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Haematology and blood biochemistry in the

horse: A guide to interpretation

Article in In practice · June 2002

DOI: 10.1136/inpract.24.6.318

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Annalisa Barrelet Sidney Ricketts

Rossdales Equine Hospital & Diagnostic Centre Rossdales Equine Hospital & Diagnostic Centre
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Laboratory tests have become an

essential part of equine practice
and, when used appropriately,
provide a valuable ancillary aid
to case management

Hmoyn ldies=

Haemnatology and blood biochemnistry

in the horse: a guide to interpretation
ANNALISA BARRELET AND SIDNEY RICKETTS

THE use of laboratory tests as an aid to the diagnosis of clinical problems has become commonplace
in equine practice, and should be considered an integral part of the total management of a case.
The indications for blood sampling are many and varied, but it is important to realise the limitations
of laboratory results - there is no substitute for a thorough clinical examination. Clinicians should
therefore guard against relying too heavily on test results for a diagnosis and, rather, clinical pathology
should be used as an ancillary aid. This article reviews the interpretation of basic haematological and
blood biochemical tests which are available to the equine practitioner.
Annalisa Barrelet
graduated from the
Royal Veterinary
College in 1986 and BLOOD SAMPLING the date of collection. To avoid sample deterioration,
completed a master's analyses should be performed as soon as possible. Where
degree in comparative
pathology in the INDICATIONS a visit is involved, some delay is inevitable, and it is
USA before joining Blood sampling is useful in the diagnosis of clinical dis- prudent to carry samples in an insulated 'cool bin' (12
Rossdale and Partners ease (whether it be infectious, parasitic or due to specific to 20°C) to avoid the deleterious effects of excessive
in Newmarket as an
assistant. She currently organ dysfunction) and also in the routine management temperature fluctuations that may occur in a vehicle.
works as a clinical of horses (eg, routine neonatal foal examinations, perfor- Whole blood samples should not be refrigerated, as this
pathologist in the
practice's Beaufort mance horse monitoring, preanaesthetic checks and pre- results in haemolysis rendering haematological tests and
Cottage Laboratories. purchase examinations). In addition, laboratory tests are many serum enzyme analyses inaccurate. Ideally, serum
She holds the RCVS
certificate in equine sometimes required for insurance examinations (eg, should be separated from clotted samples as soon as
stud medicine. immunoglobulin G [IgG] tests for neonatal foals) and practical. Centrifugation or simple standing and pipetting
often for import/export examinations. may be used; modem collection and separation tubes are
particularly convenient in this regard. Once separated,
OBTAINING SAMPLES serum or plasma may be refrigerated or frozen for stor-
In order to avoid spurious haemoconcentration ('fright age purposes.
and flight' splenic contraction), it is important to take
samples from horses and ponies in a quiet, calm
environment. The degree of excitement caused by
Sidney Ricketts venepuncture depends on the temperament of the horse
graduated from and the technique of the clinician. Most samples will be
Bristol in 1971 after Anticoagulant Vacutainer* Monovettet
intercalating a degree collected from a jugular vein, which is large and easily
in biochemistry. He visualised in most horses and ponies. If handled and Haematology EDTA Lilac top Red top
worked as an intern
in equine medicine approached properly, many animals are quite prepared to General Plain Red top Brown top
and surgery in the submit themselves to blood collection without fuss; biochemistry (serum) (with serum-
USA before joining however, for apprehensive individuals, an appropriate separating gel)
Rossdale and Partners
in Newmarket in degree of restraint is essential to ensure the safety of all White top
1972. He became (plain)
a partner in the
concerned. Samples should be collected using suitable
Heparin Green top Orange top
practice in 1975 and equipment and into appropriate containers (see table on (plasma)
is now managing the right).
partner. He holds Fibrinogen Citrate Blue top Green top
the RCVS diploma in
equine stud medicine SAMPLE HANDLING Glucose Fluoride Grey top Yellow top
and is an RCVS Once collected, samples should be indelibly and accu-
specialist in equine *Becton Dickinson, tSarstedt
stud medicine. rately labelled with the horse's and owner's names and

