 45, semen samples, endometrial smears 46
Histology
47
Necropsy (postmortem) examinations
48
Biopsy sampling
52
Tables of reference ranges
56
Adult horses 48, CMSNG sampling, neonatal foals, foeti and placentae 50
Skin, lump, liver, lung 52, kidney, endometrial 53, testicular, ileal 54, rectal 55
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introduction
In all aspects of veterinary medicine, an
accurate diagnosis is a pre-requisite for
specific treatment and appropriate case
management. Making a diagnosis involves
a challenging combination of art and science and, if
used correctly, laboratory investigations may be helpful.
Following a definitive diagnosis, follow-up clinical
and laboratory examinations can assess the effects of
treatment. Where progress is unsatisfactory, more detailed
investigations are often indicated as directed by the
characteristics of the individual case.
Making a diagnosis can be likened to ‘jigsaw puzzling’.
The more pieces become available, the easier it becomes
to solve the puzzle. Historical information and clinical
examination results remain the first steps in the diagnostic
pathway, sometimes allowing a definitive diagnosis to
be made immediately, e.g. for a mid-shaft cannon bone
fracture. More often, a differential diagnosis needs to be
refined by clinical pathological aids and further clinical
diagnostic procedures, e.g. recurrent laminitis and
hepatopathy associated with equine Cushing’s syndrome.
These aids must be applied appropriately, accurately and
efficiently, to best effect.
Incorrect results or incorrectly interpreted results may
confuse the diagnostic pathway, leading to incorrect
treatment and case management. In-house quality control
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and external quality assurance are important for all
veterinary laboratories to ensure reliability of results and
to give confidence both to laboratory staff and to their
clients.
Veterinary surgeons have a duty to relieve suffering
in animals as quickly and efficiently as possible. Ideally,
to achieve this, they should provide their patients with
the most accurate diagnosis possible, as early as possible,
using whatever aids are available and appropriate so that
early treatment is successful. This may appear costly in the
short term, but can sometimes save expense in the longer
term by avoiding delays in starting appropriate treatments
or by avoiding the use of inappropriate treatments and
thereby shortening time to recovery. In addition to welfare
considerations, early accurate diagnoses and treatments
bring significant benefits for performance horses and aid
profitability to their owners.
Clinicopathological aids may be applied in cases of
clinical disease and for the assessment of their treatment,
in preventive medicine programmes, and as management
aids. The tables on the next two pages are presented as
broad guides to the use of laboratory aids to diagnosis
in specific clinical, preventive medicine and managerial
indications.
August 2006
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Using Clinical Pathological aids to diagnosis
Clinical disease
Clinical condition
Potentially useful tests
Infection
Haematology, serum amyloid A, plasma fibrinogen, serum
protein electrophoresis, bacteriology, mycology, virology,
serology.
Intestinal parasitism
Haematology, serum amyloid A, plasma fibrinogen, serum
albumin and protein electrophoresis, tapeworm ELISA, faecal
worm egg count, rectal biopsy.
Liver disease
Haematology, serum amyloid A, plasma fibrinogen, serum
protein electrophoresis, AST, LD and isoenzymes, GGT, GLDH,
SAP, bile acids, liver scan and biopsy.
Kidney disease
Haematology, serum amyloid A, plasma fibrinogen, serum
proteins, urea, creatinine, urine analysis and electrolytes,
fractional electrolyte clearance ratios, bacteriology, kidney scan
and biopsy.
Pancreatic disease
Serum GGT, amylase and lipase.
Skin disease
Skin scrapings, biopsy, bacteriology, mycology, feed allergen
tests.
Bone metabolic/parathyroid abnormality
SAP, calcium and phosphate, urine phosphate
clearance ratios.
Weight loss/diarrhoea
Haematology, serum amyloid A, plasma fibrinogen, serum
albumin and protein electrophoresis, SAP, IAP, glucose
absorption test, peritoneal fluid cytology, faecal Rotavirus assay,
serum electrolytes, feed allergen tests. Faecal occult blood.
Pulmonary abnormality
Haematology, serum amyloid A, plasma fibrinogen, serum
proteins, blood gas analysis, faecal lungworm examination,
tracheal wash/BAL cytology, bacteriology.
Fluid/electrolyte balance
Haematology, serum amyloid A, plasma fibrinogen, serum
albumin and proteins, electrolytes, urine analysis and fractional
electrolyte clearances, blood gas analysis.
Stallion genital abnormality
Endocrinology, bacteriology, semen analysis, testicular biopsy.
Mare genital abnormality
Endocrinology, bacteriology, endometrial cytology and biopsy.
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Preventive medicine
Clinical condition
Useful tests
Intestinal parasitism
Haematology, serum amyloid A, plasma fibrinogen, serum
albumin and protein electrophoresis, tapeworm ELISA, faecal
worm egg count.
Venereal disease
Penile and preputial, clitoral and endometrial aerobic and
microaerophilic bacteriology.
Diarrhoea
Rectal/faecal bacteriology for Salmonella spp. and
Campylobacter spp. and fluid faeces for Rotavirus test. Faecal
occult blood test.
Nasal discharge
Nasopharyngeal, guttural pouch and tracheal washes for
Streptococcus equi and Rhodococcus equi bacteriology.
Skin lesions
Scrapings for dermatophytes, skin biopsy.
Laboratory Profiles
Age/type/condition
Useful tests
12-36 hours
Haematology, serum amyloid A, plasma fibrinogen, serum
proteins, IgG.
Weanling and yearling
Haematology, serum amyloid A, plasma fibrinogen, serum
proteins, SAP, calcium, phosphate, urinary phosphate fractional
clearance ratios.
Inflammatory disease
Haematology, serum amyloid A, plasma fibrinogen, serum
proteins and electrophoresis.
Horses in training
Haematology, serum amyloid A, plasma fibrinogen, serum
proteins, AST, CK.
Mature horse complete profile
Haematology, serum amyloid A, plasma fibrinogen, serum
proteins and electrophoresis, AST, CK, LD and isoenzymes, GGT,
GLDH, SAP, IAP, urea, creatinine.
Management Aids
Management requirement
Useful tests
‘Unfitness’ (horses in training)
Haematology, serum amyloid A, plasma fibrinogen, serum
proteins, AST, CK.
Mare pregnancy tests
45-95 days – serum eCG.
>120 days – serum oestrone sulphate.
>150 days – urinary oestrogens.
Mare luteal function
Plasma progesterone.
Anthelmintic programme efficacy
WEC, tapeworm ELISA
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sampling require m e n ts
Laboratory results are only as good as the samples submitted
allow them to be. Samples should be taken by the correct
techniques, with suitable equipment, into suitable containers
and media/preservatives and securely dispatched to the
laboratory in the shortest possible time.
Labelling and request forms
All specimens should be labelled legibly
with the name of the horse, date and
time of sampling and site of collection,
if appropriate, to enable proper reporting
and certification of results. A concise
case history should always be included,
to help the laboratory make sure that
the appropriate tests are performed and
reported quickly and efficiently and in order
to allow the clinical pathologist to help the
clinician interpret the results.
Dispatch
Avoid sending perishable specimens over
the weekend, where they may be held
up and deteriorate in the post. Where
possible it is sensible to centrifuge or to
allow clotted blood samples to stand until
it is possible to pour or pipette off the
serum, in order to avoid haemolysis. Do
not refrigerate EDTA samples or haemolysis
will render the results useless. Do not send
frozen or unfixed tissues for histological
examinations as they will undergo natural
autolysis and arrive in a less than suitable
or even useless condition for satisfactory
examination.
For urgent samples and for dead foals,
foeti and placentae for postmortem
examinations, we strongly recommend
personal delivery, e.g. by owner or private
courier service.
Post and Packaging
Please instruct your secretarial staff
in the proper packaging of specimens
so that disappointments do not occur.
Sound packaging is essential to avoid
the breakage of containers in the post
and the loss of, or damage to samples. Internal and external packing is essential
('Jiffy bags' alone cannot be relied upon).
The leakage of pathological specimens
can present public health hazards and
the Royal Mail may refuse to handle
soiled packages. The following notice was
published and circulated by the Royal
College of Veterinary Surgeons in August
1988:1. In general, the despatch of deleterious
substances by post is banned by the
Post Office. There are however special
exemptions for pathological material sent
to and from laboratories by veterinary
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Sampling Equipment
Test
Sample
Container/anticoagulant/preservative
Haematology
Whole blood
EDTA (lilac Vacutainer or blue Monovette)
Plasma fibrinogen
Whole blood
Sodium citrate (blue Vacutainer or green Monovette)
Clinical chemistry, serology, minerals and electrolytes
Serum
Empty tube (red Vacutainer or brown Monovette)
Plasma glucose
Whole blood
Fluoride/oxalate (grey Vacutainer or yellow Monovette)
Plasma progesterone
Plasma or serum
Lithium heparin (green Vacutainer or orange Monovette)
Mare pregnancy
Serum or plasma
Empty tube (red Vacutainer or brown Monovette)
Urinalysis Urine
Sterile, empty, leak-proof container
Blood selenium or glutathione peroxidase Erythrocytes
Lithium heparin (green Vacutainer or orange
Monovette) BacteriologySwabs
Amies charcoal transport medium
Fluids
Blood grow medium
Faeces
Sterile, leak-proof container
Blood
Virology
Rotavirus Blood grow medium
Liquid faeces in sterile leak-proof container
Swabs
Viral transport medium
ParasitologyFaeces
Sterile, leak-proof container
Dermatology
Skin scrapings
Sterile, leak-proof container
Cytology
Endometrial and other smears
Rolled onto sterile gelatine-coated slides and fixed with ‘smear fix’ or carbowax
Peritoneal, pleural, CSF, synovial fluids,
tracheal washes
and BALs One sample preserved in EDTA, another diluted
50:50 in cytospin centrifuge fluid and another
undiluted in a sterile leak-proof container
(synovial fluid also in blood grow medium for bacterial culture)
Histology
General tissue samples Thin, representative samples fixed in an ample
volume (at least 10 x) of 10% formol saline
Endometrial and testicular biopsies
Bouin’s fluid
Semen
One sample diluted 50:50 in formol citrate and another in an empty, sterile, leak-proof container.
Semen analysis
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surgeons and some others. Very highly
infected materials such as that containing
foot and mouth disease virus or some
especially dangerous human pathogens are
excluded from this exemption.
2. Members of the public may send
specimens through the post only at the
express request of a registered laboratory
or veterinary surgeon.
3. Only first-class letter post or data post
may be used. Parcel post must not be
used.
4. The Post Office requires that all samples
be packed in a particular way. These rules
must be followed otherwise the Post Office
may remove and destroy the specimen.
a. Every specimen must be enclosed in
a primary container, which is securely
sealed. This container must not exceed
50ml (although special multi-specimen
packs may be approved).
b. The primary container must be wrapped
in sufficient absorbent material to absorb all
possible leakage in the event of damage.
c. The container and absorbent material
must be sealed in a leak proof plastic bag.
d. This package must then be placed in
either:
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i. A polypropylene clip down container.
ii. A cylindrical light metal container.
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iii. A strong cardboard box with full depth lid.
iv. A specially grooved two-piece polystyrene box.
5. It is recommended that this completed
package should be placed in a padded
bag.
6. Multi-specimen packs may be used
provided that each primary container is
separated from the next by absorbent
packing.
7. Any other packaging systems must have
the prior approval of the Post Office.
8. Labelling: The outer cover must be
labelled ‘Pathological Specimen Fragile. With Care’. It must show the name and
address of the sender to be contacted in
case of leakage.
9. Therapeutic and diagnostic substances,
such as blood, serum, vaccines etc. are
classified as pathological specimens.
Ensure that anything you send by post
complies with the regulations otherwise
it may be removed from the mail and
destroyed, and you will lose a valuable
specimen. You may be prosecuted by
the Royal Mail. You may cause injury or
disease to someone handling the package
either during its transit through the mails,
or at the receiving laboratory.
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ref erence r ang e s
Clinicians and clinical pathologists must rely upon so-called
‘normal’ reference ranges to interpret laboratory results.
Unfortunately it is not possible to produce one set of ‘normals’
to suit all equine animals, as horses and ponies of different
ages, types, uses and stages of training all have significant
variations in some parameters.
Another problem is that individual horses
vary considerably not only in their own
‘normal’ reference results but also by the
effects that variations from reference range
and even clinical abnormality will have
on their clinically-apparent health and
performance. Sub-clinical disease, e.g.
low-grade anaemia, viral ‘challenges’,
low-grade myopathy, hepatopathy and
nephropathy will all produce effects on
the appropriate laboratory results although
the horse is considered clinically ‘normal’. The owner, rider, trainer or manager’s
assessment of each horse as an individual
remains essential and laboratory results
should never be used to 'train' performance
horses. Experienced interpretations are
required for different types of horses used
for different purposes.
mathematically normal distribution within
the horse population to be applicable and
this is rarely the case. Alternatives are to
work with percentiles (the range within
which the results of 90% of clinically
normal horses fall). Even then clinically
normal horses will have significant subclinical changes and individual horses will
have idiosyncratic ranges different to the
population range. Clinicians and clinical
pathologists must use reference ranges that
they are comfortable with. Reference ranges for the more commonly
used haematological and clinical chemical
parameters, for adult non-Thoroughbred
horses, neonatal and older Thoroughbred
foals, Thoroughbred yearlings and two
and three-year-old Thoroughbred horses in
training can be found on pages 56-71.
The ‘classical’ method of producing
reference ranges for laboratory tests was to
use the ‘bell-shaped curve’ of two standard
deviations either side of the mean of large
numbers of clinically normal horses. This
requires the measured parameter to have a
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Haematology
In the presence of clinical signs of disease, haematological
examinations, performed on sequestrated (EDTA)
blood samples, may reveal abnormalities suggesting
haemoconcentration, anaemia, bacterial or viral infections,
parasitic or allergic conditions.
These examinations may also be valuable
as part of a preventive medicine programme
for groups of horses, when examined on a
regular and routine basis, and interpreted
carefully. Such a sampling programme may
provide useful information that can be used
as a basis for advice to trainers of race and
performance horses.
Most clinical pathology laboratories now use
automated or semi-automated cytochemical
or particle counting haematology analysers.
These provide much more accurate and
repeatable results than the older manual
counting technologies if the analysers are
correctly calibrated for equine samples.
It is important to remember that results
from analysers that provide granulocyte/
non-granulocyte differential leucocyte
counts must be interpreted differently from
those that produce cytochemically stained
and laser scanned differential leucocyte
counts. The latter, if calibrated correctly,
can be indistinguishable from traditional
manual counts performed on stained
films. Automated differentials are highly
repeatable and are more accurate than
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manual cell counts as some analysers
differentiate and count 10,000 rather
than 100 leucocytes. Beaufort Cottage
Laboratories currently use a Bayer Advia
120 automated cytochemical haematology
analyser.
Samples for haematological examinations
should be taken from horses at rest with
minimal excitement, otherwise splenic
contraction can make the interpretation of
erythrocyte parameters impossible.
Erythrocytes
(RBC, PCV, Hb, MCV, McHc, McH)
Haemoconcentration/dehydration (raised
total erythrocyte count, haematocrit and
haemoglobin concentration) can only be
interpreted in samples taken from resting
horses that are relaxed at collection and
this can be very difficult to achieve in
some excitable individuals. Reflex splenic
contraction occurs in horses in response to
fright, excitement and exercise, increasing
numbers of young macrocytic erythrocytes
in the circulating pool. Routine sampling
sessions in performance horse stables are
therefore often conducted at standard times
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after a period of rest and quiet, e.g. early
morning or late afternoon, before mucking
out and feeding, in order to avoid sampling
excited horses and to add a degree of
standardisation. Haemoconcentration is sometimes a
feature of so-called ‘over-trained’ horses
that perform poorly, appear stressed, and
have dry scurfy skin coats, lose condition
and drink inadequately. They usually
respond well to fluid and electrolyte therapy
administered by nasogastric tube followed
by a period of rest. Chronic anhidrosis can
produce a similar picture. Dehydration is a
feature of horses who are clinically ill with
acute enteritis or colitis, requiring intensive
fluid, electrolyte, acid-base and supportive
therapy to replace losses. Haemoconcentration and dehydration are features
of exercise and heat exhaustion in horses
performing in hot dry climates and over
long distances (typically endurance races),
requiring timely diagnosis and appropriate
treatment/management. The interesting
condition of acute anhidrosis, i.e. failure
of normal sweating, occurs in some horses
who are raised in temperate climates and
then perform in hot, dry climates when
they have failed to acclimatise, resulting
in respiratory distress, laboured breathing,
pyrexia, collapse and even death.
Attempts have been made to interpret
the significance of results of post-exercise
blood samples in terms of fitness. In terms
of haematologic tests, variable degrees
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of haemoconcentration and leucocytosis
are always a feature and without rigid
standard exercise test regimes, which are
almost impossible to organise within most
performance horse training environments,
results are usually uninterpretable. Specific
muscle enzyme tests (see page 16)
performed on serum samples collected
before and after exercise, can help with the
diagnosis of exercise-induced myopathy. There has been considerable interest in
lactate assays before, during and after
exercise to determine aerobic/anaerobic
metabolic capacity, with still considerable
debate and differences of opinion.
Anaemia (low total erythrocyte count,
haematocrit and haemoglobin concentration)
in horses is less commonly a primary
condition and more often occurs secondary
to some other primary condition, e.g.
infection (bacterial or viral), parasitism (endo
or ectoparasitism) or metabolic abnormality
(e.g. hepatopathy, nephropathy), which
require specific diagnosis and treatment.
Malnutrition is rarely seen in performance
horse stables but mineral and vitamin
imbalances (involving deficiency or excess)
may occur.
Acute haemorrhage can cause acute primary
blood loss anaemia following accidental
injury involving a major blood vessel. The
haemorrhage may sometimes be visible
externally but more often occurs internally
within a body cavity, i.e. intraperitoneal,
intrapleural or intrapericardial haemorrhage.
