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Haemnatology and Blood Biochemnistry
Haemnatology and Blood Biochemnistry
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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
1 1 .0 .....11 I
Choke of laboat.ryltsts
Profile type Profile content
It is very important to use laboratory that is expe-
a
36 hours Haematology, serum proteins, fibrinogen, PV, SAA, serum IgG
rienced in testing equine samples and interpreting Weanling and yearling Haematology, serum proteins and electrophoresis, fibrinogen, SAP,
the results produced. All clinical pathology labora- serum calcium and phosphate, urine phosphate fractional clearance ratios
tories, whether in-house or extemal, should have Inflammatory profile Haematology, serum proteins, PV, fibrinogen, SAA
an active quality control programme and should
Training horse Haematology, serum proteins and electrophoresis, PV, fibrinogen, SAA,
subject themselves to external quality assessment. AST, CK, urine phosphate fractional clearance ratios
Laboratory analysis of samples has significant Mature horse Haematology, serum proteins and electrophoresis, fibrinogen, PV, SAA,
cost implications and tests should be carefully AST, CK, GLDH, LD, SAP, IAP, GGT, urea, creatinine
selected with specific objectives in mind. Most labo- Intestinal profile Haematology, serum proteins and electrophoresis, fibrinogen, PV, SAA,
ratories offer 'profiles' which will be more economi- SAP, IAP
cal than individual tests (see table on the right). If Liver profile Haematology, serum proteins and electrophoresis, fibrinogen, PV, SAA,
AST, GLDH, LD, SAP, IAP
the case history is complex, vague or otherwise not
straightforward, it is not always cost effective to AST Aspartate aminotransferase, CK Creatine kinase, GGT L-gamma glutamyl transferase,
GLDH Glutamate dehydrogenase, IAP Intestinal alkaline phosphatase, LD Lactate dehydrogenase,
minimise the initial investigations. PV Plasma viscosity, SAA Serum amyloid A, SAP Serum alkaline phosphatase
In Practice * J U N E 2002 3 19
Downloaded from http://inpractice.bmj.com/ on May 28, 2015 - Published by group.bmj.com
* Parasitic or allergic conditions (eosinophilia in some Macrocytic aniaemia indicates red blood cell regener-
cases). ation because young or newly produced red blood cells
Haemoconcentration can only be interpreted if sam- are larger than mature ones. A spurious increase in red
ples are taken from resting horses under cailm conditions. blood cell size occurs as a result of degeneration of the
This can be difficult to achieve in needle-shy, nervous cells in vitro due to osmotic swelling. Anisocytosis (vari-
or excitable animals. Post-exercise samples are usually ability in the size of red blood cells) is caused by the
impossible to interpret. Where routine blood profiling presence of macrocytes and microcytes among normnal
of competition horses is performed, samples should be red blood cells. This is a rare finding in horses and
taken regularly at the same quiet time of the day when pomies and is Lisually associated with a severe reoenera-
horses are at rest. tiv e anaemia.
ANAEMIA
Most cases of equine anaemia occur secondarily to other LEUCOCYTOSIS
conditions. A regenerative anaemia is one in which the Physiological leucocytosis (high white blood cell count)
bone marrow responds appropriately with incr-eased red occurs as a result of excitement or stress. Increased num-
blood cell production. Examples include acute blood bers of mature neutrophils and lymphocytes are released
loss, haemolytic anaemia, and mild/moderate intestinal into the circulation from their reserses and marginated
parasitism. Reticulocytes are seldom found in the periph- pools. The response occurs rapidly (in seconids) and is
eral blood of adult horses and are rarely found in foals due to increased blood pressure. heart rate and splenic
recosering from haiemolytic anaemia (neonatal isoery- contractioIl, all of which aIc associated with intense
throlysis). In other species, they are often found in asso- exercise, fear or excitement. It is important to consider
ciation with blood loss or a hypoxic insult. this response when interpreting haemnatological data
Non-regenerative anaemia, or anaemia associated from young- or excitable Individuals. The response is a
with inadequate red blood cell productioni, may occur in tranisient phenomenon and norimial resting levels are i-e
cases with generalised bone marrow suppress.ion, chronic established within about an hour.