318 In Practice 0 J U N E 2 002

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Choke of laboat.ryltsts
It is very important to use laboratory that is expe-
a
rienced in testing equine samples and interpreting
the results produced. All clinical pathology labora-
tories, whether in-house or extemal, should have
an active quality control programme and should
subject themselves to external quality assessment.
Laboratory analysis of samples has significant
cost implications and tests should be carefully
selected with specific objectives in mind. Most labo-
ratories offer 'profiles' which will be more economi-
cal than individual tests (see table on the right). If
the case history is complex, vague or otherwise not
straightforward, it is not always cost effective to
minimise the initial investigations.
SAMPLE DISPATCH
If samples are to be sent to an external laboratory by post,
it is important to avoid sending them over a weekend
as they will be delayed and may reach the laboratory
in an unsatisfactory condition for analysis. With urgent
samples, or at times of slow postal service, personal
delivery (eg, by the owner or using a private courier
service) is much better. Samples should be accompanied
by the appropriate laboratory submission form, fully
completed with the clinical history, reason for submission
and any specific requirements.
When sending samples, it is important that correct
packaging is used in order to avoid breakage and
loss of samples, and to conform to relevant current
legislation. The leakage of pathological specimens can
present a public health hazard and the postal service
may refuse to handle soiled packages. Samples should
be sealed within leak-proof inner containers and securely
enclosed in padded and crush-proof outer containers.
The outer package should be clearly labelled and
samples should be dispatched by first class or special
delivery post.
Rfr Ice rig
Clinicians and clinical pathologists must have some
basis on which to establish whether the results
obtained are normal or abnormal. It is difficult to
define 'normal' but most reference ranges relate to
data frm clinially normal horses, where the top
and bottom 10 per cent have been excluded (0th
percentiles). Thisstatisticl appro h is preferable to
the use Of tw standard deviions eithr side of the
mean, as many eu test uts do not show
a mathemacal normal dWisriobtion.
There are a number of variables that affect the
results of equine laboratory tests. These include:
* Individual variation such as age, breed, sex and
level Of fitness;
* Sample collection, timing, handling and trans-
portation factors;
* Laboratory equipment and analytical methodolo-
gy used.
It is not usually practical to produce clinically nor-
mal referenceranges for each situon and so, as a
In Practice * J U N E 2002
1 1 .0 .....11 I
Profile type Profile content
36 hours Haematology, serum proteins, fibrinogen, PV, SAA, serum IgG
Weanling and yearling Haematology, serum proteins and electrophoresis, fibrinogen, SAP,
serum calcium and phosphate, urine phosphate fractional clearance ratios
Inflammatory profile Haematology, serum proteins, PV, fibrinogen, SAA
Training horse Haematology, serum proteins and electrophoresis, PV, fibrinogen, SAA,
AST, CK, urine phosphate fractional clearance ratios
Mature horse Haematology, serum proteins and electrophoresis, fibrinogen, PV, SAA,
AST, CK, GLDH, LD, SAP, IAP, GGT, urea, creatinine
Intestinal profile Haematology, serum proteins and electrophoresis, fibrinogen, PV, SAA,
SAP, IAP
Liver profile Haematology, serum proteins and electrophoresis, fibrinogen, PV, SAA,
AST, GLDH, LD, SAP, IAP
AST Aspartate aminotransferase, CK Creatine kinase, GGT L-gamma glutamyl transferase,
GLDH Glutamate dehydrogenase, IAP Intestinal alkaline phosphatase, LD Lactate dehydrogenase,
PV Plasma viscosity, SAA Serum amyloid A, SAP Serum alkaline phosphatase
SAMPLE ANALYSIS
The laboratory, whether in-house or external, must
ensure that samples are received, unpacked, logged,
prepared, analysed and reported accurately as soon as
possible. Particular attention should be paid to in-house
quality control and external quality assurance, as unreli-
able results are misleading and, consequently, worse than
no results at all.
HAEMIATOLOGY
Quantification and examination of the cellular constitu-
ents of equine blood may reveal:
* Various types of anaemia (low red blood cell count,
packed cell volume and haemoglobin level);
* Haemoconcentration/dehydration (raised red blood
cell count, packed cell volume and haemoglobin level);
* Bacterial infections (high white blood cell count and
neutrophilia);
* Viral infections (low white blood cell count and neu-
tropenia during the acute phase);
compromise, age-specific rages for haematological
and biochemkal tests, and typ efic (eg, thorough-
bred/non-thoroughbred) ranges for haematological
tests, produced using a particular laboratory's proto-
cols, should suffice Textbook rferece ranges are
not alwaysaplcal especially for biochemical
tests, as analyta methodology varies widely bet-
ween labortordies.
Where routin prling of p horses is
carried out it is useu to establish a set of nonral para-