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Chronic haemorrhage, e.g. following
castration, may cause an anaemia and selfperpetuating thrombocytopenia. Platelet
transfusion may result in haemostasis
without further surgical interference. Guttural pouch mycosis may cause
internal carotid artery ulceration, resulting
in profound acute and sometimes fatal
epistaxis and blood-loss anaemia. Gastric
ulceration, not uncommonly seen in
performance horses, can cause anaemia
from chronic haemorrhage. Examination
of faecal samples for the presence of
occult blood can be performed but results
are frequently positive, most commonly
because of activity of intestinal parasites,
even in well-managed horses, and are
therefore not reliably diagnostic of gastric
ulceration. Unfortunately, serum pepsinogen
assays are not reliably diagnostic of gastric
ulceration in horses. Where suspected,
the diagnosis must be made and the
significance assessed by gastroscopic
examinations.
Intravascular haemolysis, i.e. erythrocyte
destruction resulting in haemolytic
anaemia, has a variety of different
causes, which require specific diagnostic
testing. Equine Infectious Anaemia
(Coggins’ agar gel immunodiffusion
test), autoimmune haemolytic anaemia
(Coombs’ antiglobulin test), Piroplasmosis
(Babesiosis) (complement fixation test),
disseminated intravascular coagulopathy
(DIC) (prolonged prothrombin and activated
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l a b o r a t o rie s
partial thromboplastin times) and a variety
of plant and environmental toxins are some
causes of intravascular haemolysis.
Equine anaemia is most commonly
macrocytic (raised McV), reflecting splenic
replacement with juvenile erythrocytes,
as seen with blood loss, infections and
parasitism. Reticulocytes are not commonly
seen in equine blood samples and so
their presence or absence is not a reliable
means of determining regeneration or
non-regeneration, as in other species. Normocytic (McV within normal range)
anaemia is sometimes seen in horses who
are being challenged by respiratory viruses
but who show no clinical signs. Microcytic
anaemia (low McV) is uncommon but is
sometimes seen in immature individuals
and has been reported in horses with iron/
folate deficiency.
Leucocytes
(WBC & Differential leucocyte count)
Leucocytosis (raised total leucocyte count)
and neutrophilia (raised segmented
neutrophil count) most commonly occurs
in performance horses in association
with septic and non-septic inflammatory
conditions. Septic inflammation is most
commonly associated with bacterial
infection. In performance horses this
may occur following an injury, e.g.
penetrating wounds followed by cellulitis,
septic arthritis or tenosynovitis, upper
respiratory infections and less commonly
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in systemic bacterial infections. Non-septic
inflammation can occur following nonpenetrating injury, e.g. bruising of soft
tissues, tendons, ligaments or periosteum
and traumatic arthritis or tenosynovitis,
sometimes associated with degenerative
joint disease. Marked leucocytosis is seen
in foals responding to bacterial infections,
notably Rhodococcus equi (‘summer
pneumonia’) and Streptococcus equi
(‘strangles’) infections.
Neutrophilic ‘shifts to the left’ (appearance of
juvenile neutrophils or ‘band’ neutrophils),
while diagnostically helpful in foal-hood
infections, are seldom seen in adult horses
unless severely and acutely infected
with conditions such as acute cellulitis
or lymphangitis. Some cases of acute
salmonellosis, endotoxaemia, peracute
enterocolitis, peritonitis and pleuritis
have neutrophilic shifts to the left, more
commonly associated with leucopenia and
neutropenia.
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Leucopenia (low total leucocyte count) and
neutropenia (low segmented neutrophil
count) is most commonly seen in adult
performance horses during the acute phase
of a viral challenge, when there may or
may not be clinical signs. These may
include lethargy, pyrexia, nasal discharge,
coughing and oedematous legs. Leucocytic
changes seen with infection very much
depend upon sampling time in relation
to the stage of the disease process (see
Fig.1).
If the early acute infectious phase has
passed then the leucopenic phase will be
missed and haematologic examinations
will reflect repair and sometimes-secondary
bacterial involvement with leucocytosis and
neutrophilia (see page 14). Therefore, blood
samples should be taken from symptomless
stablemates where viral infections are
suspected, in order to help with diagnosis,
epidemiology and assessment of recovery.
Unfortunately, unless Equine Influenza,
Fig.1:equine blood leucocyte response to viral challenge
Circulatingbloodleucocytesx
10^9/l
14
12
10
8
6
4
2
0
1
2
3
4
5
6
Days–Viralchallengestartsonday1.Basalleucocytecountis8x109/l
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8
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Herpesvirus, Rhinovirus or Adenovirus
infections are involved (for which specific
serologic assays are available), virological
‘screening’ investigations are seldom
rewarding.
Profound leucopenia is seen in neonatal
foals with either bacterial (most commonly
Escherichia coli) or viral (most commonly
Equine Herpesvirus-1) septicaemia and
is an indication for urgent intensive care.
A blood culture should be performed
immediately prior to starting broadspectrum antibiotic therapy in order to
try to identify the pathogen and clarify
antibiotic sensitivity.
Occasionally, profound leucopenia with
neutropenia and neutrophilic shifts to the
left are seen in adult performance horses
suffering from acute salmonellosis, peracute
enterocolitis, peritonitis and pleuritis or
pleuropneumonia and in newly foaled
mares with toxic metritis/laminitis, most
commonly following placental retention.
Profound leucopenia is always a sign of
severe illness, indicating the need for
intensive care and suggesting a guarded
prognosis.
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Lymphocytosis
(raised
lymphocyte
count) is seen in horses in response
to endogenous catecholamine release
during excitement or exercise. Otherwise,
it is most commonly seen in response to
some chronic viral infections and in more
rarely seen autoimmune diseases. Massive
leucocytosis (sometimes greater than 100
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x 109/l) with lymphocytosis is a feature of
generalised lymphoma, in which neoplastic
lymphocytes can be demonstrated in
peripheral blood and sometimes in body
cavity fluid samples and tissue biopsy
samples.
Lymphopenia (low lymphocyte count) is
seen in horses in response to endogenous
glucocorticoid release and in response to
exogenous corticosteroid administration. Otherwise, it may be seen in acute viral
infections, severe bacterial infections,
septicaemia, endotoxaemia and immune
deficiency conditions. Profound, persistent
leucopenia always carries a poor
prognosis.
Monocytosis (raised monocyte count)
reflects increased phagocytic demand
as may occur with chronic suppurative
conditions with tissue necrosis. In
performance horses, monocytosis is most
commonly seen during the post-acute or
recovery phases following upper respiratory
viral infections.
Eosinophilia (raised eosinophil count)
occurs with antigen-antibody response
in tissues rich in mast cells, e.g. skin,
lung, gastrointestinal tract and uterus
and in parasitically sensitised horses. In
performance horses, low-grade eosinophilia
is most commonly seen in association
with leucopenia or lymphocytosis in
the acute-phase of responses to viral
infections. In horses at pasture, the first
differential diagnosis for eosinophilia is
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e q u i n e
intestinal parasitism and warrants further
investigations with protein electrophoresis
and faecal worm egg counts. Rare cases
of eosinophilic leukaemia have been seen
with eosinophil counts as high as 2.5
x 109/l (25% of differential leucocyte
count).
Basophilia (raised basophil count) is very
uncommon in horses, as are the presence
of basophils themselves. Basophils have
been a feature in cases of hyperlipidaemia
and in some horses that were recovering
from colic.
Platelets
Thrombocytosis (raised platelet count) is
seen uncommonly in adult horses but may
occur in bacterial infections. It may occur
with bacterial infections in foals and has
been associated with Rh. equi infections.
Thrombocytopenia (low platelet count)
may be a reflection of decreased
production, increased usage or from various
spurious factors, e.g. drug administration,
the presence of cold agglutinins in the
sample or platelet clumping in EDTA).
Thrombocytopenia is sometimes seen
in horses with viral infections. Where
pseudothrombocytopenia is suspected,
platelet counts should be measured on two
blood samples collected at the same time,
one into EDTA and the other into sodium
citrate anticoagulants. If the sodium citrate
sample, after correction for dilution, is
considerably higher than the EDTA sample,
cli n ic a l
p a t h o l o g y
EDTA-induced pseudo-thrombocytopenia is
the likely answer. Decreased production
may occur with neoplasia or a toxic insult
to the bone marrow, the latter is diagnosed
by biopsy. Idiopathic thrombocytopenia
is
probably
an
immune-mediated
condition and thrombocytopenia is seen
in horses with disseminated intravascular
coagulopathy (DIC) most commonly a
serious complication of acute enterocolitis.
Haematologic examinations are often used
routinely to screen horses in training for
subclinical disease and can be helpful
when examined on a regular routine basis,
and interpreted carefully. Haematologic
signs of viral infection (leucopenia and
neutropenia or relative lymphocytosis,
depending on stage of sampling), in
a clinically normal horse may suggest
‘challenge’, i.e. the horse’s immune system
is responding but not succumbing to the
infection. In this condition, many horses
do not perform to their best athletic
potential, their recovery after exertion may
be prolonged and they may be more
likely to suffer secondary complications
such as pneumonia, lung abscess, skeletal
and/or cardiac myopathy and/or exercise-
induced pulmonary haemorrhage
Haematologic examinations, in combination
with inflammatory protein measurements
(see later) are often used to help screen
and monitor the progress of foals and
older horses at studfarms when Rh. equi
infections or Strep. equi epidemics occur.
17
T h e
B e a u f o rt
c o tt a ge
In cases of clinical disease, haematologic
variations from 'normality' are often marked
and obvious, but more sophisticated
analytical equipment is required when
screening for less obvious variations and
trends. Modern automated cytochemical
haematology analysers (e.g. Bayer Advia
120) provide accurate differential cell
counts that correlate closely with traditional
manual differential counting techniques. Their automated differentials are highly
repeatable and are likely to be more
accurate than manual cell counts as they
are performed on 10,000 rather than 100
leucocytes. These analysers provide results
that are more accurate, repeatable and
therefore reliable for the routine monitoring
of performance horses. However, when
required by clinical history or results
NOTES
18
l a b o r a t o rie s
obtained, a traditional stained smear is
still required to demonstrate erythrocyte
or leucocyte abnormalities. Red cell
abnormailities include Howell-Jolly bodies
and nucleation, fragmentation, oxidative
damage, spherocytes, basophilic stippling
or Babesia spp. parasites in piroplasmosis.
White cell changes include left shifts, toxic
degeneration, hypersegmentation, neoplastic
change or cytoplasmic inclusions as seen
for example in ehrlichiosis.
G u i d e
t o
e q u i n e
cli n ic a l
p a t h o l o g y
clinical ch emi st r y
Proteins
Total protein, albumin and globulin
estimations are useful in the assessment
of general bodily condition and nutritional
status and the response to infectious or
parasitic disease. Electrophoresis helps
determine the significance of raised total
globulin levels. Specific tapeworm ELISA
assays are now available commercially. Low
serum albumin and/or rising globulin levels
are a ‘red flag’ warning, most commonly seen
with cyathostomiasis (hypoalbuminaemia),
large strongylosis (raised beta 1 globulin),
mixed helminthiasis (hypoalbuminaemia
and raised beta 1 globulin), hepatopathy
(hypoalbuminaemia and raised beta 2
globulin), antibody response to infection
(raised gamma globulin) or abscess
formation (raised alpha 2 and gamma
globulins). Globulins can be differentiated
by electrophoresis (Fig.2).
Strep. equi infections) will often show
characteristic alpha 2 and gamma globulin
responses. Serum samples should be
used for protein electrophoresis, as raised
fibrinogen levels in heparinised plasma
samples will cause confusing rises in beta
2 globulins (Fig.7).
Occasionally, horses with generalised
lymphosarcoma or plasma call myeloma
have massively increased total protein
and globulin levels, for which protein
electrophoresis shows a massively raised,
discrete, ‘skyrocket’ peak, usually in the
beta 2 globulin range (Fig.8) suggesting
monoclonal lymphoma protein production.
Fig.2:serum protein electrophoresis
normal horse serum
Test:ELECTRGel1–803/01/2002
Protein Electrophoresis
This identifies elevations in specific globulin
fractions:1. Alpha 2 globulin - acute-phase
inflammatory protein responses (Fig.3).
2. Beta 1 globulin - Strongylus vulgaris
and mixed strongyle larval activity (Fig.4).
3. Beta 2 globulin – hepatopathy (Fig.5).
4. Gamma globulin - antibody responses
to bacterial or viral infections (Fig.6).
Horses with abscesses (e.g. Rh. equi and
Fraction
1
2
3
4
5
TotalG/L
Rel%
5.0
26.1
20.6
24.9
23.5
G/L
1.10
5.74
4.53
5.48
5.17
22.00
19
T h e
c o tt a ge
l a b o r a t o rie s
Fig.3:serum protein electrophoresis
raised alpha 2 globulin
Test:ELECTRGel2–910/01/2002
Fig.4:serum protein electrophoresis
raised alpha 2 and beta 1 globulins
Test:ELECTRGel1–308/02/2002
Fig.3:serum protein electrophoresis
raised alpha 2 globulin
Test:ELECTRGel2–910/01/2002
Fig.4:serum protein electrophoresis
raised alpha 2 and beta 1 globulins
Test:ELECTRGel1–308/02/2002
Fraction
1
2
3
4
5
Fraction
1
TotalG/L
2
3
4
5
20
B e a u f o rt
Rel%
2.8
33.7
16.4
24.5
23.4
Rel%
39.00 2.8
33.7
16.4
24.5
23.4
G/L
0.78
13.14
6.40
9.56
9.13
G/L
0.78
13.14
6.40
9.56
9.13
Fraction
1
2
3
4
5
Fraction
1
TotalG/L
2
3
4
5
Rel%
1.3
24.0
48.2
10.4
16.2
Rel%
48.00 1.3
24.0
48.2
10.4
16.2
G/L
0.62
11.52
23.14
4.99
7.78
G/L
0.62
11.52
23.14
4.99
7.78
Fig.5:serum
protein
TotalG/L
39.00 electrophoresis
raised beta 2 globulin
Test:ELECTRGel1–101/02/2002
Fig.6:serum
protein
TotalG/L
48.00 electrophoresis
raised gamma globulin
Test:ELECTRGel1–204/01/2002
Fig.5:serum protein electrophoresis
raised beta 2 globulin
Test:ELECTRGel1–101/02/2002
Fig.6:serum protein electrophoresis
raised gamma globulin
Test:ELECTRGel1–204/01/2002
Fraction
1
2
3
4
5
Fraction
1
TotalG/L
2
3
4
5
Rel%
0.9
17.0
15.2
39.3
27.6
Rel%
54.00 0.9
17.0
15.2
39.3
27.6
TotalG/L
54.00
G/L
0.49
9.18
8.21
21.22
14.90
G/L
0.49
9.18
8.21
21.22
14.90
Fraction
1
2
3
4
5
Fraction
1
TotalG/L
2
3
4
5
Rel%
2.1
18.3
17.5
17.1
45.1
Rel%
36.00 2.1
18.3
17.5
17.1
45.1
TotalG/L
36.00
G/L
0.76
6.59
6.30
6.16
16.24
G/L
0.76
6.59
6.30
6.16
16.24
G u i d e
t o
e q u i n e
cli n ic a l
Fig.7:serum protein electrophoresis
fibrinogen spike in heparinised plasma
Test:ELECTRGel2–516/05/2002
Fraction
1
2
3
4
5
TotalG/L
Rel%
5.4
19.8
17.5
36.0
21.3
G/L
2.48
9.11
8.05
16.56
9.8
46.00
p a t h o l o g y
Fig.8:serum protein electrophoresis
monoclonal beta 2 globulin 'sky rocket'
Test:ELECTRGel1–830/05/2002
Fraction
1
2
3
4
5
TotalG/L
Rel%
2.1
9.5
9.5
71.2
7.6
G/L
1.91
8.65
8.65
64.79
6.92
91.00
Serum Immunoglobulin G (IgG)
As there is no transplacental transfer of
IgG before birth, foals are born essentially
agammaglobulinaemic. During the last few
months of gestation, mares concentrate
IgG in their colostrum. Foals’ intestines are
capable of absorbing IgG for their first 12
hours of life. Providing the mare makes
colostrum of good quality in terms of IgG
concentration, she does not ‘run milk’
before foaling and the foal sucks sufficient
colostrum within the first 12 hours, the foal
acquires good circulating IgG levels and
therefore adequate passive immunity.
Foals should have their serum IgG levels
checked as a routine preventive medicine
policy. Serum samples collected from foals
after 12 hours of age should have IgG
levels of more than 4 g/l and ideally more
than 6 g/l. Levels of less than 2 g/l indicate
failure of transfer of colostral immunity and
levels of 2-4 g/l indicate partial failure. Foals with levels below 4 g/l are considered
at risk for neonatal infections and should be
transfused with hyperimmune plasma and
their serum IgG levels re-checked 24 hours
later to make sure that IgG levels have risen
to acceptable levels.
Our experience suggests that none of the
currently available ‘stable-side’ foal serum
IgG tests are reliably accurate at the 0-4 g/l
end of the scale and we measure IgG by an
immunospectrophotometric method run on
our autoanalyser.
IgG can be measured in colostrum
immediately after parturition semiquantitatively, using a refractometer
(Colostrometer) (see table on page 22). If
readings suggest an IgG level of less than
45 g/l, the foal should be considered for
donor colostrum supplementation by bottle
or stomach tube.
21
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22
B e a u f o rt
c o tt a ge
l a b o r a t o rie s
Colostrometer reading
Concentration IgG g/l
Colostrum quality
<10-15%
0-25
Poor
15-20%
28-45
Borderline
20-30%
45-80
Good
>30%
>80
Excellent
Plasma Fibrinogen
Serum Amyloid A (SAA)
This is an acute-phase reactive protein,
which increases in response to inflammation.
Elevations are found in the presence of
tissue damage and this assay may help
with diagnosis and prognosis in cases of
internal abscessation, chronic infectious or
parasitic disease and in cases of exercise
induced pulmonary haemorrhage (EIPH).
The test can be performed on fresh, paired,
non-haemolysed EDTA and serum samples
by subtraction of total serum protein from
plasma protein results, but more accurate
results are obtained from samples collected
into sodium citrate anticoagulant to
measure fibrinogen by direct coagulometric
assay. When measured serially with
serum amyloid A (see right) the kinetics
of the inflammatory response can often
be determined (Fig.9) and this can be
very helpful when monitoring response to
treatment. Fibrinogen is a very useful test
to help diagnose and monitor the response
to treatment for a number of pyogenic
conditions in foals and yearlings, e.g.