inflammation, neoplasia, chronic renal disease, or iron Pathological leucocytosis with neutrophilia occuIrs
deficiency. A specific diagnosis relies on further blood durine infectious and non-infectious inflaimmll.atory coil-
biochemistry and other diagnostic tests (eg, examiniation ditions and the differentiation can sometimies be unclear.
of bone marrow, peritoneal and pleural fluids). A positi'Ve An increased numbei of circulating immature neLitro-
direct antiglobulin (Coombs) test indicates the presence of phils (ie, metcamyelocyte and 'band' neutrophils) is the
antibody or complemiient on red blood cell surtaces, and is most sensitive differentiator of infectious inflammatorv
diagnostic of immune-mediated anaemia. This test maiy be disease, but these arc not seen as frequently in horses
positive in cases of idiopathic immune-mediated anaemia, and ponies as in somile other animals. In horses, band
neonatal isoerythrolysis and equine infectious anaemia. 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:
Blood film from a horse with * Increased utilisation in inflamed tissues:
severe haemolytic anaemia.
Note the marked variability in * Sequestration to highly vascular organs (eg, in cases
the size of the red blood cells of pneumonia);
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).
4-
kW~ ~ ~ ~ ~
2-
T~~~~~~~~~~1~~~4
0 I
- W | X r l X
I I
0 A 1 2 3 4 5 6 7
Eosinophils may be associated with allergic or parasitic Blood film showing basophils. Note the purplish cytoplasmic
conditions. Note the bilobed nucleus and prominent red granules. Basophils are rare in equine samples although
cytoplasmic granules. Eosinophilia can also be seen in they have been seen in the recovery phase in horses with
the post-acute phase of viral infections colic
blood (serum) samples should be collected between 18 therefore, a 'panel' measurement protocol is always
and 36 hours of age. The most accurate method of mea- helpful in making interpretations of diagnosis, chronicity
surement is using a specific immunoturbidometric and response to treatment.
method but, as this is not available at all laboratories, the
less sensitive ELISA test-kit methods may be used. Serum amyloid A
Serum amyloid A is a useful inflammatory marker in the
INFLAMMATORY PROTEINS horse. It is a highly sensitive, rapidly reacting, acute
A number of proteins can be measured in equine serum phase inflammatory protein which can help to detect
to help with the diagnosis of inflammatory conditions. early responses to infection and monitor the response to
They each have different responses and kinetics and, treatment. It rises within hours and peaks at two days,
Albumin
Albumin
Albumin A
A
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
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
acid results appear to correlate well with the results of sul- cellular to the extracellular compartment as a result of
phobromophthalein clearance times, and biopsy appear- acidaemia. Equine erythrocytes contain a relatively high
ance, and so are a convenient measure of liver function. potassium concentration, and haemolysis in the sample
will artificially elevate serum potassium levels.
Glucose Hypokalaemia is caused by conditions that increase
The value of a glucose test in adult horses is limited to potassium loss, including:
cases of pituitary adenoma (equine Cushing's disease). * Renal loss (due to renal disease or the use of
In neonatal foals, glucose tests form an important part of frusemide);
a septicaemia profile. Glucose estimations are also made * Intestinal loss (eg, diarrhoea, sequestration in distend-
in the oral glucose absorption test, which is used to ed bowel); or
assess small intestinal absorptive capacity. * Sweating (equine sweat has a high potassium content).
Urea Chloride
Urea is produced in the liver from the metabolism of Chloride (Cl) is a major cation in the circulation and
ammonia, and excreted by the kidneys. Elevated urea changes in its concentration tend to follow and mirror
levels are seen in the presence of abnormal renal func- the other major ions. Hypochloraemia is associated with
tion. Many cases of equine dysautonomia show a degree hyponatraemia, increased bicarbonate or an increased
of uraemia, but this may be a result of catabolism and a anion gap. Hyperchloraemia is associated with hyper-
period of anorexia can produce similar results. natraemia or decreased serum bicarbonate concentra-
tions. Equine sweat contains higher levels of chloride
Creatinine than sodium, and sweating may cause a more pro-
Creatinine is derived from the breakdown of creatine in nounced hypochloraemia than hyponatraemia.