meters for the ideally individs In this
wy early and subtle changes may be dect. This
can be done relatively simp by p inputting
results in:to i srystndard comroputeeadsheet or
database programs and appling elementary statistical
analys A compliating factor is that refer e ranges
for young performnce horses are constanty changing
as age and fitnes alters, and there is no preise method
for overcomig this problem. H iicls
or tpe-spefic rans are ton options
3 19
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* Parasitic or allergic conditions (eosinophilia in some
cases).
Haemoconcentration can only be interpreted if sam-
ples are taken from resting horses under cailm conditions.
This can be difficult to achieve in needle-shy, nervous
or excitable animals. Post-exercise samples are usually
impossible to interpret. Where routine blood profiling
of competition horses is performed, samples should be
taken regularly at the same quiet time of the day when
horses are at rest.
ANAEMIA
Most cases of equine anaemia occur secondarily to other
conditions. A regenerative anaemia is one in which the
bone marrow responds appropriately with incr-eased red
blood cell production. Examples include acute blood
loss, haemolytic anaemia, and mild/moderate intestinal
parasitism. Reticulocytes are seldom found in the periph-
eral blood of adult horses and are rarely found in foals
recosering from haiemolytic anaemia (neonatal isoery-
throlysis). In other species, they are often found in asso-
ciation with blood loss or a hypoxic insult.
Non-regenerative anaemia, or anaemia associated
with inadequate red blood cell productioni, may occur in
cases with generalised bone marrow suppress.ion, chronic
inflammation, neoplasia, chronic renal disease, or iron
deficiency. A specific diagnosis relies on further blood
biochemistry and other diagnostic tests (eg, examiniation
of bone marrow, peritoneal and pleural fluids). A positi'Ve
direct antiglobulin (Coombs) test indicates the presence of
antibody or complemiient on red blood cell surtaces, and is
diagnostic of immune-mediated anaemia. This test maiy be
positive in cases of idiopathic immune-mediated anaemia,
neonatal isoerythrolysis and equine infectious anaemia.
Blood film from a horse with
severe haemolytic anaemia.
Note the marked variability in
the size of the red blood cells
Blood film from a horse with severe septicaemia showing
a metamyelocyte (arrow). Note the lack of lobularity of
the nucleus. Metamyelocytes only appear in the circulation
when demand for polymorphonuclear neutrophils greatly
exceeds production
320
Macrocytic aniaemia indicates red blood cell regener-
ation because young or newly produced red blood cells
are larger than mature ones. A spurious increase in red
blood cell size occurs as a result of degeneration of the
cells in vitro due to osmotic swelling. Anisocytosis (vari-
ability in the size of red blood cells) is caused by the
presence of macrocytes and microcytes among normnal
red blood cells. This is a rare finding in horses and
pomies and is Lisually associated with a severe reoenera-
tiv e anaemia.
LEUCOCYTOSIS
Physiological leucocytosis (high white blood cell count)
occurs as a result of excitement or stress. Increased num-
bers of mature neutrophils and lymphocytes are released
into the circulation from their reserses and marginated
pools. The response occurs rapidly (in seconids) and is
due to increased blood pressure. heart rate and splenic
contractioIl, all of which aIc associated with intense
exercise, fear or excitement. It is important to consider
this response when interpreting haemnatological data
from young- or excitable Individuals. The response is a
tranisient phenomenon and norimial resting levels are i-e
established within about an hour.
Pathological leucocytosis with neutrophilia occuIrs
durine infectious and non-infectious inflaimmll.atory coil-
ditions and the differentiation can sometimies be unclear.
An increased numbei of circulating immature neLitro-
phils (ie, metcamyelocyte and 'band' neutrophils) is the
most sensitive differentiator of infectious inflammatorv
disease, but these arc not seen as frequently in horses
and ponies as in somile other animals. In horses, band
neutrophils are most commonly seen in severe acute
bacterial infections or septicaemiiic processes, and toxic
band cells may be a feature (eg, in the case of neonatal
septicaemia and adult colitis or enterotoxaemia).
LEUCOPENIA
Leucopenia (low white blood cell count) occurs if the
supply of white blood cells to the circulationi is out-
stripped by the demand or their exit from the circulation
and is therefor-e anI indicator of pathology. Increased
egress from the circulation may be a result of:
* Increased utilisation in inflamed tissues:
* Sequestration to highly vascular organs (eg, in cases
of pneumonia);
I A A- m O- u
Blood film showing white blood cells associated with a
severe septicaemic process (eg, Salmonella enterotoxaemia,
neonatal septicaemia). (A) Band neutrophil and (B) toxic
band neutrophils (note the ring-like nuclear configuration)
In Practice * J U N E 2002
 Downloaded from inpractice.bmj.com on March 9, 2013 - Published by group.bmj.com