Rh. equi and Strep. equi.
This is a highly sensitive, rapidly reacting
inflammatory protein, which can be very
helpful in monitoring early responses to
infection and their response to treatment. Most normal horses have zero measurable
levels and in the face of acute, particularly
septic inflammation, levels increase quickly
(within 24 hours) to over 20 mg/l and
often more than 100 mg/l. Levels peak
and fall similarly quickly with subsidence
of inflammation when the infection is
controlled (Fig.9). SAA is a very useful
addition to routine neonatal foal ‘profiles’
to help identify those who may have or
may be developing septicaemia and require
antibiotic therapy.
Aspartate Aminotransferase
(AST, AAT, SGOT)
Elevations are seen in the presence of acute
myopathy or hepatopathy. After myopathy,
levels peak at 24-48 hours and return to
baseline by 10-21 days, assuming that no
further damage occurs. This test, taken
with CK at first visit and then 10-14 days
later, can therefore be a useful guide to
recovery from acute myopathy (Fig.10).
G u i d e
t o
e q u i n e
cli n ic a l
p a t h o l o g y
Fig.9:schematic diagram showing equine inflammatory protein dynamics
following an inflammatory challenge
Magnitudeofresponse(%)
Magnitudeofresponse(%)
Fig.9:schematic diagram showing equine inflammatory protein dynamics
SAA
Pfib
following an inflammatory challenge
100
80
SAA
Pfib
100
60
80
40
60
20
40
1
2
3
4
5
6
7
8
9
10 11
20
12
13
14
15
16
17
18
19
20
21
12
13
14
15
16
17
18
19
20
21
Days
1
2
3
4
5
6
7
8
9
10 11
Days
Fig.10:schematic diagram showing equine serum muscle enzyme levels during
a relatively mild episode of exertional rhabdomyolysis
3500
Fig.10:schematic diagram showing equine serum muscle enzyme levels during
CK
a relatively mild episode of exertional rhabdomyolysis
AST
3500
2500
3500
iu/i
iu/i
CK
AST
2000
3500
1500
2500
2000
1000
1500
500
0
1000
0
1
2
3
4
5
6
7
0
8
9
10
11
12
13
14
15
9
10
11
12
13
14
15
Days
500
0
1
2
3
4
5
6
7
8
Days
23
T h e
B e a u f o rt
c o tt a ge
Creatine Kinase (CK, CPK)
Elevations are specifically seen in the
presence of acute myopathy. CK isoenzyme
analysis was used in human medicine
to help differentiate cardiac and skeletal
myopathy from brain pathology, but has
never become routinely established in
equine sports medicine. Cardiac troponin
(see page 25) assays are now used to
differentiate skeletal from cardiac myopathy
in horses.
CK levels peak at 6-12 hours and return
to baseline by 3-4 days, assuming that no
further myopathy occurs. When measured
alongside AST (see page 22), which takes
longer to rise, peak and return to normal,
the timing and response to treatment
of myopathy in horses can be usefully
monitored (Fig.10). Paired CK assays
taken before and 2-3 hours after strenuous
exercise, can form a useful diagnostic test
for exercise-induced myopathy, in horses
l a b o r a t o rie s
than in fit horses. The conditioning process
protects equine muscle cells from exerciseinduced injury.
Very high CK levels are seen in young foals
with nutritionally associated myopathy
(‘white muscle disease’) associated with
selenium deficiency.
Lactate Dehydrogenase (LD)
A variety of disease conditions can cause
elevations in total LD and more useful
differentiation can sometimes be provided
by isoenzyme analysis (Fig.11):■ LD isoenzyme 1 - most dramatically
increased by intravascular haemolysis
(Fig.12).
Fig.11:serum ld isoenzymes
normal horse
Test:LDGel2–516/01/2002
where the diagnosis may be in doubt. Some
respiratory virus infections, notably Equine
Herpesvirus-1, appear to increase muscle
cell membrane fragility and predispose
to exercise-induced myopathy in horses
in training. This condition is sometimes
associated with clinical signs of fatigue and
stiffness in performance horses.
Significant myopathy, demonstrable by
higher serum CK and AST levels, occurs
more often after exercise in unfit rather
Fraction
LD1
LD2
LD3
LD4
LD5
TotalIU/L
24
Rel%
9.2
26.6
41.6
18.4
4.2
500
IU/L
46
133
208
92
21
LD1/LD2:0.35
G u i d e
t o
e q u i n e
■ LD isoenzyme 2 - elevated in some
cases of cardiac pathology, an indication
for cardiac troponin assay (see below)
(Fig.13).
■ LD isoenzyme 3 – no known disease
association in the horse.
■ LD isoenzyme 4 - most commonly
elevated by intestinal pathology (Fig.14).
■ LD isoenzyme 5 - rises seen with
skeletal myopathy and hepatopathy,
requiring further differentiation with CK
(see page 24) and liver enzyme (see
pages 24-28) assays (Fig.15).
Total LD levels are age-dependent to
maturity and reference ranges must be
consulted when interpreting results for
young horses (see pages 56-69).
Cardiac Troponin (cTnI)
Two proteins (tropomyosin and troponin)
working in concert with calcium, regulate
muscle contraction. Troponin is a globular
protein complex composed of three
single chain polypeptide subunits: TnI
(troponin inhibitory component), which
prevents muscle contractions in the
absence of calcium; TnT (tropomyosinbinding component), which connects
the troponin complex with tropomyosin;
and TnC (calcium binding component),
which binds calcium. The cardiac musclespecific isoform cTnI (24 kDa) exhibits
approximately 60% homology with the
skeletal isoforms (sTnI) and has a unique
cli n ic a l
p a t h o l o g y
31 amino acid extension of the N-terminus.
Experience in human medicine has shown
that after acute myocardial infarction
(AMI), elevated cTnI levels appear in the
circulation within 3-6 hours. Serum levels
peak within 14-20 hours and return to
normal after 5-7 days. The measurement
of cTnI can therefore be a useful diagnostic
aid for AMI and an aid for the monitoring
of recovery.
Myocardial infarction is rarely diagnosed in
the horse but myocardial necrosis is seen in
conditions such as atypical myoglobinuria.
Myocarditis is sometimes suspected on the
basis of arrhythmias, echocardiographic
abnormalities and/or raised serum lactate
dehydrogenase isoenzyme 2 levels. This
can follow upper respiratory viral infections.
Horses who have suffered in this way will
need more rest and supportive treatment
followed by follow-up to normality before
return to strenuous exercise if potentially
serious complications are to be avoided.
Raised cTnI levels are an indication for
cardiac ultrasound examinations and
ambulatory ECG monitoring.
25
T h e
26
B e a u f o rt
c o tt a ge
l a b o r a t o rie s
Fig.12:serum ld isoenzymes
raised ld1
Test:LDGel2–516/01/2002
Fig.13:serum ld isoenzymes
raised ld2
Test:LDGel2–1029/06/2002
Fig.12:serum ld isoenzymes
raised ld1
Test:LDGel2–516/01/2002
Fig.13:serum ld isoenzymes
raised ld2
Test:LDGel2–1029/06/2002
Fraction
LD1
LD2
LD3
LD4
Fraction
LD5
LD1
LD2
TotalIU/L
LD3
LD4
LD5
Rel%
IU/L
44.4
316
34.1
243
16.7
119
3.3
23
Rel%
IU/L
1.5
11
44.4
316
34.1
243
712
LD1/LD2:1.3
16.7
119
3.3
23
1.5
11
Fraction
LD1
LD2
LD3
LD4
Fraction
LD5
LD1
LD2
TotalIU/L
LD3
LD4
LD5
Rel%
IU/L
22.0
180
39.6
324
27.1
221
7.6
62
Rel%
IU/L
3.6
29
22.0
180
39.6
324
817
LD1/LD2:0.56
27.1
221
7.6
62
3.6
29
TotalIU/L
712
TotalIU/L
817
LD1/LD2:1.3
LD1/LD2:0.56
Fig.14:serum ld isoenzymes
raised ld
Test:LDGel1–331/07/2002
Fig.15:serum ld isoenzymes
raised ld
Test:LDGel1–310/05/2002
Fig.14:serum ld isoenzymes
raised ld
Test:LDGel1–331/07/2002
Fig.15:serum ld isoenzymes
raised ld
Test:LDGel1–310/05/2002
Fraction
LD1
LD2
LD3
LD4
Fraction
LD5
LD1
LD2
TotalIU/L
LD3
LD4
LD5
Rel%
IU/L
4.9
39
12.1
96
29.4
233
41.4
328
Rel%
IU/L
12.2
97
4.9
39
96
792 12.1
LD1/LD2:0.40
29.4
233
41.4
328
12.2
97
Fraction
LD1
LD2
LD3
LD4
Fraction
LD5
LD1
LD2
TotalIU/L
LD3
LD4
LD5
Rel%
IU/L
9.3
205
12.7
280
15.8
348
7.3
161
Rel%
IU/L
54.8
1207
9.3
205
12.7
280
2203 LD1/LD2:0.73
15.8
348
7.3
161
54.8
1207
TotalIU/L
792
TotalIU/L
2203
LD1/LD2:0.40
LD1/LD2:0.73
G u i d e
t o
e q u i n e
cli n ic a l
Cardiac troponin (cTnI) levels are measured
in serum samples (lower results may be
found in plasma samples). Clinically
normal horses have serum cTnI levels
of less than 0.2 ng/ml. Experience so
far suggests that greater than 0.3 ng/
ml is abnormal, i.e. suggests myocardial
pathology and 0.2-0.3 ng/ml is currently
a ‘grey’ zone. Levels of 0.9-5.4 ng/ml
have been measured in horses with
ultrasound-confirmed cardiomyopathy.
Nevertheless, results greater than 0.2 ng/
ml are considered ‘red flags’ for expert
cardiological appraisals.
Sorbitol Dehydrogenase (SDH)
This is an enzyme found in the cytoplasm
of hepatocytes and is therefore virtually
liver-specific, although rises are sometimes
seen in horses with skin conditions and
enteropathy. It is useful for the identification
of acute hepatocellular damage for in-house
laboratory conditions, but the enzyme is
highly labile. Therefore, samples must be
handled with care and assays must be
performed within 8-12 hours of sampling
thus SDH assays are unsatisfactory for
transported samples. SDH is not assayed at
our laboratories - the more stable GLDH assay
is now used more frequently than SDH.
Glutamate Dehydrogenase (GLDH)
Elevations are seen in the presence
acute hepatocellular damage. This is
mitochondrial enzyme found mainly
liver, heart muscle and kidney. It is
of
a
in
a
p a t h o l o g y
relatively stable enzyme and is a suitable
replacement for sorbitol dehydrogenase
(SDH) (see left) in transported samples. GLDH rises are sometimes seen in horses
with skin conditions and enteropathy.
L-Gamma
Glutamyltransferase (GGT)
GGT is found in cell membranes of
hepatocytes and biliary epithelial cells,
but the enzyme is also found in the
pancreas and kidney. Elevations in serum
levels are seen in the presence of acute
hepatitis, chronic liver cirrhosis and in very
rare cases of pancreatitis. Nephropathy
does not usually result in significantly
raised serum GGT levels so high levels
measured in the horse are usually a sign
of biliary or cholestatic disease. Chronic
pyrrolizidine alkaloid toxicity (ragwort, i.e.
Senecio jacobea poisoning) causes bile
duct hyperplasia and biliary stasis and
therefore typically results in raised serum
GGT and SAP (see below) levels. This
remains an important cause of hepatopathy
in horses and ponies in UK, who ingest
the plant unknowingly in poor-quality hay. Toxicity is uncommon in well-managed
horses.
Idiopathic GGT elevations are sometimes
seen in horses in training that appear
otherwise healthy, but perform poorly. The
cause of these GGT rises has not yet been
satisfactorily defined although plant and
fungal hepatotoxins have been suspected. In most cases, other liver enzymes are
27
T h e
B e a u f o rt
c o tt a ge
within normal range as are urea and
creatinine levels, and liver biopsy reveals
insignificant histopathological findings.
It is therefore not certain that primary
hepatopathy or nephropathy is involved. Some cases have raised muscle enzyme
levels suggesting an association with
myopathy, either directly or secondarily,
again perhaps via respiratory virus-induced
increased muscle cell membrane fragility. Most cases respond (GGT levels return
to normal) to a period of reduction in the
training programme.
Urine GGT:creatinine ratios are elevated
(>4.0) in renal tubular pathology.
Alkaline Phosphatase (SAP)
Elevations in this brush border enzyme are
most commonly seen in the presence of
chronic biliary obstructive liver pathology
(e.g. chronic pyrrolizidine alkaloid toxicity). High levels are also seen with abnormalities
of bone metabolism and intestinal
malfunction, a useful assay in growing
foals and yearlings with clinical signs of
developmental orthopaedic disease, where
SAP results may be very high and will
respond to restricted exercise and mineral,
vitamin and trace element supplementation.
SAP levels are age-dependent to maturity
and appropriate reference ranges must
be consulted when interpreting results for
young horses (see pages 56-69).
28
l a b o r a t o rie s
Intestinal Phosphatase (IAP)
Elevations in IAP relative to total SAP
are seen in the presence of intestinal
pathology.
References ranges for serum SAP and IAP
levels are very age-related and apparently
high results must be interpreted carefully in
foals, yearlings and immature performance
horses.
Bilirubin
The analysis of bilirubin levels is seldom
useful in the horse but may aid the
classification of anaemia and jaundice
in some cases. Owing to the horse's
unusual biliary excretion system, indirect
(unconjugated) bilirubin levels, may be
higher than those in other species, without
clinical disease, and the significance of
elevations without other abnormalities may
therefore be difficult to interpret. A period of
anorexia, inanition or intestinal malfunction
typically increases indirect bilirubin levels
spuriously.
Bile acids
This is a much better guide to hepatobiliary
status than bilirubin assays. High bile acid
levels occur with embarrassed hepatic
function and are a useful diagnostic and
prognostic liver function and prognostic
guide in horses. It is important to remember that none
of the liver enzymes, measured singly
or in a profile, give useful information
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about liver function. The liver has a large
functional reserve and compensatory
capacity. Liver enzyme rises suggest
hepatopathy which can be differentiated
to a degree into acute, chronic, biliary
obstructive or mixed pathology, but bile
acid assays and bromsulphalein clearance
test results reveal either adequate (normal
levels) or impaired (high levels) functional
compensation. Impaired hepatic function
suggests a guarded prognosis. Liver biopsy
and ultrasound examinations are required
to confirm an etiologic diagnosis and to
refine prognosis.
Amylase
Elevations occur in the presence of
pancreatitis, but this condition is rarely
diagnosed in the horse.
Glucose
Other than for oral glucose and xylose
absorption tests, useful for the diagnosis
and evaluation of intestinal malabsorption
cases, the value of this assay in adult
horses is limited to cases of pituitary
pars intermedia dysfunction (equine
Cushing’s syndrome) which are frequently
hyperglycaemic. Measurement of blood
glucose is invaluable in the management of
critically ill foals as profound hypoglycaemia
is often seen in neonatal septicaemia. Samples for glucose assay must be taken
into fluoride anticoagulant or must reach
the laboratory within hours of collection.
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Oral Glucose Absorption Test
The horse to be tested should be fasted
for 18-24 hours. 1 g glucose per kg body
weight is administered by stomach tube. Blood samples are collected into fluoride
anticoagulant at times 0, 30, 60, 90, 120,
150, 180, 210 and 240 mins. Glucose
levels should peak at double resting (0
mins) levels at 60-120 mins and return to
resting levels by 240 mins.
Cholesterol and Triglycerides
Elevations are seen in the presence
of abnormal lipid metabolism and
hyperlipidaemia. These conditions are
typically seen in the Shetland and other
small ponies, donkeys and occasionally as
a secondary complication in horses that
are anorexic or unable to eat. Pregnant
Shetland pony mares appear particularly
susceptible following a managemental and
nutritional change/challenge.
Urea
Urea is produced in the liver from the
metabolism of ammonia. Elevations are
seen in the presence of abnormal renal
function. Urea levels may also rise in
the haemoconcentrated and ‘over-trained’
horse, associated with fluid balance shifts
rather than renal disease. Many cases
of equine dysautonomia (‘grass sickness’)
have a degree of uraemia but this is
usually caused by catabolism. A period
of anorexia can have a similar result.
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Nephrosis and nephritis are important
conditions in neonatal and older foals and
are occasionally seen in other age groups.
Creatinine
Creatinine is formed in muscles from
creatine breakdown and is excreted via
the kidneys. In normal horses, daily
production and excretion are remarkably
constant, leading to its use as an arithmetic
constant for use with urinary fractional
excretion rates (see below). Therefore
serum elevations reflect renal malfunction
(reduced glomerular filtration), levels being
controlled by excretion rate. This may occur
in horses with pre-renal (e.g. circulatory
disturbances, dehydration or shock), renal
(insufficiency) or post-renal conditions. Measurement of urine specific gravity and
fractional excretion of electrolytes may
help to differentiate pre-renal and renal
azotaemia. When uroperitoneum (postrenal azotaemia) is suspected it can be
useful to compare peritoneal fluid and
serum creatinine concentrations as a ratio
of greater than 3:1 confirms uroperitoneum.
For both renal and post-renal azotaemia,
ultrasonography can be very helpful.