muscle and excreted via the kidneys. Creatinine levels
are an important marker of renal function, with levels Calcium
controlled by the excretion rate. The daily production Calcium (Ca) exists in the circulation in its free ionised
and excretion is remarkably constant. Elevated levels state, complexed with cations, and bound to albumin (40
of creatinine are seen in association with reduced per cent). Routine assays measure total calcium concen-
glomerular filtration and may result from prerenal tration. Given the high degree of albumin binding, results
(eg, circulatory disturbances, dehydration, shock), renal should be interpreted with a knowledge of the protein sta-
(insufficiency) or postrenal (obstructive) conditions. tus of the horse. Calcium is primarily excreted in the kid-
neys, and so disorders that reduce glomerular filtration
ELECTROLYTES cause hypercalcaemia. Other conditions which can affect
Sodium calcium levels are pseudohyperparathyroidism of neo-
Serum sodium (Na) concentrations reflect the ratio of the plasia, and vitamin D toxicosis. Hypocalcaemia may be
total body sodium content in relation to the total body caused by, or associated with, hypoalbuminaemia, inade-
water, so interpretation of sodium levels should be per- quate dietary calcium intake, lactation and liver disease.
formed with knowledge of the hydration status of the horse. An imbalance of calcium and phosphorus in the diet
Hyponatraemia associated with dehydration indicates can lead to hypocalcaemia and secondary hyperpara-
sodium depletion which may occur as a result of: thyroidism, particularly in growing horses. Assessment
* Renal loss (due to renal disease, the use of frusemide, of the renal fractional excretion of phosphorus can be
or osmotic diuresis); helpful in clarifying the diagnosis when physiological
* Intestinal loss (diarrhoea, sequestration in distended processes have been able to maintain serum calcium and
bowel); or phosphorus values within normal ranges.
* Excessive sweating.
Hyponatraemia is also a feature of uroperitoneum, Phosphate
which is most commonly seen in neonates with a rup- Hyperphosphataemia is most often seen as a result of
tured bladder. If hyponatraemia is seen in an oedematous leakage from erythrocytes in haemolysed samples.
horse, congestive heart failure, renal or liver failure Hypophosphataemia can be caused by increased loss or
should be considered. decreased intake of phosphate (P04). Renal disease
Hypematraemia occurs where there is decreased results in hypophosphataemia in the horse.
water uptake or loss of hypotonic fluid (eg, respiratory
loss, diabetes insipidus).
SUMMARY
Potassium
Potassium (K) is primarily an intracellular ion and The use of laboratory tests has become an essential part
consequently serum potassium concentration does not of equine practice. Clients, insurance companies and Acknowledgement
reflect the whole body concentration of potassium. Acid/ colleagues expect the modem practitioner to use clinical The authors would like to thank
Alison Haslam for her help and
base status further complicates the interpretation of pathology as an aid to diagnosis in addition to the various expertise with the haematology
serum potassium concentration. A decrease in pH of 0-1 other clinical diagnostic tests which are now available. illustrations.
unit causes an increase in serum potassium concentration When used appropriately, the results of laboratory tests
Reference
of 0.6 mmolllitre. Therefore, acidosis can mask potassi- will be helpful to the patient and its owner in terms of PEPYS, M. B., BALTZ, M. L.,
um depletion by increasing the concentration of potassi- making a definitive diagnosis, assessing the prognosis TENNANT, G. A., KENT, J.,
OUSEY, J. C. & ROSSDALE, P. D.
um in the extracellular fluid. and monitoring the response to treatment and progression (1989) Serum amyloid A (SAA)
Hyperkalaemia is primarily associated with reduced of a case. The expense of carrying out laboratory tests is in horses: objective
renal excretion (abrupt reduction in glomerular filtration, measurement of the acute
often outweighed by the costs of a missed or delayed phase response. Equine
uroperitoneum) or a shift of potassium from the intra- diagnosis, both in financial and welfare terms. Veterinary Journal 21, 106-109
These include:
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Notes