* Sequestration to body cavities (eg, in cases of suppu-

rative peritonitis or pleuritis); or
* Destruction in the microcirculation.
Reduced neutrophil production may occur in bone
marrow suppression (eg, in viral infections). Marked
leucopenia and neutropenia is seen in association with
challenge by endotoxin. In adults, this may be associated
with an acute abdominal crisis (eg, strangulation, intussus-
ception, salmonellosis) or exhaustion/shock; in neonates,
it is associated with bacteraemia and septicaemia. In such
Abnormal monocyte from
cases, the appearance of band (juvenile) neutrophils (some a horse with colic and
of which show toxic changes) is a feature. In animals with endotoxaemia. Note the
increased lobularity of the
acute abdominal crisis, abnormal monocytes and reactive nucleus and the darker
lymphocytes are sometimes seen. staining cytoplasm

Leucocytes
There are five types of leucocytes (white blood cells):
neutrophils, eosinophils, basophils, monocytes and
lymphocytes. All, except the lymphocytes, are pro-
duced in the bone marrow. Lymphocytes are pro-
duced in the lymph nodes, spleen and lymphoid
follicles distributed throughout the tissues. Circulat-
ing granulocytes (neutrophils, eosinophils and baso-
phils) enter the circulation as mature cells. Circulating
monocytes are immature and undergo one or two
more divisions after entering the tissues where they
become macrophages.
Total and differential white blood cell counts and
careful microscopic observation of leucocyte morphol-
ogy should be performed to fully evaluate leucocytes.
Some modern automated cytochemical haematologi-
cal analysers provide accurate differential leucocyte
counts which correlate closely with traditional manual
differential counting techniques. These automated dif-
ferentials are performed on 10,000 leucocytes rather Automated
haematological analyser
than 100, providing more accurate results which are trophils). A suppurative condition is usually associated
repeatable and, therefore, reliable. with a moderate to marked leucocytosis and neu-
The leucocytic response is a dynamic process and trophilia. The appearance of band (juvenile) neu-
results will depend greatly on the sampling time in trophils indicates increased neutrophil production
relation to the stage of the physiological or disease where the supply of mature forms is unable to match
process. Depending on the time of sampling, marked tissue demand.
leucopenia (low white blood cell count), a normal In cases of overt clinical disease, haematological
leucocyte count, or leucocytosis (high white blood cell changes are likely to be marked and obvious. Con-
count) may be seen in inflammatory responses to versely, when sampling is being used for screening
infectious or toxic conditions. The total leucocyte and purposes in apparently healthy animals, changes are
neutrophil counts depend on the balance of demand likely to be more subtle and interpretive success
(tissue requirement and/or endotoxin-induced seques- depends on identifying early changes (eg, reactive
tration) and supply (bone marrow production of neu- monocytes and lymphocytes).

Blood film showing cells

associated with a long-
standing infectious process.
(A) Band (immature)
polymorphonuclear
neutrophils; Al is less
mature than A2. (B) Mature
polymorphonuclear
neutrophils. (C) Reactive
lymphocyte (associated with
antibody production).
(D) Normal monocyte
Blood film showing neutrophilia. Note the presence (associated with macrophage
of increased numbers of mature polymorphonuclear activity). (E) Small clump of
neutrophils platelets

In Practice JUNE 2002 321

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Blood film showing a normal lymphocyte (A) and a reactive

or stimulated lymphocyte (B). The reactive lymphocyte is
larger and has increased blueish cytoplasm. These cells may An abnormal lymphocyte, as may be seen in viral infections.
be associated with post-acute phase inflammatory processes Note the increased cytoplasm which appears clear blue, the
and endotoxaemia (eg, colic) large cell size and relatively non-uniform shape

Reactive monocyte (A) which is typically seen in long-

. :wM -x'IL IO-,wI SW DA Af standing infections in the post-acute phase. Note the
Blood film showing a normal equine monocyte vacuolation of the nucleus

In horses with viral infections, there is an initial leu- LYMPHOCYTOSIS

copenia (usually without the appearance of toxic band
cells) in the first 24 to 48 hours after challenge, followed
by a reflex leucocytosis with lymphocytosis. Large reac-
tive lymphocytes may be a feature. In the post-acute and
recovery phase, a relative monocytosis may be seen. In
an uncomplicated case, the haemogram may return to
normal after five to seven days but, in cases which suc-
cumb to secondary bacterial infection, there will be a
persistent leucocytosis and neutrophilia.
14-
12-
10-
8-
A high lymphocyte count may be seen in some chronic
siral infections or in cases with chronic immune stimula-
tion or lymphoid neoplasia. It should be remembered
that none of these conditions consistently produces a
lymphocytosis. In horses with lymphosarcoma or chron-
ic inflamiimation, peripheral lymphocyte counts are not
often elevated, even though the lymph nodes ar-e
enlarged, but may be dramatically elevated (as high as
100 x 109/litre) in cases of generalised lymphoma.
EOSINOPHILIA
A high eosinophil count suggests an antigen-antibody
response in tissues rich in mast cells (ie, skin, lung,
gastrointestinal tract and female genital tract) and para-
sitism in horses which have become sensitised. The
authors have seen a few cases of eosinophilic leukaemia
with eosinophil counts as high as 2 5 x 1()9/litre (25 per
cent on differential leucocyte count).

6- White blood (cellIs x 1 09/l itre >O:

w

4-

kW~ ~ ~ ~ ~
2-
T~~~~~~~~~~1~~~4
0 I
- W | X r l X
I I
0 A 1 2 3 4 5 6 7

Days Pleomorphic lymphocytes from a horse with lymphoid

Leucoccyte response neoplasia. Note the prominent nucleoli and increased
Viral challenge to vira31 challenge cytoplasm, which is dark blue staining