Urinary Fractional Electrolyte
and Mineral Clearance Ratios
30
In horses with normal renal tubular
function, urinary excretion rate of creatinine
is almost constant. It can therefore be
used as an arithmetic constant to produce
a measure of the fractional excretion of
electrolytes and minerals in equine urine. l a b o r a t o rie s
Concentrations of electrolytes or minerals
are measured in serum and urine samples
collected at the same time or at least within
1 hour of one another. Diuretics must not
be used to stimulate urination or spurious
results will be obtained. The percentage excretion of an electrolyte
is calculated from the following equation:-
Where: (E)u = Concentration of electrolyte in urine
(E)u
(E)s
X
(Cr)s
(Cr)u
X100=
Fractionalurinary
excretion(%)
(E)s = Concentration of electrolyte in serum
(Cr)u =Concentration of creatinine in urine
(Cr)s = Concentration of creatinine in serum
Fractional excretion rates for sodium,
potassium, chloride, magnesium, calcium
and phosphate are most commonly
measured. The result provides an
assessment of the horse’s homeostatic
regulatory status for that electrolyte or
mineral, e.g. sodium and chloride may be
selectively excreted at an increased rate
because of excessive dietary salt intake. Phosphate may be excreted at an increased
rate to try to maintain a normal serum
calcium:phosphate ratio in the face of
inadequate calcium intake. The use of this
method applies only to horses with normal
renal function. Impaired renal tubular
function results in high urine excretion
rates for all the electrolytes and minerals,
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from failure of retention - in such cases,
creatinine is not a valid arithmetic constant.
Electrolyte imbalance may predispose
exercise-induced myopathy in horses
in training and so fractional clearance
ratios may sometimes be helpful in the
investigation and management of recurrent
cases. Dietary deficiencies, excesses or
imbalances can be corrected and return
to normal excretion rates monitored,
sometimes with useful results in terms of
resolution of myopathy.
In secondary nutritional hyperparathyroidism, which occurs in horses on a
high phosphate diet, phosphate excretion
rate is high, indicating the need for oral
calcium supplementation and phosphate
reduction, to restore balance. Experience
has shown that in UK, many healthy,
fit, stabled and well performing horses,
receiving high cereal training rations, have
urinary fractional phosphate excretion rates
in excess of 9%, emphasising the need for
calcium supplementation. Higher excretion
rates are seen in horses with clinical
manifestations of secondary nutritional
hyperparathyroidism, which include shifting
lameness, periosteal thickening, facial and
mandibular swelling.
Urinary phosphate clearance ratios are useful
measures of calcium:phosphate balance in
weanlings and yearlings, important for
bone growth and development. Calcium:
phosphate imbalance can predispose to
physitis.
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Calcium, Potassium and Chloride
Electrolyte imbalance and fluid loss may
occur with diarrhoea, endotoxaemia,
intestinal crises and exertional exhaustion. The latter is of particular importance for
endurance horses and for other horses
performing in hot and humid weather
conditions. Serial assays are helpful with
intensive care cases. In other cases,
fractional urinary electrolyte excretion
rates (see page 30) are a much better
assessment than single serum assays.
Electrolyte assays are very important to
the assessment of neonatal foals under
critical care, with specific supplementation,
where indicated and monitoring of return
to normality.
Calcium, Phosphate and Magnesium
Mineral analysis may be helpful in young
horses, i.e. yearlings and two-year-olds
coming into training, with signs suggesting
abnormalities of bone metabolism. As
homeostatic mechanisms are efficient,
serum levels are often normal even in the
face of whole body abnormality, and thus
urinary fractional excretion rates (see page
30) are of greater value.
Hypocalcaemia is a cause of synchronous
diaphragmatic flutter in performance horses
and of uterine inertia in pregnant mares
at full term, requiring cautious corrective
therapy.
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Plasma Lactate
A number of hand-held and bench top blood
lactate analysers have been used in horses.
Most have been designed to help assess
the human response to exercise. Results
have been used to describe and predict
aerobic endurance capacity in horses and
it has been suggested that the rate of
decline in blood lactate concentration,
which occurs after exercise, may be a
useful index of fitness. Persistent high
blood lactate concentrations may suggest
metabolic fatigue. Anaerobic capacity is
essential for sprinters and so increases in
blood lactate concentrations for fast, short
distance Thoroughbred flat racehorses may
be an advantage. Successful long distance
endurance racehorses need to be able to
maintain mainly aerobic metabolism (low
blood lactate levels) rather than switching
to increased dependence on anaerobic
metabolism (high blood lactate levels) for
prolonged periods of time.
In horses, high circulating erythrocyte
counts, particularly in response to exercise,
makes measurement of whole blood
lactate levels less accurate, repeatable
and meaningful than plasma lactate levels. This necessitates deproteinisation and
centrifugation before analysis of plasma
samples, which is much less convenient
than whole blood analyses, which can be
performed for human athletes with instantreading hand-held machines.
32
For racehorses, results are uninterpretable
without carefully controlled standard exercise
testing conditions, which are often difficult
to organise, especially under Thoroughbred
racehorse training conditions.
Plasma lactate is also used for monitoring
cardiovascular status in horses receiving
critical care. It has been shown to be a useful
prognostic tool in surgical colic patients
and is particularly helpful in monitoring
circulatory support in compromised foals.
If you wish to assay plasma lactate, please
contact Beaufort Cottage Laboratories for
sample handling guidance.
Therapeutic drug monitoring
Therapeutic drug monitoring (TDM) is
increasingly used in equine medicine and
cardiology and is an invaluable guide to
treatment regimens. The aminoglycosides,
gentimicin and amikacin, have the potential
to cause nephrotoxicity in horses while
under-dosing can lead to treatment failure.
Serum samples are drawn 30 minutes and
8, 12 or 24 (amikacin only) hours later to
identify the peak and trough levels. Peak
levels should be at least 8 times the MIC of
the organism in question (up to 4 for some
gentimicin-sensitive equine pathogens)
whereas a delay in excretion is indicative
of renal tubular dysfunction and should
prompt discontinuation of aminoglycoside
therapy. Several of the drugs that are useful
in equine cardiac patients have a low
therapeutic index. Assays for phenytoin and
digoxin are available for close monitoring for
potential drug toxicity in patients receiving
these drugs.
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blood gas an alys is
Analyses are essential for monitoring
respiratory function under anaesthesia
in cases of neonatal septicaemia or
maladjustment under critical care and
for older horses with respiratory and
intestinal abnormality, where acidosis or
alkalosis are suspected, prior to therapeutic
correction. Analyses are helpful, alongside
fluid and electrolyte balance assessments,
for enterotoxaemia and other criticallyill horses under intensive care and for
endurance horses and others performing in
hot, humid conditions, suffering exertional
exhaustion and/or heat stress. Samples must be taken in heparinised,
airtight syringes (preferably glass), and
transported on ice to the laboratory
within an hour or two of collection, so are
practically limited to immediate in-house
testing.
Endocrino logy
Pregnancy Tests
Serum gonadotrophins (eCG) may
be detected in mares where functional
endometrial cups are present. For accurate
results, serum samples should be collected
between 45 and 95 days since the last
date of mating. False negative results are
unusual inside this period, but can occur
in rare cases where eCG levels are below
test 'threshold'. False positive tests are more
common and may occur when early foetal
death has left residual functional cups. In
such cases the mare’s serum may remain
eCG positive for the functional life of the
cups, sometimes up to 100 days.
Oestrone sulphate may be detected in the
serum/plasma of mares over 120 days
pregnant. At that time, levels of >100
ng/ml are usually found (0-25 ng/ml in
non-pregnant mares). Most of the oestrone
sulphate peak originates from the foetal
gonads so this may be a useful test of
foetal viability as well as a pregnancy test. Levels fall during the last few weeks of
pregnancy.
Urinary oestrogens, of placental origin,
may be detected in mares after 150 days
of gestation. Urine samples are required. In some mares the required fluorescent
response may be unreliable to detect and
the serum oestrone sulphate test is now a
much more reliable test.
Progestagens
The analysis of plasma progesterone levels
is a useful guide to diagnosis and treatment
in the acyclic or irregularly cyclic mare. In
the non-pregnant mare, levels >2 ng/l (6.3
nmol/l) indicate functional luteal tissue
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Oestrone sulphate rig test
Normal male
Cryptorchid
‘False rig’
Oestrone sulphate
10-50 ng/ml
0.1-10 ng/ml
0-0.02 ng/ml
hCG/testosterone rig test
Testosterone stimulation test should be used for all donkeys and in horses under 3 years old.
Testosterone levels are measured in serum (clotted) or heparinised plasma samples taken before
and then 30-120 minutes after the intravenous injection of 6000 iu hCG.
Normal male
Cryptorchid
‘False rig’
Testosterone (1)
5-30 nmol/l
0.3-4.3 nmol/l
0.03-0.15 nmol/l
Testosterone (2)
5-30 nmol/l
1.0-12.9 nmol/l
0.05-0.19 nmol/l
and suggest that prostaglandin treatment
should induce luteolysis, providing that the
corpus luteum is more than 4 days old. In the pregnant mare, there is no proven
relationship between progesterone levels
and the integrity of pregnancy. Mares with
levels <2 ng/ml are unlikely to be pregnant. Most normally pregnant mares have levels
>4 ng/ml but there is considerable daily
and individual variation. The progesterone
assay is in no way an accurate pregnancy
test but may be helpful, or reassuring to
managers, in monitoring individual mares
with histories of repeated pregnancy failure.
Granulosa Cell Tumour (GCT)
The most common ovarian tumour in the
mare is the granulosa cell tumour (GCT). GCT’s produce steroid hormones which
34
can be detected in elevated quantities
in the peripheral blood. In cases where
the tumour has a significant theca cell
component (approximately 54% of cases),
serum testosterone is elevated. These
mares are more likely to be aggressive
and stallion like. GCT mares showing
anoestrus or persistent oestrus may have
normal testosterone concentrations. Inhibin
concentrations have been found to be
elevated above normal in approximately
87% of mares with GCT’s, and therefore
inhibin appears to be a more accurate
indicator of the presence of a GCT than
testosterone alone. We recommend
measurement of serum testosterone and
inhibin to support an ultrasonographic
diagnosis of GCT, or in cases in which
rectal palpation is undesirable. G u i d e
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Cryptorchidism
Oestrone sulphate assay may be used as
a cryptorchid or 'rig' test for horses (not
for donkeys) that are over 3 years old (see
table on left).
Thyroid function
Thyroid hormones, i.e. thyroxine (T4) and
tri-iodothyronine (T3) are measured in
equine serum samples. As diurnal rhythms
are involved, two samples should be
collected ideally early in the morning and
late in the afternoon. Low levels (T4 <7.7
nmol/l, T3 <0.48 nmol/l in adult horses)
may indicate hypothyroidism, which is
sometimes seen in overweight, lethargic
horses and ponies that may be prone to
laminitis. Hypothyroidism may respond to
T4 supplementation.
Pituitary function
The most common indication to
assess pituitary gland function is equine
Cushing’s syndrome, associated with
pituitary adenoma formation. Glucose
Classical cases of equine Cushing’s
syndrome are hyperglycaemic (>5.5
mmol/l) and glucosuric.
Cortisol
Basal serum cortisol levels are often
high (>240 nmol/l) in classical cases
(aged, hirsute, polydipsic, polyuric,
glucosuric). However, basal levels are
p a t h o l o g y
often within normal range for younger,
developing cases and so TRH stimulation
test, overnight dexamethasone suppression
test or combinations of such tests are
needed to help define pituitary normality
or abnormality.
Insulin
Serum insulin is sometimes a useful
‘screening’ test for equine Cushing’s
syndrome. Results <50 miu/ml are
considered normal in grazing or fasting
stabled horses and ponies. Samples should
not be collected from stabled horses within
two hours following a feed. Raised insulin
levels are seen in horses with peripheral
insulin resistance, which can occur in
cases of Equine Cushing’s syndrome. Many
classical cases have serum insulin levels of
>1000 miu/ml.
Overnight dexamethasone
suppression test
This test is based upon the fact that
dexamethasone administration suppresses
plasma cortisol in normal horses because of
feedback on ACTH release. A resting serum
sample is collected at 5 pm and 40 µg/kg
DXM (Azium, Gist-Brocades, 20 mg/500
kg horse) is injected im. A follow-up serum
sample is collected at 12 pm the following
day. Normal horses show a suppression of
serum cortisol levels to <10% of baseline
levels whereas cushingoid horses show no
significant suppression.
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TRH stimulation test
This is considered the safest of the dynamic
cortisol tests with the least risk of inducing
laminitis. Protocol is to collect a baseline
serum sample, inject 1 mg Thyroid Releasing
Hormone (TRH; Roche) iv and then collect
further serum samples at 15 and 60
minutes thereafter. Typical Cushing’s cases
show an increase in cortisol of greater
than 20% (up to 90%) above baseline
at 15 minutes, compared to a rise of
approximately 17% in the normal horse.
Cortisol concentrations return to baseline
values at 60 minutes in the normal horse,
but may remain at around 55% above
baseline in Cushing’s cases.
Combined DXM suppression,
TRH stimulation test
This test has been recently recommended,
although there is dispute about whether
it is more helpful than the overnight
DXM suppression described above. It is
indicated when the results of overnight
DXM suppression or TRH stimulation tests
are inconclusive. A protocol is to collect
a baseline serum sample and then inject
40g/kg DXM iv. A follow-up serum sample
is collected 3 hours later and then 1.0
mg TRH is injected iv. A follow-up serum
sample is collected 30 minutes later and
a final serum sample is collected 24 hours
after the DXM injection. Cushingoid horses
show suppression of serum cortisol level by
36
l a b o r a t o rie s
3 hours after DXM injection and return to
baseline by 30 minutes after TRH injection
and by 24 hours after DXM injection.
Conclusions are that while typical aged cases
of equine Cushing’s syndrome are easy to
diagnose, often on grounds of clinical signs,
hyperglycaemia and glucosuria, younger,
early and atypical cases are often extremely
difficult to confirm, with marginal clinical
abnormality and often conflicting insulin
and dynamic cortisol testing results. Basal
cortisol and insulin is a useful screening
test in horses grazing at pasture and in
stabled horses providing their samples
are not collected after a feed. The most
practical and probably reliable dynamic test
is currently considered to be the overnight
dexamethasone suppression test.
ACTH stimulation test
This test is primarily used to identify
adrenal insufficiency. In human critical care
it is recognised that many patients with
Systemic Inflammatory Response Syndrome
(SIRS) have adrenal insufficiency and may
benefit from appropriate treatment. Adrenal
insufficiency occurs commonly in premature
foals but its prevalence in adult horses is
unknown. The ACTH stimulation test is NOT
recommended for the diagnosis of equine
pituitary pars intermedia dysfunction where
the combined dexamethasone suppressionTRH stimulation test is currently considered
the gold standard.
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urine co llection & a naly sis
Urine analysis is useful to help detect renal
or bladder pathology and to investigate
cases of septic nephritis, cystitis or urethritis.
Mid-stream samples should be collected
without the use of diuretics (which alter
urine composition) into a sterile, empty
universal container. Beware of owners
collecting samples into used jam jars or
milk bottles before pouring the urine into
the provided universal container, resulting
in spurious glucosuria and bacterial culture
results. For fractional urinary electrolyte
and mineral clearance ratio measurements
(see page 30), paired urine (not following
diuretic administration) and serum samples
should be collected simultaneously (ideally)
or within one hour of each other.
Urine samples should be examined grossly
for colour and consistency, the presence
of blood (either fresh or changed), pus
or excessive crystalline material. Horse
urine is highly variable in colour from
near colourless to golden or brownish
and in its density, turbidity and mucinous
content. Specific gravity (1.008-1.040
in adult horses, 1.001-1.025 in foals)
should be measured with a refractometer.
Dipsticks are commonly used to measure
pH (normally 7.5-8.5 in adult horses,
5.5-8.0 in foals) and to detect other
abnormalities. Urine pH reflects diet and
horses grazing pasture will normally have
alkaline urine whereas those on a cerealbased performance-type diet will normally
have slightly acidic urine. Proteinuria
may occur with renal tubular pathology.
Glucosuria may be seen in classical
cushingoid horses and ponies. Haematuria
and sometimes haemoglobinuria may
occur following traumatic injury, or
with renal or cystic calculus formation. Haemoglobinuria may occur with
haemolytic conditions.Myoglobinuria is
seen with myopathies. Bilirubinuria may
occur with choleliths or other causes of
bile duct obstruction. Ketonuria is rarely
seen in horses. Microscopic examinations
should be used to detect casts (protein
and cellular masses), which suggest renal
tubular pathology, leucocytes, which
suggest inflammation/infection, bacteria,
which if seen following Gram’s stain in
association with leucocytes may indicate
infection and erythrocytes, which indicate
haemorrhage and crystals. Horse urine is
fundamentally a supersaturated solution of
calcium carbonate and will normally contain
variable amounts of predominantly calcium
carbonate crystals. Urolithiasis cases usually
have a degree of proteinuria and haematuria
and may exhibit dysuria. Large amounts
of sabulous material are not necessarily
an indication of abnormality. Further
investigations include bladder and kidney
palpation, ultrasound scan and cystoscopic
examinations, looking for sabulous (bladder)
or discrete calculus formation.
Fractional urinary electrolyte and mineral
clearance ratios (see p30) are significantly
increased in horses with nephropathy, in
particular with renal tubular malfunction.
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parasito logy
Faeces Collection
Faecal analysis is helpful in providing worm
egg counts to help monitor parasite control
programmes and to investigate cases or
diarrhoea and septic enterocolitis. Freshly
produced or rectal faecal samples should
be collected into an inverted clean rectal
sleeve so that environmental contamination
and alteration is minimised and there is
no doubt about the identity of the horse
that produced the sample. Fluid diarrhoea
samples should be submitted in sterile
universal containers and on sterile swabs
immersed in Amies’ charcoal transport
medium. It is often difficult to collect
diarrhoea samples from foals but digital
stimulation of the rectum sometimes
precipitates production of a sample.
Faecal Worm Egg Counts
These remain the basis of equine intestinal
parasitic surveillance and monitoring of
worm control programmes. They are not
always a reliable means of assessing an
individual horse, for which haematological
and serum protein (albumin and protein
electrophoresis) investigations are more
reliable (see page 19). 38
Testing methods vary between laboratories. Our worm egg counts are performed using
the ‘Ovassay’ method, a flotation method
which is particularly suitable for use in
horse samples as it is extremely sensitive
at detecting low numbers of strongyle
and related species eggs. Ascarid and
Strongyloides spp. eggs are also detected.
We believe that other methods (Stoll and
McMaster) are more suitable for counting
worm eggs in samples where high counts
are expected (e.g. farm animals) as these
methods involve dilution rather than
concentration of the eggs into the counting
area. Our experience is that well-managed
horses under good endoparasite control
regimes consistently have zero strongyle
eggs/g of faeces, using the Ovassay
technique. For example, the presence, of
egg laying strongyles in the intestinal lumen
represents the final stage of the protracted
migration of the developing larvae through
the horse’s tissues. Using this method, a
positive worm egg count of any magnitude
in the faeces is a significant finding and
indicates that the horse needs anthelmintic
treatment, the parasite control programme
requires review and the horse would benefit
from a more in-depth haematological and
serum protein appraisal.
Tapeworm segments may sometimes be
seen in the faeces by gross examination
and are often seen in the caecal content
of tapeworm-related intussusception cases
at surgical correction. A serological (ELISA)
test is available for detecting tapeworm
infestations in horses. Titres of <0.2, 0.20.6 and >0.6 are considered negative or
low-intensity, moderate and high intensity
infestations, respectively.
Faecal Lungworm Larval Counts
Dictyocaulus arnfieldi larvae may be
detected in fresh rectal-collected faeces
of infested horses using the modified
Baermann funnel gravitation method. A
severe eosinophilic bronchitis may be
detected by cytological examination of
tracheal washes (see page 44).
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micro biology
Bacteriology
Swabs from any site should be taken into
Amies charcoal transport medium before
transport to the laboratory where they may
be cultured under aerobic, microaerophilic
and/or anaerobic conditions. Where potential pathogens are isolated,
antibiotic sensitivity tests should be
performed. In acute septicaemias, before
antibiotic treatment has been started,
jugular venous blood samples may be
taken, using sterile techniques, and
inoculated into blood culture media, before
transport to the laboratory.
All genital swabs should be cultured,
unless specifically requested otherwise,
aerobically and microaerophilically. Aerobic results are available after 24/48
hours culture. Contagious Equine Metritis
(Taylorella equigenitalis) microaerophilic
culture results are available after 7
days incubation, but most specifically
experienced laboratories are able to
culture positive isolates by 3/4 days.
Klebsiella pneumoniae and Pseudomonas
aeruginosa isolates should be confirmed
by biochemical tests and K. pneumoniae
isolates capsule typed for the recognised
K1, K2 or K5 types using the 'Quellung'
or counter current immunoelectrophoretic
methods. Anaerobic culture may be performed where
indicated, using standard techniques.
Endometrial swabs should be accompanied
by a concurrently sampled endometrial
smear to aid the interpretation of culture
results (see page 46).
In addition to routine aerobic culture,
special methods should be used routinely
to screen rectal swabs and biopsies (in
transport media) and faeces for Salmonella
spp. and Campylobacter spp. pharyngeal
swabs,
Nasal
and
submandibular and parotid abscess
swabs should be routinely screened
for Streptococcus equi and, in foals,
Rhodococcus equi. Tracheal wash and bronchoalveolar
lavage (BAL) samples, when submitted
for bacteriological examinations, should
be routinely screened for Streptococcus
pneumoniae, Bordetella bronchiseptica
and Pasteurella spp. Blood culture medium should be used for,
in addition to blood samples, other body
fluid samples, e.g. peritoneal and pleural
fluids and especially for synovial fluid and
cerebrospinal fluid (CSF) from which it is
often difficult to isolate pathogens.
Skin scrapings
Skin scraping samples are collected from
horses and ponies for the confirmation
of suspected dermatophyte infections or
ectoparasite infestations. Samples should
be collected from fresh skin lesions, from
the edges of the lesions if extensive, using
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a fresh sterile scalpel blade. The hairs
and the scraped skin layers, down to
haemorrhage, should be collected into a
sterile universal container and submitted
for laboratory examination without delay.
The hairs and scraped skin layers should
be incubated for 15 minutes in warm
40% potassium hydroxide (KOH) and then
examined for clear ringworm spores in
broken hair shafts and free whole or
fragmented ectoparasitic mites, including
Sarcoptes, Psoroptes, Chorioptes and
Demodex spp. (differentiated on the basis
of their anatomical appearance).
If negative for dermatophyte spores after
KOH incubation, skin scrapings are
incubated overnight in blue/black ink
and then examined for blue/black stained
spores in clear broken hairs.
Skin scraping samples should be incubated
for bacterial and fungal growth. The latter
(taking up to 3 weeks) will differentiate the
different ringworm-causing dermatophytes,
including Trichophyton and Microsporum
spp.
Microscopic and fungal culture results for
dermatophytes do not always correlate. Positive microscopic findings but negative
culture results may suggest that the spores
were not viable at the time of scraping. Negative microscopic findings but positive
culture results may suggest that too few
spores were present in the sample to be
detected in spite of careful examination.
40
l a b o r a t o rie s
If Dermatophilus congolensis (‘rain scald’
or ‘mud fever’) infection is suspected on
clinical grounds, moist lesions should be
collected and submitted for impression
smear stained with methylene blue to look
for characteristic chains (‘piles of pennies’)
of flattened spores, sometimes in branched
conformation.
clostridial toxin tests
ELISAs to detect the toxins produced
by pathogenic forms of Clostridium
perfringens and Clostridium difficile are
indicated in horses and foals with acute
diarrhoea, particularly where the problem
may be associated with antimicrobial
administration. Faecal samples should be
taken before the drug metronidazole is
given and should be chilled if delivery to
the laboratory is likely to take more than
an hour or two.
Virology
Fluid diarrhoea samples may be examined
by latex particle agglutination for the
presence of Rotavirus antigen.
Specialist virology laboratory facilities and
expertise are required for testing for the
following equine viruses:Equine Influenza: paired serum samples
(acute and convalescent phases, collected
14 days apart) are examined for signs
of seroconversion (a four-fold or greater
rise in titres between the acute and
convalescent samples). Nasal discharge or
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cli n ic a l
nasopharyngeal swabs collected during the
acute phase into viral transport medium are
cultured specifically for the virus. A direct
ELISA test is available for the rapid detection
of the virus in acute-phase nasal discharge
or nasopharyngeal swab samples.
Equine Herpesviruses (EHV-1, EHV-3,
EHV-4): paired serum samples (acute
and convalescent phases, collected 14
days apart) are examined for signs of
seroconversion (significant rise in titres
between the acute and convalescent
samples).
Nasal
discharge
or
nasopharyngeal swabs collected during the
acute phase into viral transport medium are
cultured specifically for EHV-1 and EHV-4. Aborted foetal, placental and neurological
tissues may be examined for EHV-1 and
EHV-4 DNA by specific polymerase chain
reaction (PCR) test.
p a t h o l o g y
Equine Viral Arteritis (EVA): paired serum
samples (acute and convalescent phases,
collected 14 days apart) are examined
for signs of seroconversion (significant
rise in titres between the acute and
convalescent samples). Nasal discharge or
nasopharyngeal swabs collected during the
acute phase into viral transport medium
are cultured specifically for the virus.
Aborted foetal and placental tissues may
be examined for the virus DNA by specific
PCR test.
For the screening of equine aborted foeti
for EHV and EVA infections, please see
postmortem examinations (see page 48).
NOTES
41
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cyto logy
Diagnostic cytological examinations are valuable diagnostic
tools following the collection of appropriate and good quality
samples. Safe, reliable and appropriate techniques should be
used to collect either fluid or smear samples.
Fluid samples
These should, in general, be submitted in
sequestrene (EDTA) for a nucleated cell
count, and fixed with a suitable fixative
(e.g. cytospin fixation fluid) for specific
cytological processing. Another undiluted
and unfixed sample should be submitted
in a sterile container or on a sterile swab
in transport medium or ideally in blood
culture medium (particularly for synovial
fluid samples) for concurrent bacterial
culture.
Fluid samples with low total nucleated
cell counts (<5 x 109/l) require careful
cell counting for accuracy and will greatly
benefit from cytocentrifugation, producing a
concentrated monolayer smear for staining
and examination to allow a confident
cytopathological appraisal. Modified
Papanicolau (e.g. Pollack’s rapid trichrome
stain) or Romanowski-type (e.g. May
Grunwald Giemsa or Wright’s stains or
‘Testsimplets’ pre-stained slides or ‘DiffQuik’) stains are most commonly used
for cytopathological examinations. Each
stain has its advantages and disadvantages
and choice is usually determined by the
cytopathologist’s personal preference. We
use a modified Papanicolau stain, which
42
produces particularly good cytological
detail. For this method, samples need
to be wet-fixed (as opposed to air dried)
and an aliquot of the sample should be
submitted in cytopreservative (e.g. cytospin
fixation fluid).
In general terms, total nucleated cell counts
rise when leucocytes move through into the
cavity fluid in response to inflammation,
with or without the stimulus of infection, and
when reactive or degenerate cavity lining
cells proliferate and exfoliate. Non-septic
inflammation may produce a predominantly
reactive cavity lining cell response and
non-degenerate
polymorphonuclear
leucocytes whereas infection usually
produces degenerate and ‘toxic’-looking
polymorphonuclear leucocytes (karyorrhexis
and karyolysis) and active macrophages.
Chronic inflammation may produce a
significant mononuclear cell (lymphocytes/
histiocytes, i.e. ‘round’ cells) response. If
ulcerative neoplasia is present in the cavity
then neoplastic cells may be exfoliated
and recognised in collected fluid samples.
The most common neoplasm encountered
in equine cytopathological examinations is
lymphosarcoma/lymphoma.
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cli n ic a l
Peritoneal fluid
Analysis of peritoneal fluid is particularly
useful as a diagnostic aid in cases of colic,
weight loss and other suspected abdominal
disease. It may be of particular value in
helping to make the decision for surgical
intervention. To perform a peritoneal tap, the skin over
the site of puncture should be clipped
and prepared as for surgical intervention. With the horse restrained in the standing
position, a 19 gauge, 1.5 or 2.0 inch
needle or following local anaesthesia and a
stab incision, a 7.5 cm. blunt teat cannula
(operator preference) is carefully advanced
through the skin at the lowest part of the
abdomen and then through the linea alba. If fluid is not immediately forthcoming, the
needle may be rotated or the tap may be
repeated at other sites.
In foals, prior ultrasound scan examination
of the abdomen is recommended to
help visualise abnormalities and prevent
inadvertent penetration of the intestine. Examination of the ventral midline will help
to locate the spleen and to find a ‘pocket’ of
peritoneal fluid to guide productive needle
puncture. Similarly, if fluid is not obtained
in adult horses using the blind technique
described above, ultrasonic guidance may
be helpful.
A turbid and homogeneously blood stained
sample may indicate abdominal vascular
embarrassment. A white, turbid fluid may
suggest peritonitis. A brown, foul smelling
p a t h o l o g y
fluid may indicate intestinal rupture or an
intestinal tap. A thick and heavily blood
stained sample suggests a splenic tap. Total nucleated cell counts <5 x 109/l
suggest the presence of peritonitis or an
intestinal or peritoneal lesion, which may
warrant surgical investigation. Cytological
examination may suggest acute or chronic
infection, inflammation or neoplasia.
Pleural fluid
Pleural fluid analysis may help with the
diagnosis of pleuritis or pleuropneumonia. To perform a pleural tap, ultrasound scan
examination is recommended to confirm
the presence of pleural effusion prior to
tap (very little, if any, fluid is seen in a
normal horse), to determine its nature and
severity (fibrinous, particulate fluid with
adhesion formation is often seen in cases
of pleuropneumonia) and to locate pockets
of fluid for productive sampling. The skin
over the site of puncture should be clipped
and prepared as for surgical intervention. A 7.5 cm. blunt teat cannula is inserted,
with a 35 ml. syringe attached to prevent
aspiration of air into the pleura, through
a small skin incision, routinely between
the 6th or 7th intercostal space, 15 cm.
dorsal to the olecranon, or where guided by
ultrasound scan for specific fluid pockets. Pleural fluid is collected by suction. Total
nucleated cell counts <5 x 109/l suggest
the presence of pleuritis. Cytological
examination may reveal neoplasia, e.g.
most commonly lymphosarcoma.
43
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Synovial fluid
Analysis of Synovial Fluid is useful for the
diagnosis of reactive or septic arthritis in
horses after injury, when a joint becomes
distended, with or without lameness, and to
differentiate septic ('joint-ill') from traumatic
arthritis in foals. To perform a joint tap, septic techniques
for needle puncture should be used. The
joint to be sampled and its anatomical
relationships govern the site of puncture
and to a degree the techniques used. Total
nucleated cell counts <5 x 109/l suggest an
inflammatory lesion and cytopathological
examination allows the differentiation of
acute from chronic and reactive (nonseptic) from septic inflammatory changes.
Septic arthritis usually produces cell counts
>10 x 109/l, with degenerative and toxic
cytopathological changes.
Tracheal washes or aspirations
These samples can be extremely useful
in confirming and characterising airway
inflammation (leucocytes) in cases of
septic tracheobronchitis and in reactive and
obstructive small airway disease. Samples can be collected via a suitablelength sterile endoscope. The endoscope
is passed through the pharynx, larynx and
into the trachea. A long sterile polyethylene
tube is passed down the instrument
channel and accumulated secretions may
be aspirated directly. Alternatively, and
more usually, 50 ml sterile saline may be
44
l a b o r a t o rie s
injected as quickly as possible and then
aspirated while withdrawing the tube. If
a suitable endoscope is not available, a
sterile polyethylene tube may be passed
down a trochar inserted surgically between
two tracheal rings at the mid lower third
of the cervical trachea. This is clearly a
more invasive technique that may lead
to local wound complications but has the
advantage that there is less likelihood
of contamination of the sample with
commensal bacteria from the oropharynx. Trans-tracheal aspirates, collected by this
method, are particularly useful in cases
of pleuropneumonia where causative
organisms must be specifically identified to
direct antimicrobial therapy.
Bronchoalveolar lavage
(BAL) samples
BAL samples can be helpful in the
diagnosis of small airway disease. One
must remember that the technique samples
a focal area only and does not help with
the diagnosis of tracheobronchitis. It is
therefore more commonly used in older
horses for the characterisation of reactive
airway obstruction.
Cytological examinations of tracheal
wash or BAL samples may help in the
characterisation of mucous production and
acute and chronic, septic and reactive
(non-septic) inflammatory responses. The
demonstration of significant numbers of
eosinophils in association with signs of septic
bronchitis/bronchiolitis may be a useful
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diagnostic aid for lungworm (Dictyocaulus
arnfieldi) infestation, in horses and ponies
coughing at pasture. Bacterial culture can
be useful in identifying specific pathogens
and guiding antimicrobial therapy.
Cerebrospinal (CSF) samples
CSF samples are most commonly collected
in horses showing neurological signs
for the diagnosis and differentiation of
meningitis or traumatic injury. In horses
imported from countries where equine
protozoal myeloencephalitis occurs and
who develop neurological signs, CSF
analysis is indicated.
For a CSF tap, samples are collected from
the atlanto-occipital space with the horse
restrained in lateral recumbency with the
poll flexed. In foals it may be possible to
perform this under heavy sedation whereas
in adults, general anaesthesia is mandatory.
The skin is clipped and prepared as if for
surgical intervention and a bleb of local
anaesthetic is placed in the midline of the
dorsal neck at a level defined by a line
joining the cranial borders or the atlas,
which may be clearly palpated. A sterile
18 gauge 2 inch needle is then inserted
through the skin at 90° and advanced
until it ‘pops’ through the meninges and
CSF drips or pours from the needle into a
sterile container or is aspirated into a sterile
syringe.
Alternatively, in adult horses, CSF may
be collected via the lumbosacral (LS)
cli n ic a l
p a t h o l o g y
space, by lumbosacral tap. The horse is
restrained, sedated, in stocks. The skin
between the tuber coxae is clipped and
prepared as if for surgical intervention. A
sizeable (3-4 ml) bleb of local anaesthetic
is placed in and under the skin in the
midline at a line bisecting the caudal
borders of the tuber coxae. With the
horse standing ’square’ with weight evenly
distributed on both hind legs, a sterile 18
gauge 6 inch spinal needle with stylette in
place is then inserted though the skin in
the midline at the line bisecting the tuber
coxae and down through the palpable
depression just caudal to the sixth lumbar
spinous process. The horse will often flinch
when the subarachnoid space is penetrated
and this is an indication to start aspiration
into a sterile syringe for a fluid sample.
Gross examination of CSF reveals a clear
almost colourless fluid in normality and
a turbid and/or bloodstained fluid with
meningitis or following traumatic injury. Laboratory examinations of CSF need to be
able to measure very low total nucleated
cell counts (<0.2 x 109/l in normality)
accurately and cytocentrifuge-prepared
smears are essential to provide adequate
numbers of cells with which to make
a confident cytopathological appraisal.
In horses from endemic areas that are
suspected of protozoal myeloencephalitis,
CSF samples may be submitted to specialist
laboratories for serological and DNA (PCR)
testing.
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Bone marrow aspirates
These are not commonly collected from
horses - only in cases that may have severe,
undefined anaemia, severe persistent
leucopenia or thrombo-cytopenia and in
some cases of leukaemia.
Samples are most commonly collected
from the wing of the ilium, the ribs or the
sternum. Sternal tap is recommended
because it is most reliably productive.
The ventral midline, under the sternum
is clipped and prepared as if for surgical
intervention. Local anaesthetic is infused
into and under the skin of the ventral
midline, just caudal to the olecranon, with
the horse standing normally, restrained,
sedated, in stocks. A bone marrow
collection needle or an 18 gauge 3.5 inch
spinal needle is introduced through a stab
incision in the skin upwards to contact
the sternum. The needle is then rotated
with upward pressure until it enters the
sternum, the stylette is removed and firm
suction is used to aspirate a sample into a
sterile 10 or 20 ml syringe, pre-treated with
a few drops of 15% tripotassium EDTA to
prevent clotting.
The sample is transferred to EDTA tubes
and labelled for laboratory examination,
without delay. If delay is inevitable, smears
should be made and fixed, to be sent
alongside the wet samples, in case they
are needed.
46
The cytopathological examination of bone
marrow smears requires specific experience.
Normal and abnormal cell types are classified
l a b o r a t o rie s
and their relative proportions assessed. The
normal equine myeloid:erythroid (M:E) ratio
should be 0.5-1.5.