322 In Practice * J U N E 2002

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Eosinophils may be associated with allergic or parasitic
conditions. Note the bilobed nucleus and prominent red
cytoplasmic granules. Eosinophilia can also be seen in
the post-acute phase of viral infections
BASOPHILIA
Basophils are rarely encountered in equine blood sam-
ples. Increased numbers may be seen in cases of hyper-
lipaemia, conditions that cause eosinophilia or in animals
recovering from colic.
MONOCYTOSIS
A high monocyte count may occur during disease
processes characterised by increased tissue demand for
phagocytosis of macromolecular particles, such as tissue
necrosis, and in chronic suppurative diseases. Mono-
cytosis is often seen in the post-acute or recovery phase
following a viral infection.
THROMBOCYTOPENIA
Increased platelet destruction is usually immune mediat-
ed. This may be primary or occur secondarily to other
disease processes including infections, neoplasia and
drug treatments. Increased platelet usage occurs as a
result of haemorrhage or trauma and leads to a transient
and reversible thrombocytopenia. Disseminated intra-
vascular coagulopathy causes a severe and prolonged
thrombocytopenia.
In some horses, an EDTA-related pseudothrombo-
cytopenia may be seen. The EDTA anticoagulant causes
clumping of the platelets which results in erroneously
low platelet counts being recorded. Examination of a
blood film reveals the presence of platelet clumps, often
at the extremities of the blood film. In such cases,
heparinised or citrated blood should give normal platelet
numbers.
THROMBOCYTOSIS
High platelet counts are seen in some cases of bacterial
infection, notably (but not specifically) Rhodococcus equi
infection in foals.
Platelets
A low platelet count may result from decreased
production, increased usage, or from various spuri-
ous factors (eg, drug administration, presence of
cold agglutinins). Decreased production of platelets
may be associated with neoplasia of, or toxic insult
to, the bone marrow. Pancytopenia occurs in some
of these diseases (eg, lymphoma, myeloma). Bone
marrow aspirates or core biopsies should be exam-
ined for the presence of megakaryocytes.
In Practice * JUNE 2002
Blood film showing basophils. Note the purplish cytoplasmic
granules. Basophils are rare in equine samples although
they have been seen in the recovery phase in horses with
colic
BLOOD BIOCHEMISTRY
PROTEINS
Estimations of total protein, albumin and globulin are
useful in the assessment of general bodily condition and
the response to infectious or parasitic disease.
Albumin
Raised albumin levels occur as a result of haemoconcen-
tration and dehydration while low albumin levels are due
to protein loss from the intestine or kidney, or reduced
albumin production by the liver.
The most common cause of hypoalbuminaemia in the
horse is protein-losing enteropathy. Acute protein-losing
enteropathy is associated with an acute-onset entero-
colitis, such as salmonellosis, or cyathostomiasis during
the larval emergence phase. Chronic protein-losing
enteropathy may be seen with heavy parasitism (cyatho-
stomiasis or mixed large and small stronglye burden) or
a progressive infiltrative lesion of the intestinal mucosa
resulting in chronic malabsorption.
Albumin is lost through the kidney in cases of
renal failure, including pigment-induced nephropathy,
glomerulonephritis and neoplasia.
Liver failure results in reduced production of albumin;
the diagnosis is confirmed by tests showing increased
serum liver enzymes and bile acids.
Globulin
Total globulin is made up of alpha (o), beta (l) and
gamma (y) globulin fractions. The fractions are separated
by electrophoresis and evaluation of the resulting pro-
portions gives considerably more specific information
than an estimation of total globulin alone.
The oc2-globulin range predominantly includes acute
phase tissue proteins and is frequently elevated in cases of
acute inflammation and parasitism (especially cyathosto-
miasis). PI-globulin is often elevated in cases of large and
mixed strongyle larval migration. P2-globulin is usually
raised in horses with hepatopathy. It is of note that fibrino-
gen runs in the same electrophoretic range as 52-globulin,
which makes interpretation of 32-globulin levels difficult
in heparinised plasma samples. Dramatic J32- or y-globulin
monoclonal 'spikes' are sometimes seen in cases with
lymphoma or plasma cell myeloma. y-globulin is raised in
response to antibody production, which may be seen in
horses with chronic infection or inflammation.
Hypogammaglobulinaemia is seen in neonatal foals
in which passive immunity has not been transferred. For
routine passive immunity screening of neonates' IgG,
3 23
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blood (serum) samples should be collected between 18
and 36 hours of age. The most accurate method of mea-
surement is using a specific immunoturbidometric
method but, as this is not available at all laboratories, the
less sensitive ELISA test-kit methods may be used.
INFLAMMATORY PROTEINS
A number of proteins can be measured in equine serum
to help with the diagnosis of inflammatory conditions.
They each have different responses and kinetics and,
therefore, a 'panel' measurement protocol is always
helpful in making interpretations of diagnosis, chronicity
and response to treatment.
Serum amyloid A
Serum amyloid A is a useful inflammatory marker in the
horse. It is a highly sensitive, rapidly reacting, acute
phase inflammatory protein which can help to detect
early responses to infection and monitor the response to
treatment. It rises within hours and peaks at two days,