Semen samples
Semen samples should be collected into
an artificial vagina to allow a meaningful
interpretation. In addition to a full case
history, details of methods of collection, the
colour, consistency, volume and motility of
the ejaculate should be submitted with the
sample. An undiluted semen sample should
be sent in a sterile container for sperm
density and bacteriological examinations,
and a sample diluted immediately 1:1 with
formol citrate solution for sperm live:dead
and morphology examinations.
Smear Samples
Smear samples should, in general, be
submitted already rolled onto a gelatincoated slide and fixed (‘Smear-fix’, carbowax
or even hair spray) for specific cytological
processing. Another undiluted and unfixed
sample should be submitted in a sterile
container or on a sterile swab in transport
medium for concurrent bacterial culture.
Endometrial smears
Endometrial Smears are simple and quick to
perform and provide a much more accurate
and direct test for the diagnosis of acute
endometritis in mares, pre-coitus than with
swab examinations alone. When used in
conjunction with endometrial swabs for
bacteriological examinations, results are
infinitely more meaningful, interpretable
and therefore valuable. The presence of
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endometrial epithelial cells is used as a
test of smear quality and the presence or
absence of polymorphonuclear leucocytes
is used as the diagnostic test for acute
endometritis. Smears may be collected during oestrus
by a variety of methods, but we favour
a simple non-guarded technique. An
extended, sterile, large tipped swab is
passed via a sterile speculum, through
the relaxed cervix and rotated onto the
endometrial lining. The swab is withdrawn
and should be rolled onto gelatine-coated
cli n ic a l
p a t h o l o g y
slides immediately and fixed with 'Smearfix', carbowax or even hair spray prior to
transport to the laboratory for staining.
Excellent results have been obtained by
rolling smears onto 'Testsimplets' (Boehringer
Mannheim UK Ltd.) pre-stained slides,
then after two to three minutes at room
temperature, washing off the background
blue colour, drying and cover slipping. This
technique provides an excellent permanent
smear sample for immediate reading and/or
for sending for a second opinion.
Hi sto logy
Using our state-of-the-art microwave
processing system, same day results can be
provided for most fixed tissue specimens.
In general terms, equine lesions are best
sampled, if possible and appropriate,
by total removal and submission
for histopathological processing and
examination. If total removal is not possible,
samples of representative size and location
should be collected by wedge resection.
Fine needle aspirates should only be
collected if it is not possible to collect
larger samples. Experience suggests that
they often result in a traumatised lesion
without a conclusive diagnosis and with
a recommendation for the collection of a
larger and more representative sample,
delaying resolution. Total lesion removal is
always preferable, if possible.
Removed lesions or biopsies should be
placed immediately into an adequate
volume (10 x sample volume) of 10%
formol saline. Reproductive tissues are
best fixed in Bouin’s fluid because of their
higher water content. Large lesions should
be sectioned before submersion to allow
adequate penetration of fixative. Sectioning
should be performed with a thought to how
the sample will need to be trimmed and
orientated for histopathological processing
to allow the pathologist to make a complete
and meaningful appraisal. When sampling
organs and tissues, we recommend that
samples are taken from adjacent, grossly
normal sites as well as from lesional
sites. Notes of your own postmortem
examination findings should be sent to aid
histopathological interpretations. Samples
should be carefully packed for transport to
avoid leakages and breakages.
47
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necro psy (p os tmorte m) ex ami nat i o ns
Full necropsy examinations should be
performed on all adult and younger horses
who die unexpectedly, those who may pose
a risk to in-contact horses, or for whom
confirmation of pathology is required,
and those who are the subject of an
insurance claim. Ideally, the examination
should be performed, without delay, under
conditions of adequate facilities, by suitably
qualified and experienced pathologists,
so that the end result is satisfactory in
terms of answering the questions posed. Nevertheless, for a variety of reasons, it
may not always be possible for dead horses
to be transported to a specialised equine
pathology facility and examinations may
need to be performed ‘in the field’. Faced
with this need, those responsible should
consider what conditions are required
to enable a productive examination to
be performed. Also, waste and carcase
disposal, prevention of environmental
contamination and spread of infectious
agents to humans and other animals must
be considered. Occasionally, it may be
possible to refer part of a carcase for
specific necropsy investigations, e.g. a foot
or a leg only for orthopaedic examinations
or the head and neck to thoracic vertebra
3 in cases of ataxia for cervical cord
compression examination.
48
Adult horses
Where full post-mortem facilities, including
hoists and tables are not available,
experience suggests that the horse should
be positioned on the ground in right lateral
recumbency. If an incline is available, the
horse’s back should be positioned up the
hill. The horse should be identified and
an external examination performed and
findings recorded.
The first cut is made deeply under the left
axilla to lay the left front leg upward and
flat back over the horse’s withers. The
second cut is made deeply under the left
groin, aiming caudally towards the anus,
through the left hip joint, to lay the left hind
leg upwards and flat back over the horse’s
pelvis. The abdomen is then opened along
the midline, from xiphisternum to pubis and
the left abdominal wall is laid back over the
horse’s back by cutting up adjacent to the
last rib and up into the groin. The caecum,
large colon and small intestines are then
removed from the abdomen while looking
for signs of displacement or gross pathology.
The small intestines are removed carefully,
examining the cranial mesenteric root for
verminous pathology and the pancreas,
and taking care not to remove the kidneys
and adrenal glands from their attachments
below the spine. The spleen, stomach and
liver are then removed and examined. The
kidneys are then carefully removed for
examination to leave the adrenal glands
attached either side of the aorta. A block
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of tissue is then removed to include both
adrenal glands and the adjacent aorta,
for overnight fixation, prior to dissecting
out the coeliaco-mesenteric sympathetic
nerve ganglia, which lie between the
adrenal glands and the aorta, but which
are less easy to find in fresh condition.
The bladder and internal genitalia are then
removed and examined. The diaphragm
is then examined and opened to reveal the
thoracic viscera. The ribs are separated
through their costochondral junctions,
the intercostal muscles are cut and the
ribs are broken back over the horse’s
back. The ‘pluck’ including the trachea,
heart and lungs are then removed for
examination. The thymus is examined and
removed in immature horses. The head
is removed through the atlanto-occipital
joint and ideally sectioned longitudinally
with a band saw. The brain, meninges,
guttural pouches, paranasal sinuses, teeth,
pharynx and larynx are then examined
thoroughly. The limb joints are opened
for examination of the synovial fluid and
surfaces. If indicated by the clinical signs,
the neck and back vertebrae are dissected
out and opened for examination of their
articulations and the spinal cord. It is clear
that a satisfactory examination of the head
and spine of an adult horse is difficult to
achieve under field conditions.
Samples for bacterial or viral examination
should be collected from abscesses, areas
of inflammation, body cavities or seared
cli n ic a l
p a t h o l o g y
viscera, using sterile swabs and placed
without delay into Amies’ charcoal or
specific viral transport media.
Fluid samples from abscesses, cysts or
body cavity fluids should be aspirated
with a sterile syringe and then submitted
in sequestrene (EDTA) for a nucleated cell
count, and fixed with a suitable fixative
(e.g. cytospin fixation fluid) for specific
cytological processing. Another undiluted
and unfixed sample should be submitted
in a sterile container or on a sterile swab
in transport medium or ideally into blood
culture medium for concurrent bacterial
culture.
Samples for histopathological processing
and examination should be carefully
selected to provide thin and small, but
representative tissue wedges, and totally
immersed in 10% formol saline. Large
thick samples fail to fix adequately and
histopathological examinations are then
unnecessarily complicated by autolytic
changes, abused tissues may be ruined by
artefactual damage and unrepresentative
samples may not include the primary
pathological changes.
It is often wise to retain appropriate samples,
e.g. gastric and intestinal content, urine and
cubes of liver and kidney, carefully labelled,
frozen, in case toxicological studies are
required at a later date.
49
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Coelioacomesenteric sympathetic
nerve ganglion (CMSNG) sampling
These tissues are specifically required for
the diagnosis of equine dysautonomia
(‘grass sickness’). In addition to performing a full necropsy
examination (see above) to investigate
other pathological conditions, the CMSNG
require a specific approach to sampling. With the horse in right lateral recumbency
and the left front and left hind legs cut
back, the abdomen is opened along the
midline, from xiphisternum to pubis and
the left abdominal wall is laid back over the
horse’s back. The caecum, large colon and
small intestines are then removed from the
abdomen. The small intestines are removed
carefully, sectioning the cranial mesenteric
root and taking care not to remove the
kidneys and adrenal glands from their
attachments below the spine. The spleen,
stomach and liver are then removed. The
kidneys are then removed carefully for
examination to leave the adrenal glands
attached either side of the aorta. A block
of tissue is then removed to include both
adrenal glands and the adjacent aorta, for
overnight fixation in an adequate volume of
10% formol saline. The following day, the tissues are examined
and oriented to identify the two adrenal
glands on the section of aorta. The adrenal
glands are then carefully dissected off
the aorta and the CMSNG are identified,
removed and sectioned transversely and
longitudinally for further fixation prior to
50
l a b o r a t o rie s
processing. When fixed they appear greycoloured, solid and clearly neurological,
rather than vascular (tubular on transverse
section) or lymph node-like. They are
much less easy to find and identify in fresh
unfixed condition.
After further fixation and processing, the
ganglia are examined for the specific
neuronal histopathological degenerative
changes that appear characteristic of
equine dysautonomia.
Neonatal Foals, Foeti and
Placentae
These should be examined to determine
the cause of foetal death and abortion,
specifically looking for signs of EHV or EVA
infections, important infectious causes of
potential epidemic abortion in mares, as
specifically recommended by the Horserace
Betting Levy Board’s Code of Practice.
The carcase and placental membranes
should be examined together to find signs
of foetal/foal, placental and maternal
pathology. The foetus or foal may be
opened in right lateral recumbency in a
manner similar to that described above
for adult horses. Although they may not
be clearly seen in all cases and although
combinations differ in different cases, the
important gross pathological abnormalities
of EHV infection to look for are:-
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cli n ic a l
1.A freshly aborted foetus enclosed in
its placental membranes, suggesting
‘explosive’ abortion or a septicaemic
neonate.
2. Jaundiced hooves, mucous membranes
and/or subcutaneous tissues.
3.Excess, jaundiced peritoneal, pleural
and/or pericardial fluids.
4.Jaundiced visceral serosae and gelatinous
jaundiced peri-renal fat.
5. Pleural oedema and pneumonia.
6.Multiple, pale, pinpoint foci visible on
the liver capsule.
The foetus should be weighed, the
crown rump length and the umbilical
cord length measured and recorded. The
placenta should be spread out, checked for
completeness and abnormalities recorded. Samples for laboratory investigation should
include:1.Small but representative slices of liver,
lung, thymus, spleen, adrenal gland and
chorioallantois (horns and posterior pole),
plus any other interesting abnormality,
in carefully packed leak-proof sealed
containers, in an adequate volume
of 10% formol saline for histological
examinations.
p a t h o l o g y
3.Swab samples of peritoneal fluid, liver,
lung, heart blood, gastric contents,
the cervical pole of the chorioallantois
and areas of possible placentitis in
Amies charcoal transport medium for
bacteriological examinations.
4.We recommend that small fresh foetal
liver and lung samples are stored at –
20°C (deep-freeze), in case viral isolation
studies are required at a later stage.
In addition, the carcase should be thoroughly
examined for other abnormalities in case an
infectious cause is not involved. Maternal
uterine and placental incompetence often
results in a grossly impoverished foetus.
Umbilical cord vascular embarrassment (an
important cause of equine foetal death and
abortion, often associated with excessive,
i.e. more than 90 cm, cord length) may
be grossly obvious and, if significant, will
be accompanied by signs of foetal vascular
engorgement, haemorrhagic peritoneal and
pleural fluids, congested and autolysed
viscera and meconium staining/foetal
diarrhoea.
Whole carcases and whole placentae to be
referred for necropsy investigations should
be sent to a laboratory that is specifically
experienced with equine neonatal
pathology.
2.Small (0.5 cm) cubes of liver,
lung, thymus, spleen and pieces of
chorioallantois in viral transport medium
for EHV and EVA DNA (PCR) tests.
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l a b o r a t o rie s
bio psy samplin g
Skin Biopsy
These are best obtained by full-thickness
wedge incision. While skin punch biopsy
techniques may be simpler to obtain,
they are seldom as rewarding to examine. Tissues obtained should be fixed
immediately in 10% formol saline.
Lump biopsy
Biopsy is recommended for masses that
appear in or under the skin and sometimes
for deeper masses that may be palpated
or imaged by ultrasound or laparoscopic
examination. If a total lesion resection is
not possible, as is the case for many skin
and subcutaneous masses, representative
biopsies are best obtained by full-thickness
wedge incision, and fixed immediately in
10% formol saline.
Liver Biopsy
Liver biopsy is recommended where serum
biochemical examinations have suggested
hepatic pathology. With the horse restrained
standing, ideally in stocks, the liver on the
right side of the horse is examined with
ultrasound to determine the ideal site
for puncture and to detect signs of gross
pathology, e.g. choleliths or Echinococcus
spp. cysts. Biopsies are best obtained with a 6' 'Trucut' biopsy needle inserted between the
14th and 15th ribs, on the right side of the
horse, along a straight line drawn between
the point of the hip and the shoulder,
52
or elsewhere, if suggested by ultrasound
examination. Specific lesion biopsies are
best obtained by ultrasound guidance. The
tissue sample should be carefully removed
from the needle and fixed in 10% formol
saline. The needle channel should then be
carefully swabbed for bacterial culture.
Lung Biopsy
Lung biopsy may be indicated where there
is clear ultrasonic evidence of pulmonary
pathology. As biopsy of a lung abscess
or focus of infection is contraindicated,
evidence should suggest neoplasia or
focal non-septic pathology before biopsy
is attempted. Pulmonary neoplasia is very
rare in horses so lung biopsy is seldom
indicated.
Needles specifically designed for lung
biopsy should be used and the specific
lesion to be biopsied should be imaged by
ultrasound scan examination. The overlying
intercostal skin site identified, infiltrated
with local anaesthetic and clipped and
prepared as for surgical intervention. With
the transducer coupled in a sterile surgical
glove and sterile coupling gel on its surface,
the lung lesion is imaged and a lung biopsy
device is introduced through the skin and
into the lesion to collect the biopsy.
The tissue sample is fixed in 10% formol
saline without delay and the needle channel
may, if required, be swabbed immediately
for bacterial culture.
G u i d e
t o
e q u i n e
cli n ic a l
Kidney Biopsy
Renal biopsy may be indicated where
there is clinicopathological and ultrasonic
evidence of kidney pathology. The
technique is not without risk of injury
to the horse and therefore should only
be contemplated if clearly indicated and
should only be performed with great care,
using an ultrasound-guided technique.
The right kidney is more easily accessible. Ultrasound scan examination is used to
identify a suitable site, free from large blood
vessels, in the posterior poles. The horse
should be restrained, sedated in stocks. The skin over the kidney is clipped for
imaging to identify the site for penetration,
local anaesthesia is induced and the skin is
then prepared as for surgical intervention. With the transducer coupled in a sterile
surgical glove and sterile coupling gel on
its surface, the kidney is imaged to find the
ideal site for puncture. The biopsy needle is
then introduced through the skin and into
the renal parenchyma, sampling specific
pathological features if visible.
If biopsy of the left kidney is to be
attempted, it is helpful for an assistant to
palpate the kidney per rectum in order to
stabilise it against the body wall, facilitating
the ultrasound-guided biopsy technique.
The tissue sample is fixed in 10% formol
saline without delay and the needle channel
may be swabbed immediately for bacterial
culture.
p a t h o l o g y
The horse should be stable-rested for at
least 48 hours after biopsy and should be
monitored for signs of ill health, particularly
associated with renal haemorrhage.
Endometrial Biopsy
Endometrial biopsies are indicated for the
routine investigation of barren mares and
for the investigation of specific endometrial
pathology. Ideally, they are more easily
obtained and interpreted when the mare
is in mid-dioestrus, but samples may be
safely obtained at any stage of the oestrous
cycle from any non-pregnant mare. Biopsies are obtained with special forceps
(Kruuse UK or Rocket of London Ltd.),
via the vagina and cervix. The mare is
restrained as for routine gynaecological
examinations with her tail bandaged,
rectum evacuated of faeces, tail bandaged
and perineum hygienically prepared. The
mare is re-confirmed not pregnant. The
sterile biopsy forceps are introduced into
the mare’s vagina with a gloved hand, the
cervix is identified (making sure that the
urethral opening has not been accidentally
entered) and the index finger is placed
through the mare’s cervix into the uterine
body. The forceps are then advanced along
the index finger, as a guide, through the
cervix and into the uterine body. The finger
is then ‘hooked’ in the cervix, which may
then be retracted caudally. This straightens
the cervix and the uterine body, allows the
biopsy forceps to fully enter the uterine
body in a cranial direction and prevents
53
T h e
B e a u f o rt
c o tt a ge
accidental cervical injury. With the hand
holding the forceps held against the mare’s
buttocks (to avoid injury to the uterus if
the mare moves suddenly) maintaining
the forceps in the uterus, the manipulating
hand and arm are then withdrawn from
the vagina and placed in the rectum. The
forceps are then palpated and advanced
into one or other of the uterine horns. The
jaws of the forceps are then opened and
a fold of endometrium is gently pushed
into the jaws, which are then closed, the
biopsy is completed and the forceps are
withdrawn from the uterus and vagina. Tissues are carefully removed from the
forceps with fine forceps to avoid artefactual
damage and are fixed immediately in
Bouin's fluid. A fine-tipped sterile swab may
be used to sample the inside of the jaws of
the biopsy instrument for bacterial culture.
Sometimes more than one endometrial
to provide adequately interpretable tissues
and to avoid accidental injury to the
testicular artery, by blind needle biopsy,
which may have fatal consequences. The
testicle is examined by ultrasound scan for
visible pathology and best site for sampling
and the scrotum is prepared as for surgical
intervention. Automated punch biopsy and
ultrasound guided techniques are available
but the relatively small tissue samples
are better suited to specific research
requirements. Great care must be taken to
avoid injuring the testicular artery. Testicular tissues should
immediately in Bouin's fluid.
be
fixed
Ileal biopsy
Using our state-of-the-art microwave
processing system, same day results can
be provided for fixed tissue specimens.
fold is removed and then the smaller one
may be placed into transport medium for
bacterial culture.