Albumin

Fraction Rel% g/litre Fraction Rel% g/l itre

1 5-2 1.50 1 2-6 1-56
2 215 624 2 19-2 11-52
3 255 7.40 3 35-2 21-12
4 176 5 10 4 16-9 10-14
5 30 2 8-76 5 26.1 1566
Total globulin = 29 g/litre Total globulin = 60 g/litre
Normal protein electrophoresis trace. The fractions Protein electrophoresis trace from a horse with significant intestinal
represent (from left to right) albumin (large, unshaded parasitic larval migration. The albumin level is relatively low, c2-globulin
area), a1-globulin, a2-globulin, P1-globulin, 0B2-globulin is slightly raised, P1-globulin is markedly raised, and y-globulin is modestly
and y-globulin raised

Albumin

Albumin A
A

Fraction Rel% g/litre Fraction Rel% g/litre

1 2-0 1 50 1 3-5 2 30
2 114 8-55 2 17-9 11-46
3 98 735 3 12-6 8-06
4 728 54-60 4 16-5 10-50
5 40 3-00 5 49-5 31-68
Total globulin = 75 g/litre Total globulin = 64 g/litre
Protein electrophoresis trace from a horse with Protein electrophoresis trace from a horse with a chronic inflammatory
lymphoid neoplasia (plasma cell myeloma). Note response (internal abscess). Note the modest elevation in a2-globulin and
the low albumin and marked monoclonal spike in the marked elevation in y-globulin, indicating tissue damage and antibody
the 02-globulin range response

324 In Practice * JUNE 2002

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Plasma viscosity/cX2-globulin
Plama - Serum amyloid A
Plasma viscosity is a crude measure of acute tissue Plasma fibrinogen
damage and protein production, which can help
in the diagnosis and prognosis of an inflammatory
response. Its use has superseded the measurement
of the erythrocyte sedimentation rate which is
an inherently inaccurate test due to the marked
tendency of horse erythrocytes to form rouleaux.
Plasma:viscosity rises to a peak by five to seven
days, after which it returns to normal, with a satis-
factory response to treatment seen within 10 days
to two weeks.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
A Days
after which it returns to normal, with a satisfactory Inflammatory reaction
response to treatment seen within one week (Pepys and Inflammatory protein dynamics during an inflammatory response
others 1989). iu/litre
- Creatine kinase
3500 -
Plasma fibrinogen - Aspartate aminotransferase
Elevations in plasma fibrinogen, an acute phase reactive 3000 -
protein, are found in horses with tissue damage. An 2500 -
assay of this parameter may help in the diagnosis and
prognosis of cases with more chronic inflammation (eg, 2000 -
internal abscessation, chronic parasitism or infections). 1500 -
The test can be performed crudely by subtraction of
protein levels between fresh paired serum and plasma 1000 -
samples, but more accurate results are obtained by using 500
direct coagulometry on a citrated blood sample. Plasma Iv l
fibrinogen rises to a peak by 10 days and returns to o l l l l l l l l l l l l l l l l l l l l l
normal slowly, with a satisfactory response to treatment 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
seen by three weeks.
Days
t Myopathy occurs

SERUM ENZYMES Serum muscle enzyme levels following a relatively mild episode
Aspartate aminotransferase of exertional rhabdomyolysis
Elevations in aspartate aminotransferase (AST, AAT;
also called serum glutamic-oxaloacetic aminotransferase, cardiomyopathy has now been superseded by the avail-
SGOT) are seen in the presence of acute hepatopathy or ability of serum troponin assays.
myopathy. Levels peak at around 24 to 48 hours after a
myopathic episode and then return to baseline by 10 to Lactate dehydrogenase
21 days, assuming that no further muscle cell damage Lactate dehydrogenase (LD) occurs in a variety of body
occurs. Serial AST results on serum samples, collected tissues, and increases in its total level must be interpret-
after the acute insult, at 10 to 14 days and again three ed after fractionation of the isoenzymes. Specific iso-
weeks later, can provide a useful guide to recovery from enzyme elevations are helpful in the differentiation of
myopathy. some equine conditions (see table below). The most
dramatic isoenzyme abnormalities are seen in cases of
Creatine kinase skeletal myopathy.
Elevations in creatine kinase (CK; also called creatine
phosphokinase, CPK) are seen specifically in the pres- Glutamate dehydrogenase
ence of acute myopathy. Levels peak at six to 12 hours Elevations in glutamate dehydrogenase (GLDH) are seen
and return to baseline by three to four days, assuming in the presence of acute hepatocellular damage (eg,
that no further damage occurs. Paired serum samples ischaemia, halothane toxicity). This is a mitochondrial
collected before, and two to three hours after, submaxi- enzyme found mainly in the liver, kidney and heart mus-
mal exercise and assayed for creatine kinase levels can cle. Experience suggests that levels may also rise in cases
form a useful diagnostic test for exercise-induced myo- of enteropathy and in some horses with skin disease.
pathy in horses where the diagnosis may be in doubt.
Some equine viral infections appear to increase muscle I *S I
membrane permeability/fragility, and predispose to low-
grade increases in circulating muscle enzyme levels. LD isoenzyme
elevation Clinical significance
This is sometimes associated with clinical signs such as
fatigue and stiffness. LD1 elevation Haemolysis
Creatine kinase can be fractionated into its iso- LD2 elevation Myocardial damage
LD3 elevation No known clinical significance in the horse
enzymes of skeletal muscle, cardiac muscle and brain
LD4 elevation Intestinal pathology
origin, but this assay is not widely available in veterinary LD5 elevation Skeletal myopathy or hepatopathy
laboratories. Creatine kinase isoenzyme analysis for