Ileal biopsies may be indicated to attempt
to confirm or deny a clinical diagnosis of
equine dysautonomia (‘grass sickness’),
where exploratory laparotomy is indicated. Endometrial biopsy samples should be
referred to a laboratory that is specifically
experienced in both equine endometrial
histopathology and equine gynaecology,
in order to receive a meaningful
interpretation.
At exploratory laparotomy, a transmural,
i.e. full-thickness, longitudinal ellipse of
ileum, approximately 15mm by up to
10mm is excised at the proximal end of
the ileocaecal fold, midway between the
mesenteric and antimesenteric borders.
Testicular Biopsy
The tissue is placed immediately into
10% formol saline and processed for
histopathological examination. The ganglia
in the myenteric plexus between the
Testicular biopsies are best obtained by
conventional wedge resection with the
horse under general anaesthesia in order
54
l a b o r a t o rie s
G u i d e
t o
e q u i n e
muscular layers are examined for normal
and abnormal neurones. In ‘typical’ cases
of equine dysautonomia, these ganglia are
devoid of normal neurones and appear
‘skeletal’. The occasional degenerate
neurone, with nuclear degeneration and
cytoplasmic vacuolar degenerative changes
are seen in some cases. Also, clusters
or normal neurones are seldom seen in
the submucosal layers and again, the
occasional degenerate neurone, with
nuclear degeneration and cytoplasmic
vacuolar degenerative changes are seen in
some cases.
Ileal biopsies should be referred to a
laboratory that is specifically experienced
with examining these tissues.
cli n ic a l
p a t h o l o g y
Rectal biopsy
Rectal biopsies are useful for the
investigation of weight loss and diarrhoea
cases or where haematological and serum
biochemical results suggest enteropathy. Samples are best obtained with mare
endometrial biopsy forceps (Yeoman's
basket-jawed), inserted just a hand's length
through the anus. A rectal fold (mucosa
and submucosa only) is removed laterally on either side (dorsal or ventral biopsies are
more prone to puncture blood vessels and
result in worrying haemorrhage).
One sample is fixed in 10% formol saline
and the other placed into Amies’ charcoal
transport medium for bacteriological
examinations.
NOTES
55
T h e
B e a u f o rt
c o tt a ge
l a b o r a t o rie s
reFerence r ange table s
The data in the following tables are from Beaufort Cottage
Laboratories (www.rossdales.com/laboratory) for:
Adult non-Thoroughbred horses
Neonatal Thoroughbred foals (24-48 hours old)
Older Thoroughbred foals (approximately three weeks old)
Yearling Thoroughbred horses
Two-year-old Thoroughbred horses in training
Three-year-old Thoroughbred horses in training and
Adult Thoroughbred horses at stud.
Adult Non-Thoroughbred Horses
56
Test
Abbrev.
Total erythrocytes
RBC
Packed cell volumePCV
Haemoglobin
Hb
Mean cell volume
MCV
Mean cell haemoglobin. conc.
McHc
Mean cell haemoglobin
McH
Total leucocytes
WBC
Segmented neutrophils
Segs
Segs
Lymphocytes
Lymphs
Lymphs
Monocytes
Monos
Monos
Eosinophils
Eos
Eos
PlateletsPlts
Total Protein
TSP
AlbuminAlb
GlobulinGlob
Alpha 1 globulin
α1 glob
Alpha 2 globulin
α2 glob
Beta 1 globulin
β1 glob
Beta 2 globulin
β2 glob
Gamma globulin
γ glob
Plasma fibrinogen
Fib
Units
x1012/l
l/l
g/dl
fl
g/dl
pg
x109/l
x109/l
%
x109/l
%
x109/l
%
x109/l
%
x109/l
g/l
g/l
g/l
g/l
g/l
g/l
g/l
g/l
g/l
Mean
8.2
0.37
13.5
46.0
36.1
16.6
7.5
4.4
58
2.6
35
0.3
4
0.2
2
156
63
35
28
1.2
5.8
7.4
5.3
9.0
2.1
Range
6.2-10.2
0.31-0.43
11.1-15.9
40.0-50.0
33.5-38.7
15.2-19.0
6.0-10.0
3.4-5.4
51-65
2.0-3.2
29-41
0.2-0.4
2-6
0-0.4
1-3
100-250
53-73
29-41
18-38
0.4-2.0
3.2-8.4
4.0-10.8
1.7-8.9
4.6-13.4
0.3-3.9
G u i d e
t o
e q u i n e
cli n ic a l
Test
Abbrev.
Serum amyloid A
SAA
Aspartate amino transferaseAST
Creatinine kinaseCK
Lactate dehydrogenase
LD
LD isoenzyme 1
LD1
LD isoenzyme 2
LD2
LD isoenzyme 3
LD3
LD isoenzyme 4
LD4
LD isoenzyme 5
LD5
Gamma glutamyl transferaseGGT
Glutamate dehydrogenaseGLDH
Serum alkaline phosphatase
SAP
Intestinal alk. phosphataseIAP
IAP
Urea
Urea
CreatinineCreat
GlucoseGlu
Total bilirubin
TBili
Direct bilirubin
DBili
Bile acids
BAcids
CholesterolChol
Triglycerides
Trigs
Lipase
Lip
AmylaseAmyl
CalciumCa
Fractional urinary clearanceCa
PhosphatePO4
Fractional urinary clearancePO4
Magnesium
Mg
Fractional urinary clearance
Mg
Copper (serum)Cu
Copper (plasma)Cu
Zinc
Zn
SodiumNa
Fractional urinary clearanceNa
PotassiumK
Fractional urinary clearanceK
ChlorideCl
Fractional urinary clearanceCl
CortisolCort
Fasting InsulinIns
Tri-iodothyronine
T3
Thyroxine
T4
Cardiac Troponin
cTnI
Selenium
Se
p a t h o l o g y
Units
Mean
mg/l
1.3
iu/l
263
iu/l
186
iu/l
525
% total LD 14
% total LD 26
% total LD 38
% total LD 18
% total LD 4
iu/l
16
iu/l
3
iu/l
204
iu/l
47
% total SAP 22.6
mmol/l
5.2
mmol/l
125
mmol/l
4.9
mmol/l
20
mmol/l
9
mmol/l
5.1
mmol/l
2.45
mmol/l
0.7
mmol/l
30
iu/l
9
mmol/l
3.1
%
6.2
mmol/l
1.4
%
0.3
mmol/l
0.8
%
11.7
mmol/l
18.2
mmol/l
23.0
mmol/l
12.7
mmol/l
138
%
0.09
mmol/l
4.0
%
32.8
mmol/l
99
%
0.72
nmol/l
136
miu/ml
25.5
nmol/l
1.0
nmol/l
22.7
ng/ml
0.1
mmol/l
3.0
Range
0-20
102-350
110-250
225-700
10-18
22-30
34-42
13-23
1-7
1-40
1-10
147-261
13-87
10-34
2.5-10.0
85-165
4.3-5.5
13-34
4-16
1-8.5
2.0-3.3
0.2-1.2
8-50
3-15
2.9-3.3
2.6-15.5
0.9-1.9
0.02-0.53
0.6-1.0
3.8-21.9
14.0-22.0
18.0-28.0
10.0-15.0
134-142
0.02-1.0
3.0-5.0
15-65
95-103
0.04-1.6
71-240
8.0-47.5
0.48-1.46
7.7-42.8
0.05-0.2
0.7-8.4
57
T h e
B e a u f o rt
c o tt a ge
l a b o r a t o rie s
Neonatal Thoroughbred Foals (24-48 hrs old)
Test
58
Abbrev.
Units
Mean
Range
Total erythrocytesRBC
x1012/l
9.4
Packed cell volumePCV
l/l
0.36
Haemoglobin
Hb
g/dl
13.2
10.2-15.4
Mean cell volume
MCV
fl
38.8
31.7-44.9
Mean cell haemoglobin. conc.
McHc
g/dl
36.1
31.7-39.4
Mean cell haemoglobin
McH
pg
14.0
11.2-16.4
Total leucocytes
WBC
x109/l
8.8
6.2-12.4
Segmented neutrophils
Segs
Segs
x109/l
%
6.7
75
4.1-9.5
58-85
Lymphocytes
Lymphs
Lymphs
x109/l
%
1.8
21
1.0-3.1
14-37
Monocytes
Monos
Monos
x109/l
%
0.19
2
0.1-0.5
0.5-5
Eosinophils
Eos
Eos
x109/l
%
0.1
1
0.1-0.2
1-2
PlateletsPlts
x109/l
220
140-315
Total Protein
g/l
54
41-66
AlbuminAlb
g/l
31
25-35
GlobulinGlob
g/l
24
15-36
TSP
6.9-11.8
0.30-0.44
Alpha 1 globulin
α1 glob
g/l
0.9
0.5-1.5
Alpha 2 globulin
α2 glob
g/l
5.0
4.0-5.6
Beta 1 globulin
β1 glob
g/l
5.1
3.1-7.2
Beta 2 globulin
β2 glob
g/l
3.3
1.3-6.0
Gamma globulin
γ glob
g/l
6.5
4.8-10.1
Immunoglobulin GIgG
g/l
11.7
6.9-18.6
Plasma fibrinogen
Fib
g/l
1.9
0.5-3.9
Serum amyloid A
SAA
mg/l
2.2
0-26
Aspartate amino transferaseAST
iu/l
157
Creatinine kinaseCK
iu/l
414
165-761
Lactate dehydrogenase
iu/l
860
615-1110
LD
111-206
LD isoenzyme 1
LD1
% total LD
5
2-7
LD isoenzyme 2
LD2
% total LD 20
17-24
LD isoenzyme 3
LD3
% total LD 42
36-46
LD isoenzyme 4
LD4
% total LD 28
21-34
LD isoenzyme 5
LD5
Gamma glutamyl transferaseGGT
% total LD
iu/l
5
2-10
21
10-32
G u i d e
t o
e q u i n e
Test
cli n ic a l
Abbrev.
Glutamate dehydrogenaseGLDH
Serum alkaline phosphatase
SAP
Units
p a t h o l o g y
Mean
Range
iu/l
25
8-43
iu/l
3341
2424-4544
Intestinal alk. phosphataseIAP
IAP
iu/l
824
% total SAP 23.7
Urea
5.7
528-1200
<26
Urea
mmol/l
CreatinineCreat
mmol/l
GlucoseGlu
mmol/l
Total bilirubin
TBili
mmol/l
55
Direct bilirubin
DBili
mmol/l
20
6-34
Bile acids
BAcids
mmol/l
0-8.0
CholesterolChol
mmol/l
5.5
4-7.4
Triglycerides
Trigs
mmol/l
0.8
0.4-1.9
Lipase
Lip
mmol/l
85 63-113
133
3.1
2.9-8.4
97-188
2.1-4.1
16-94
AmylaseAmyl
iu/l
0.4
0-2.8
CalciumCa
mmol/l
2.9
2.7-3.2
Fractional urinary clearanceCa
%
PhosphatePO4
mmol/l
2.0
1.6-2.5
Fractional urinary clearancePO4
%
0.3
0.02-0.53
Magnesium
Mg
mmol/l
0.8
0.6-1.1
Fractional urinary clearance
Mg
%
Copper (serum)Cu
mmol/l
Copper (plasma)Cu
mmol/l
Zinc
mmol/l
Zn
SodiumNa
mmol/l
Fractional urinary clearanceNa
%
135
PotassiumK
mmol/l
4.3
Fractional urinary clearanceK
%
ChlorideCl
mmol/l
Fractional urinary clearanceCl
%
CortisolCort
nmol/l
Fasting InsulinIns
miu/ml
97
Tri-iodothyronine
T3
nmol/l
Thyroxine
T4
nmol/l
8.4
Cardiac Troponin
cTnI
ng/ml
Selenium
Se
mmol/l
400
0.46
129-140
3.8-5.0
90-103
<14
<800
0.18-0.79
59
T h e
B e a u f o rt
c o tt a ge
l a b o r a t o rie s
Older Thoroughbred Foals (approx. 3 weeks old)
Test
Units
Mean
Range
x1012/l
Packed cell volumePCV
l/l
Haemoglobin
Hb
g/dl
12.4
10.6-13.6
Mean cell volume
MCV
fl
34.3
30.6-39.3
Mean cell haemoglobin. conc.
McHc
g/dl
36.1
34.4-38.9
Mean cell haemoglobin
McH
pg
12.4
11.1-14.5
Total leucocytes
WBC
x109/l
9.3
6.9-15.2
Segmented neutrophils
Segs
Segs
x109/l
%
5.4
58
4.1-9.1
42-88
Lymphocytes
Lymphs
Lymphs
x109/l
%
3.3
38
0.9-5.9
22-54
Monocytes
Monos
Monos
x109/l
%
0.31
3
0.2-0.6
1-7
Eosinophils
Eos
Eos
x109/l
%
0.1
1
0.1-0.3
1-3
PlateletsPlts
x109/l
Total Protein
60
Abbrev.
Total erythrocytesRBC
TSP
10.1
0.34
228
8.8-11.8
0.30-0.38
133-325
g/l
55
42-66
AlbuminAlb
g/l
31
26-37
GlobulinGlob
g/l
24
15-33
Alpha 1 globulin
α1 glob
g/l
1.1
0.9-1.6
Alpha 2 globulin
α2 glob
g/l
6.3
4.1-8.9
Beta 1 globulin
β1 glob
g/l
6.1
4.4-7.3
Beta 2 globulin
β2 glob
g/l
4.0
2.8-5.8
Gamma globulin
γ glob
g/l
5.3
4.1-6.8
Plasma fibrinogen
Fib
g/l
3.0
1.5-4.2
Serum amyloid A
SAA
mg/l
1.2
0-5.4
Aspartate amino transferaseAST
iu/l
333
329-337
Creatinine kinaseCK
iu/l
233
204-263
Lactate dehydrogenase
LD
iu/l
920
631-1199
LD isoenzyme 1
LD1
% total LD 12
11-13
LD isoenzyme 2
LD2
% total LD 29
27-33
LD isoenzyme 3
LD3
% total LD 40
38-41
LD isoenzyme 4
LD4
% total LD 16
13-18
LD isoenzyme 5
LD5
% total LD
3
2-4
G u i d e
t o
e q u i n e
Test
cli n ic a l
Mean
Range
Gamma glutamyl transferaseGGT
iu/l
22
13-30
Glutamate dehydrogenaseGLDH
iu/l
19
8-31
iu/l
1840
1195-2513
Serum alkaline phosphatase
Abbrev.
SAP
Units
p a t h o l o g y
Intestinal alk. phosphataseIAP
IAP
iu/l
366
% total SAP 20.3
Urea
3.4
260-471
18.7-22.2
Urea
mmol/l
CreatinineCreat
mmol/l
GlucoseGlu
mmol/l
Total bilirubin
TBili
mmol/l
38
22-54
Direct bilirubin
DBili
mmol/l
12
5-18
Bile acids
BAcids
mmol/l
0-8.0
123
4.2
CholesterolChol
mmol/l
Triglycerides
Trigs
mmol/l
Lipase
Lip
mmol/l
AmylaseAmyl
iu/l
CalciumCa
mmol/l
Fractional urinary clearanceCa
%
3.0
2.8-4.1
97-138
3.2-6.2
2.9-3.1
PhosphatePO4
mmol/l
2.4
2.2-2.7
Fractional urinary clearancePO4
%
0.3
0.02-0.53
Magnesium
Mg
mmol/l
0.8
0.7-1.0
Fractional urinary clearance
Mg
%
Copper (serum)Cu
mmol/l
Copper (plasma)Cu
mmol/l
Zinc
mmol/l
Zn
SodiumNa
mmol/l
141
Fractional urinary clearanceNa
%
PotassiumK
mmol/l
Fractional urinary clearanceK
%
4.8
ChlorideCl
mmol/l
Fractional urinary clearanceCl
%
99
CortisolCort
nmol/l
Fasting InsulinIns
miu/ml
Tri-iodothyronine
T3
nmol/l
Thyroxine
T4
nmol/l
Cardiac Troponin
cTnI
ng/ml
Selenium
Se
mmol/l
12.7
130
135-145
4.1-5.5
96-102
0.5-4.2
60-320
61
T h e
B e a u f o rt
c o tt a ge
l a b o r a t o rie s
Yearling Thoroughbred Horses
Test
62
Abbrev.
Units
Mean
Range
Total erythrocytesRBC
x1012/l
9.5
Packed cell volumePCV
l/l
0.35
Haemoglobin
Hb
g/dl
12.9
10.0-14.3
Mean cell volume
MCV
fl
37.0
33.6-40.0
Mean cell haemoglobin. conc.