In Practice * JUNE 2002 3325

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Fraction Rel% iu/litre Fraction Rel% iu/litre

LD1 150 101 LD1 3-2 299
LD2 29 8 201 LD2 45 420
LD3 41 4 280 LD3 7-1 654
LD4 11.8 80 LD4 13 2 1233
LD5 2-0 14 LD5 72 0 6725
Total lactate dehydrogenase = 676 iu/litre Total lactate dehydrogenase = 9331 iu/litre
LD1/LD2 =0 5 LD1/LD2 = 0 71
Lactate dehydrogenase isoenzyme trace showing normal distribution Lactate dehydrogenase isoenzyme trace from a horse with recent severe
of fractions rhabdomyolysis. Note the marked elevation in the LD5 fraction

L-gamma glutamyl transferase Sorbitol dehydrogenase

L-gamma glutamyl transferase (GGT) is fh
membranes of hepatocytes and biliary epi
Elevated levels of this enzyme are found in
presence of chronic liver cirrhosis, cholestasis
cyte necrosis. Other sources of L-gamma glu
ferase in the horse include the pancreas
However, because pancreatitis is a very ra
in this species and renal disease does nc
to raised serum L-gamma glutamyl transfei
this enzyme is a relatively specific indicator c
cholestatic disease. Nevertheless, idiopathi
glutamyl transferase elevations are sometimes
formance horses in full work and are often ass
group poor performance. The precise cause c
L-gamma glutamyl transferase syndrome rem
and, following liver biopsy examinations,
means clear that significant hepatopathy is i
association with 'over-training' is suspected. t
ly return to within normal limits after a redL
training schedule.
Automated biochemical
analyser
Sorbitol dehydrogenase (SDH) is a hepatocyte cytoplas-
mic enzyme and, clinically, it is virtually liver-specific.
It is the ideal enzyme for the detection of acute hepato-
cellular damage but, unfortunately, has a relatively short
halt:life (it has to be tested within eight to 12 hours of
sampling) and is therefore inappropriate ftor samples that
need to be dispatched to an external laboratory.
Serum alkaline phosphatase
Serum alkaline phosphatase (SAP) is a brush border
enzyme, elevations of which are most often seen in asso-
ciation with chronic biliary obstructive liver pathology,
abnormalities of bone metabolism and intestinal mal-
function. Reference ranges vary with skeletal maturity
and, therefore, age.
Intestinal alkaline phosphatase
Elevations of intestinal alkaline phosphatase (IAP) rela-
tive to total alkaline phosphatase are seen in the presence
of intestinal pathology in adult horses. Interpretation is
unclear in skeletally immature young horses.
OTHER BIOCHEMICAL TESTS
Bilirubin
Analysis of bilirubin may aid the classification of anaemia
and jaundice but, owing to the horse's unusual biliary
excretion system, indirect (unconjugated) bilirubin levels
may be high in the absence of clinical disease and the sig-
nificance of elevations without other liver enzyme abnor-
malities may be difficult to determine. A period of anorexia
or inanition typically increases indirect bilirubin levels.

Bile acids
Increased serum bile acid levels suggest impaired biliary
function and, therefore, provide a useful prognostic guide.
It is important to remember that the liver has a large
functional reserve and none of the liver enzyme results
give any information about its functional capacity. Bile

326 In Practice * JUNE 2002

 Downloaded from inpractice.bmj.com on March 9, 2013 - Published by group.bmj.com
acid results appear to correlate well with the results of sul-
phobromophthalein clearance times, and biopsy appear-
ance, and so are a convenient measure of liver function.
Glucose
The value of a glucose test in adult horses is limited to
cases of pituitary adenoma (equine Cushing's disease).
In neonatal foals, glucose tests form an important part of
a septicaemia profile. Glucose estimations are also made
in the oral glucose absorption test, which is used to
assess small intestinal absorptive capacity.
Urea
Urea is produced in the liver from the metabolism of
ammonia, and excreted by the kidneys. Elevated urea
levels are seen in the presence of abnormal renal func-
tion. Many cases of equine dysautonomia show a degree
of uraemia, but this may be a result of catabolism and a
period of anorexia can produce similar results.
Creatinine
Creatinine is derived from the breakdown of creatine in
muscle and excreted via the kidneys. Creatinine levels
are an important marker of renal function, with levels
controlled by the excretion rate. The daily production
and excretion is remarkably constant. Elevated levels
of creatinine are seen in association with reduced
glomerular filtration and may result from prerenal
(eg, circulatory disturbances, dehydration, shock), renal
(insufficiency) or postrenal (obstructive) conditions.
ELECTROLYTES
Sodium
Serum sodium (Na) concentrations reflect the ratio of the
total body sodium content in relation to the total body
water, so interpretation of sodium levels should be per-
formed with knowledge of the hydration status of the horse.
Hyponatraemia associated with dehydration indicates
sodium depletion which may occur as a result of:
* Renal loss (due to renal disease, the use of frusemide,
or osmotic diuresis);
* Intestinal loss (diarrhoea, sequestration in distended
bowel); or
* Excessive sweating.
Hyponatraemia is also a feature of uroperitoneum,
which is most commonly seen in neonates with a rup-
tured bladder. If hyponatraemia is seen in an oedematous
horse, congestive heart failure, renal or liver failure
should be considered.
Hypematraemia occurs where there is decreased
water uptake or loss of hypotonic fluid (eg, respiratory
loss, diabetes insipidus).
Potassium
Potassium (K) is primarily an intracellular ion and
consequently serum potassium concentration does not
reflect the whole body concentration of potassium. Acid/
base status further complicates the interpretation of
serum potassium concentration. A decrease in pH of 0-1
unit causes an increase in serum potassium concentration
of 0.6 mmolllitre. Therefore, acidosis can mask potassi-
um depletion by increasing the concentration of potassi-
um in the extracellular fluid.
Hyperkalaemia is primarily associated with reduced
renal excretion (abrupt reduction in glomerular filtration,
uroperitoneum) or a shift of potassium from the intra-
In Practice J U N E 2002
cellular to the extracellular compartment as a result of
acidaemia. Equine erythrocytes contain a relatively high
potassium concentration, and haemolysis in the sample
will artificially elevate serum potassium levels.
Hypokalaemia is caused by conditions that increase
potassium loss, including:
* Renal loss (due to renal disease or the use of
frusemide);
* Intestinal loss (eg, diarrhoea, sequestration in distend-
ed bowel); or
* Sweating (equine sweat has a high potassium content).
Chloride
Chloride (Cl) is a major cation in the circulation and
changes in its concentration tend to follow and mirror
the other major ions. Hypochloraemia is associated with
hyponatraemia, increased bicarbonate or an increased
anion gap. Hyperchloraemia is associated with hyper-
natraemia or decreased serum bicarbonate concentra-
tions. Equine sweat contains higher levels of chloride
than sodium, and sweating may cause a more pro-
nounced hypochloraemia than hyponatraemia.
Calcium
Calcium (Ca) exists in the circulation in its free ionised
state, complexed with cations, and bound to albumin (40
per cent). Routine assays measure total calcium concen-
tration. Given the high degree of albumin binding, results
should be interpreted with a knowledge of the protein sta-
tus of the horse. Calcium is primarily excreted in the kid-
neys, and so disorders that reduce glomerular filtration
cause hypercalcaemia. Other conditions which can affect
calcium levels are pseudohyperparathyroidism of neo-
plasia, and vitamin D toxicosis. Hypocalcaemia may be
caused by, or associated with, hypoalbuminaemia, inade-
quate dietary calcium intake, lactation and liver disease.
An imbalance of calcium and phosphorus in the diet
can lead to hypocalcaemia and secondary hyperpara-
thyroidism, particularly in growing horses. Assessment
of the renal fractional excretion of phosphorus can be
helpful in clarifying the diagnosis when physiological
processes have been able to maintain serum calcium and
phosphorus values within normal ranges.
Phosphate
Hyperphosphataemia is most often seen as a result of
leakage from erythrocytes in haemolysed samples.
Hypophosphataemia can be caused by increased loss or
decreased intake of phosphate (P04). Renal disease
results in hypophosphataemia in the horse.
SUMMARY
The use of laboratory tests has become an essential part
of equine practice. Clients, insurance companies and Acknowledgement
colleagues expect the modem practitioner to use clinical The authors would like to thank
Alison Haslam for her help and
pathology as an aid to diagnosis in addition to the various expertise with the haematology
other clinical diagnostic tests which are now available. illustrations.
When used appropriately, the results of laboratory tests
Reference
will be helpful to the patient and its owner in terms of PEPYS, M. B., BALTZ, M. L.,
making a definitive diagnosis, assessing the prognosis TENNANT, G. A., KENT, J.,
OUSEY, J. C. & ROSSDALE, P. D.
and monitoring the response to treatment and progression (1989) Serum amyloid A (SAA)
of a case. The expense of carrying out laboratory tests is in horses: objective
measurement of the acute
often outweighed by the costs of a missed or delayed phase response. Equine
diagnosis, both in financial and welfare terms. Veterinary Journal 21, 106-109
327
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Haematology and blood biochemistry in the

horse: a guide to interpretation
Annalisa Barrelet and Sidney Ricketts

In Practice 2002 24: 318-327

doi: 10.1136/inpract.24.6.318

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http://inpractice.bmj.com/content/24/6/318

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