McHc
g/dl
36.6
34.5-40.0
Mean cell haemoglobin
McH
pg
13.5
12.6-15.0
7.5-11.5
0.30-0.41
Total leucocytes
WBC
x109/l
9.5
6.0-15.0
Segmented neutrophils
Segs
Segs
x109/l
%
4.9
51
3.7-5.4
40-53
Lymphocytes
Lymphs
Lymphs
x109/l
%
3.9
41
3.5-4.9
35-49
Monocytes
Monos
Monos
x109/l
%
0.3
3
0.2-0.5
2-5
Eosinophils
Eos
Eos
x109/l
%
0.2
2
0.1-1.0
1-5
PlateletsPlts
x109/l
Total Protein
g/l
TSP
174
118-243
59
50-70
AlbuminAlb
g/l
32
20-40
GlobulinGlob
g/l
27
20-50
Alpha 1 globulin
α1 glob
g/l
1.3
0.7-2.0
Alpha 2 globulin
α2 glob
g/l
7.6
6.0-9.3
Beta 1 globulin
β1 glob
g/l
6.7
4.8-8.3
Beta 2 globulin
β2 glob
g/l
5.0
4.3-5.9
Gamma globulin
γ glob
g/l
7.1
4.6-8.4
Plasma fibrinogen
Fib
g/l
3.3
2.7-4.4
Serum amyloid A
SAA
mg/l
1.2
0-10
Aspartate amino transferaseAST
iu/l
381
Creatinine kinaseCK
iu/l
267
190-370
Lactate dehydrogenase
iu/l
874
476-1200
LD
323-441
LD isoenzyme 1
LD1
% total LD 11
5-15
LD isoenzyme 2
LD2
% total LD 25
20-30
LD isoenzyme 3
LD3
% total LD 36
30-44
12-30
LD isoenzyme 4
LD4
% total LD 20
LD isoenzyme 5
LD5
% total LD
Gamma glutamyl transferaseGGT
iu/l
5
2-6
23
10-30
G u i d e
t o
e q u i n e
Test
cli n ic a l
Abbrev.
p a t h o l o g y
Units
Mean
Glutamate dehydrogenaseGLDH
iu/l
9
7-18
Serum alkaline phosphatase
iu/l
611
500-1200
Intestinal alk. phosphataseIAP
IAP
iu/l
140
% total SAP 23
100-250
<25
Urea
SAP
Urea
mmol/l
CreatinineCreat
mmol/l
GlucoseGlu
mmol/l
Total bilirubin
TBili
mmol/l
34
Direct bilirubin
DBili
mmol/l
12
Bile acids
BAcids
mmol/l
5.1
CholesterolChol
mmol/l
2.45
2.0-3.3
Triglycerides
Trigs
mmol/l
1.06
0.26-2.6
Lipase
Lip
mmol/l
AmylaseAmyl
5.6
Range
118
4.7
4.6-8.0
93-125
3.4-5.9
22-46
5-18
0-8.0
iu/l
CalciumCa
mmol/l
3.0
2.7-3.3
Fractional urinary clearanceCa
%
6.2
2.6-15.5
PhosphatePO4
mmol/l
1.7
1.5-2.0
Fractional urinary clearancePO4
%
1.9
0.0-3.2
Magnesium
Mg
mmol/l
0.8
0.6-0.9
Fractional urinary clearance
Mg
%
11.7
3.8-21.9
Copper (serum)Cu
mmol/l
17.1
13.5-20.3
Copper (plasma)Cu
mmol/l
Zinc
mmol/l
Zn
SodiumNa
mmol/l
Fractional urinary clearanceNa
%
134
PotassiumK
mmol/l
Fractional urinary clearanceK
%
ChlorideCl
mmol/l
Fractional urinary clearanceCl
%
CortisolCort
nmol/l
Fasting InsulinIns
miu/ml
Tri-iodothyronine
T3
nmol/l
Thyroxine
T4
nmol/l
Cardiac Troponin
cTnI
ng/ml
Selenium
Se
mmol/l
0.14
3.8
36.5
100
0.90
1.4
21
130-140
0.02-1.2
3.6-4.0
12-70
97-105
0.03-2.0
0.4-2.6
5-40
63
T h e
B e a u f o rt
c o tt a ge
l a b o r a t o rie s
Two-Year-Old Thoroughbred Horses in Training
Test
64
Abbrev.
Units
Mean
Range
Total erythrocytesRBC
x1012/l
Packed cell volumePCV
l/l
Haemoglobin
Hb
g/dl
15.0
12.8-16.6
Mean cell volume
MCV
fl
39.9
37.0-42.1
Mean cell haemoglobin. conc.
McHc
g/dl
36.8
35.9-37.9
Mean cell haemoglobin
McH
pg
14.7
13.7-15.7
Total leucocytes
WBC
x109/l
9.6
7.3-12.7
Segmented neutrophils
Segs
Segs
x109/l
%
5.1
53
4.0-6.0
42-63
Lymphocytes
Lymphs
Lymphs
x109/l
%
3.6
38
2.7-4.4
28-46
Monocytes
Monos
Monos
x109/l
%
0.4
4
0.26-0.56
2-6
Eosinophils
Eos
Eos
x109/l
%
0.2
2
0-0.3
1-3
PlateletsPlts
x109/l
170
127-206
Total Protein
g/l
62
59-66
AlbuminAlb
g/l
37
35-39
GlobulinGlob
g/l
25
TSP
10.2
0.40
8.7-11.7
0.34-0.45
21-28
Alpha 1 globulin
α1 glob
g/l
1.1
0.6-1.4
Alpha 2 globulin
α2 glob
g/l
6.6
5.4-7.8
Beta 1 globulin
β1 glob
g/l
6.9
5.7-8.5
Beta 2 globulin
β2 glob
g/l
4.2
1.8-6.8
Gamma globulin
γ glob
g/l
5.6
3.7-8.2
Plasma fibrinogen
Fib
g/l
1.9
1.5-2.3
Serum amyloid A
SAA
mg/l
1.3
0-20
Aspartate amino transferaseAST
iu/l
545
308-820
Creatinine kinaseCK
iu/l
354
166-572
Lactate dehydrogenase
LD
iu/l
793
569-917
LD isoenzyme 1
LD1
% total LD 10
7-12
LD isoenzyme 2
LD2
% total LD 28
24-34
LD isoenzyme 3
LD3
% total LD 40
35-44
LD isoenzyme 4
LD4
% total LD 17
11-23
LD isoenzyme 5
LD5
Gamma glutamyl transferaseGGT
% total LD
iu/l
5
1-10
24
12-40
G u i d e
t o
e q u i n e
Test
cli n ic a l
Abbrev.
Units
p a t h o l o g y
Mean
Range
Glutamate dehydrogenaseGLDH
iu/l
10
4-11
Serum alkaline phosphatase
iu/l
452
293-672
SAP
Intestinal alk. phosphataseIAP
IAP
iu/l
109
% total SAP 23.2
Urea
4.8
64-171
<25
Urea
mmol/l
CreatinineCreat
mmol/l
GlucoseGlu
mmol/l
Total bilirubin
TBili
mmol/l
26
Direct bilirubin
DBili
mmol/l
10
Bile acids
BAcids
mmol/l
4.6
CholesterolChol
mmol/l
2.45
2.0-3.3
Triglycerides
Trigs
mmol/l
1.06
0.26-2.6
Lipase
Lip
mmol/l
134
4.7
3.8-6.0
110-160
3.4-5.9
13-39
4-15
0-8.0
42.5
23.8-124.5
3.3-22.1
AmylaseAmyl
iu/l
8.9
CalciumCa
mmol/l
3.1
2.9-3.3
Fractional urinary clearanceCa
%
6.2
2.6-15.5
PhosphatePO4
mmol/l
1.4
1.1-1.6
Fractional urinary clearancePO4
%
0.3
0.02-0.53
Magnesium
Mg
mmol/l
0.8
0.6-0.9
Fractional urinary clearance
Mg
%
11.7
3.8-21.9
Copper (serum)Cu
mmol/l
17.1
13.5-20.3
Copper (plasma)Cu
mmol/l
Zinc
mmol/l
Zn
SodiumNa
mmol/l
Fractional urinary clearanceNa
%
136
PotassiumK
mmol/l
Fractional urinary clearanceK
%
ChlorideCl
mmol/l
Fractional urinary clearanceCl
%
CortisolCort
nmol/l
Fasting InsulinIns
miu/ml
Tri-iodothyronine
T3
Thyroxine
T4
Cardiac Troponin
cTnI
ng/ml
0.19
Selenium
Se
mmol/l
3.5
0.04
3.6
29.7
102
0.86
136
130-142
0.02-1.0
2.8-4.2
15-65
95-107
0.04-1.6
71-240
25.5
8.0-47.5
nmol/l
1.0
0.48-1.46
nmol/l
22.7
7.7-42.8
0.10-0.36
1.6-5.3
65
T h e
B e a u f o rt
c o tt a ge
l a b o r a t o rie s
Three-Year-Old Thoroughbred Horses in Training
Test
Units
Mean
Total erythrocytesRBC
x1012/l
9.7
Packed cell volumePCV
l/l
0.40
Haemoglobin
Hb
g/dl
14.9
13.0-16.1
Mean cell volume
MCV
fl
40.7
39.5-43.0
Mean cell haemoglobin. conc.
McHc
g/dl
37.8
36.6-38.8
Mean cell haemoglobin
McH
pg
15.4
14.9-16.2
Total leucocytes
WBC
x109/l
8.2
6.9-9.8
Segmented neutrophils
Segs
Segs
x109/l
%
4.6
56
3.9-5.2
48-64
Lymphocytes
Lymphs
Lymphs
x109/l
%
2.8
35
2.3-3.4
28-42
Monocytes
Monos
Monos
x109/l
%
0.36
4
Eosinophils
Eos
Eos
x109/l
%
0.1
1
PlateletsPlts
x109/l
Total Protein
66
Abbrev.
TSP
Range
8.6-10.5
0.35-0.43
0.2-0.56
2-6
0-0.2
1-2
170
127-206
58-67
g/l
62
AlbuminAlb
g/l
38
35-40
GlobulinGlob
g/l
25
22-29
Alpha 1 globulin
α1 glob
g/l
1.3
0.9-1.7
Alpha 2 globulin
α2 glob
g/l
6.3
5.1-7.8
Beta 1 globulin
β1 glob
g/l
6.7
4.5-8.0
Beta 2 globulin
β2 glob
g/l
3.3
2.5-5.1
Gamma globulin
γ glob
g/l
7.0
5.4-9.6
Plasma fibrinogen
Fib
g/l
1.8
14-2.3
Serum amyloid A
SAA
mg/l
1.3
0-20
Aspartate amino transferaseAST
iu/l
467
289-630
Creatinine kinaseCK
iu/l
390
156-875
Lactate dehydrogenase
LD
iu/l
638
430-883
LD isoenzyme 1
LD1
% total LD 11
6-17
LD isoenzyme 2
LD2
% total LD 24
19-27
LD isoenzyme 3
LD3
% total LD 39
33-43
LD isoenzyme 4
LD4
% total LD 20
16-23
LD isoenzyme 5
LD5
% total LD
6
1-18
G u i d e
t o
e q u i n e
Test
cli n ic a l
Abbrev.
Units
p a t h o l o g y
Mean
Range
Gamma glutamyl transferaseGGT
iu/l
27
13-47
Glutamate dehydrogenaseGLDH
iu/l
7
3-12
Serum alkaline phosphatase
iu/l
375
277-451
SAP
Intestinal alk. phosphataseIAP
IAP
iu/l
89
% total SAP 22.8
Urea
5.4
65-107
<24
Urea
mmol/l
CreatinineCreat
mmol/l
GlucoseGlu
mmol/l
Total bilirubin
TBili
mmol/l
26
13-39
Direct bilirubin
DBili
mmol/l
10
4-15
Bile acids
BAcids
143
4.7
3.9-6.4
118-167
3.4-5.9
mmol/l
5.2
CholesterolChol
mmol/l
2.45
2.0-3.3
0-8.0
Triglycerides
Trigs
mmol/l
1.06
0.26-2.6
Lipase
Lip
mmol/l
42.5
23.8-124.5
AmylaseAmyl
iu/l
8.9
3.3.-22.1
CalciumCa
mmol/l
3.1
2.9-3.3
Fractional urinary clearanceCa
%
6.2
2.6-15.5
PhosphatePO4
mmol/l
1.2
1.0-1.5
Fractional urinary clearancePO4
%
0.3
0.02-0.53
Magnesium
Mg
mmol/l
0.8
0.6-0.9
Fractional urinary clearance
Mg
%
11.7
3.8-21.9
Copper (serum)Cu
mmol/l
17.1
13.5-20.3
Copper (plasma)Cu
mmol/l
Zinc
mmol/l
Zn
SodiumNa
mmol/l
143
Fractional urinary clearanceNa
%
0.05
PotassiumK
mmol/l
3.9
3.4-4.3
Fractional urinary clearanceK
%
32.8
15-65
ChlorideCl
mmol/l
Fractional urinary clearanceCl
%
CortisolCort
nmol/l
Fasting InsulinIns
miu/ml
25.5
8.0-47.5
Tri-iodothyronine
T3
nmol/l
1.0
0.48-1.46
Thyroxine
T4
nmol/l
22.7
7.7-42.8
Cardiac Troponin
cTnI
ng/ml
0.19
Selenium
Se
mmol/l
3.5
103
0.78
136
130-160
0.02-1.0
96-108
0.04-1.6
71-240
0.10-0.36
1.6-5.3
67
T h e
B e a u f o rt
c o tt a ge
l a b o r a t o rie s
Adult Thoroughbred Horses at Stud
Test
Units
Mean
Range
x1012/l
Packed cell volumePCV
l/l
Haemoglobin
Hb
g/dl
14.0
11.1-16.9
Mean cell volume
MCV
fl
36.0
31.0-41.0
Mean cell haemoglobin. conc.
McHc
g/dl
34.0
32.0-36.0
Mean cell haemoglobin
McH
pg
12.1
10.0-14.2
Total leucocytes
WBC
x109/l
7.1
4.1-10.1
Segmented neutrophils
Segs
Segs
x109/l
%
3.6
52
1.4-5.8
36-68
Lymphocytes
Lymphs
Lymphs
x109/l
%
3.0
45
1.4-4.7
39-61
Monocytes
Monos
Monos
x109/l
%
0.4
4
0.36-0.56
2-6
Eosinophils
Eos
Eos
x109/l
%
0.2
3
0-0.5
0-5
PlateletsPlts
x109/l
Total Protein
68
Abbrev.
Total erythrocytesRBC
TSP
11.5
0.41
9.0-14.0
0.35-0.47
170
127-206
65-75
g/l
70
AlbuminAlb
g/l
36
34-38
GlobulinGlob
g/l
34
29-39
Alpha 1 globulin
α1 glob
g/l
1.6
Alpha 2 globulin
α2 glob
g/l
6.9
5.5-7.9
Beta 1 globulin
β1 glob
g/l
9.2
6.8-11.4
1.1-2.1
Beta 2 globulin
β2 glob
g/l
5.5
3.5-7.2
Gamma globulin
γ glob
g/l
11.3
8.3-14.4
Plasma fibrinogen
Fib
g/l
2.2
1.5-3.3
Serum amyloid A
SAA
mg/l
1.3
0-20
Aspartate amino transferaseAST
iu/l
309
256-369
Creatinine kinaseCK
iu/l
216
154-270
Lactate dehydrogenase
LD
iu/l
525
383-664
LD isoenzyme 1
LD1
% total LD 13
10-17
LD isoenzyme 2
LD2
% total LD 30
24-37
LD isoenzyme 3
LD3
% total LD 43
38-49
LD isoenzyme 4
LD4
% total LD 13
9-18
LD isoenzyme 5
LD5
% total LD
2
1-3
G u i d e
t o
e q u i n e
Test
cli n ic a l
Abbrev.
Units
p a t h o l o g y
Mean
Range
Gamma glutamyl transferaseGGT
iu/l
20
14-28
Glutamate dehydrogenaseGLDH
iu/l
8
4-14
Serum alkaline phosphatase
iu/l
403
286-571
SAP
Intestinal alk. phosphataseIAP
IAP
iu/l
103
% total SAP 24.3
77-157
<28
Urea
5.9-9.5
Urea
mmol/l
CreatinineCreat
mmol/l
7.6
GlucoseGlu
mmol/l
Total bilirubin
TBili
mmol/l
26
13-39
Direct bilirubin
DBili
mmol/l
10
4-15
Bile acids
BAcids
99
4.7
87-112
3.5-5.9
mmol/l
4.7
CholesterolChol
mmol/l
2.45
2.0-3.3
0-8.0
Triglycerides
Trigs
mmol/l
1.06
0.26-2.6
Lipase
Lip
mmol/l
42.5
23.8-124.5
AmylaseAmyl
iu/l
8.9
3.3-22.1
CalciumCa
mmol/l
3.1
3.0-3.3
Fractional urinary clearanceCa
%
6.2
2.6-15.5
PhosphatePO4
mmol/l
1.0
0.8-1.3
Fractional urinary clearancePO4
%
0.3
0.02-0.53
Magnesium
Mg
mmol/l
0.8
0.6-0.9
Fractional urinary clearance
Mg
%
11.7
3.8-21.9
Copper (serum)Cu
mmol/l
17.1
13.5-20.3
Copper (plasma)Cu
mmol/l
Zinc
mmol/l
Zn
SodiumNa
mmol/l
142
Fractional urinary clearanceNa
%
0.09
PotassiumK
mmol/l
3.8
3.3-4.1
Fractional urinary clearanceK
%
32.8
15-65
ChlorideCl
mmol/l
Fractional urinary clearanceCl
%
CortisolCort
nmol/l
Fasting InsulinIns
miu/ml
25.5
8.0-47.5
Tri-iodothyronine
T3
nmol/l
1.0
0.48-1.46
Thyroxine
T4
nmol/l
22.7
7.7-42.8
Cardiac Troponin
cTnI
ng/ml
0.1
0.05-0.2
Selenium
Se
mmol/l
3.5
1.6-5.3
104
0.72
136
135-149
0.02-1.0
100-108
0.04-1.6
71-240
69
T h e
B e a u f o rt
c o tt a ge
l a b o r a t o rie s
NOTES
Copies of the Reference Range Tables can be downloaded (as pdf files) from our website:
www.rossdales.com/laboratory
While we have made every endeavor to ensure the accuracy of information in this Guide, Rossdale &
Partners cannot be held liable for any inaccuries which may inadvertently occur.
70
G u i d e
t o
e q u i n e
cli n ic a l
p a t h o l o g y
The aim of this Guide is to help equine practitioners use
clinical pathological aids to diagnosis to good effect, to provide
‘normal’ reference ranges for horses of different ages and
types, and to give advice for the safe and satisfactory collection
and handling of samples for laboratory investigation.
The Guide was produced by Sidney W. Ricketts, LVO, BSc,BVSc,
DESM, DipECEIM, FRCPath, FRCVS
with the help of
Annalisa Barrelet, BVetMed, MS, CertESM, MRCVS
Celia M.Marr, BVMS, MVM, PhD, DEIM, DipECEIM, MRCVS
Sarah S.Stoneham, BVSc, CertESM, MRCVS
Katherine E. Whitwell, BVSc, DiplECVP, FRCVS
Robert S.G.Cash BSc, FIBiol
Mary Ashpole.
© 2006 Rossdale & Partners
Sidney Ricketts
Beaufort Cottage laboratories
High Street, Newmarket, Suffolk CB8 8JS
Tel +44 (0)1638 663017 Fax +44 (0)1638 560780
Email bcl@rossdales.com
www.rossdales.com