Download (Diagnostic-Techniques-for-Liver-Disease-in-Dogs-Cats

november 08
Diagnostic
Update
DIAGNOSTIC TECHNIQUES FOR LIVER DISEASE
IN DOGS, CATS (PART 2/2) AND HORSES
The liver is strategically positioned between the digestive tract and the systemic circulation. Arterial blood flows directly
from the aorta to the liver, ensuring a good oxygen supply.
The liver receives venous blood from the abdominal organs (stomach, pancreas, spleen and intestines) via the portal vein.
Amongst other compounds, metabolites and foreign substances are delivered to the liver in this venous blood for detoxification or biotransformation. The liver and portal circulation also play a central role in the metabolism of bile acids, ammonia and bilirubin.
This section of the diagnostic update will discuss pathophysiol-
The categorisation of icterus into a pre-hepatic/haemolytic, a
ogy and the meaning of variations in values for bilirubin, bile acid
hepatic or a post-hepatic/obstructive icterus is of key importance
and ammonia concentrations.
in considering the possible differential diagnoses
Following this, some principles for the interpretation of cytological
and histological findings in liver samples will be discussed, as the
1.1.1 Pre-hepatic hyperbilirubinaemia
cyto- and histopathological investigations of liver samples are key
Destruction of the erythrocytes (intravascular or extravascular
to the early diagnosis of liver disease.
haemolysis) is central to pre-hepatic icterus. The liver cannot
cope with the uptake and processing of the additional bilirubin.
1 Physiology and pathology of bilirubin, bile
acid and ammonia metabolism
Erythrocyte breakdown that is sufficiently acute to cause hyperbilirubinaemia is usually associated with an obvious anaemia (haematocrit < 25%). The anaemia is regenerative with haemolysis.
1.1 Bilirubin
Reticulocytosis may be mild at first (the first 2 – 4 days).
Bilirubin is a breakdown product of haemoproteins. Its main
origin is the haemoglobin from aging erythrocytes. Unconjugated
Perhaps unexpectedly, dogs suffering from haemolysis often also
bilirubin is first produced by the reticuloendothelial system, which
exhibit bilirubinuria. Urine chemistry reagent strips (“DipStix”)
is then bound to albumin and transported in the plasma. Bilirubin
detect only conjugated bilirubin and not the unconjugated form
is then conjugated with glucuronic acid in the liver, transported
increased by the breakdown of the haemoproteins. Furthermore,
through the biliary system with other components in the bile, and
unconjugated bilirubin cannot pass through the glomerular mem-
excreted into the digestive system. The bilirubin is then deconju-
brane. However, small amounts of free bilirubin may be conju-
gated to urobilinogen by bacteria in the ileum and colon.
gated by the renal tubules and then excreted in the urine.
80 – 90% of the urobilinogen is eliminated in the faeces as stercobilin, while the remaining 10 – 20% is reabsorbed (enterohepatic
Causes of pre-hepatic icterus may be, for example, an immune-
circulation).
mediated haemolytic anaemia, (either primary, or secondary to
drugs, infection or neoplasia); infections (FeLV, Mycoplasma
Icterus is a syndrome that is characterised by hyperbilirubinaemia
and a yellow colouration of the skin, mucosa and sclera due to
the deposition of bile pigments. Serum bilirubin concentration
in dogs and cats should not normally exceed 7 micromol/l. The
separated plasma becomes icteric once serum bilirubin concentrations exceed approximately 17 micromol/L. Obvious icterus is
present when serum bilirubin concentrations exceeds approximately 30 micromol/L. The yellow discolouration is most clearly
recognised under natural light and in the following organs: sclera,
oral mucosa, the pinnae of the ears, the skin around the navel
and the penile and vaginal mucosae.
haemofelis, others); oxidative damage to the erythrocytes (on-
Clinical distinction between hepatic and post-hepatic icterus can
ions, zinc, hypophosphataemia); or the resorption of blood (large
be very difficult. However this is important, as the therapeutic
haematoma). In the case of hyperbilirubinaemia, a pre-hepatic
strategies are different for the two forms of the disease. Ultra-
cause should always be looked for first in patients showing both
sound investigation can be a useful aid in the distinction between
hyperbilirubinaemia and anaemia.
these forms of icterus.
1.1.2 Hepatic hyperbilirubinaemia
1.2 Bile acids
In cases of icterus of hepatic origin, one of the stages of hepato-
Bile acids (BA) are synthesised from cholesterol solely in the liver.
cellular bilirubin transport is impaired. For example, inflammatory
After conjugation with taurine or glycine, the BAs are secreted
processes may result in swelling of the liver cells and impaired
into the bile and stored in a concentrated form in the gall blad-
biliary flow, which leads to obstruction of the intra-hepatic biliary
der. After food intake, cholecystokinin triggers the contraction of
system (cholestasis).
the gall bladder and the release of BAs into the intestines. BAs
play an important role in the digestion and absorption of fat in
However, serum bilirubin is not a good indicator of hepatocellular
the intestines. The BAs are transported into the portal circulation
function. The liver can continue to metabolise bilirubin long after
in the ileum and thus return to the liver. The BAs are once more
other liver functions are already impaired. Thus hyperbilirubinae-
extracted from the portal circulation in the liver.
mia of hepatic origin indicates liver pathology is already moderate
to severe.
Although BA synthesis can be severely compromised in patients
with severe liver disease, different factors can cause increased
Frequent causes of hepatic icterus in cats are cholangitis/cholan-
BA concentrations in the blood. These include changes to the
giohepatitis, lymphosarcoma, hepatic lipidosis and FIP.
enterohepatic circulation, a portosystemic shunt (PSS), reduced
In dogs, triggers for hepatic icterus include chronic hepatitis (idi-
extraction of BAs from the blood by the liver, or regurgitation of
opathic, hereditary), lymphosarcoma, acute hepatic necrosis and/
BAs into the systemic circulation due to cholestasis. This is why
or cirrhosis. Drugs (anti-epileptic drugs, trimethoprim-sulphona-
many animals suffering from chronic hepatitis, extensive hepatic
mide, others) and systemic diseases with a hepatic component
necrosis, or liver neoplasia often exhibit raised BA values. BAs,
may also cause icterus. Bacterial toxins and antibodies against
together with ammonia, are among the most sensitive biochemi-
components in the bacterial cell walls can also cause cholestasis
cal indicators of a congenital PSS. Most patients suffering from
(cholestasis due to sepsis). In contrast, hepatic atrophy, as is often
a congenital portal vascular anomaly have increased values for
seen in animals with a congenital portosystemic vascular anomaly,
postprandial bile acid concentrations.
is not normally associated with a hyperbilirubinaemia, even though
In contrast, BA values are often not greatly raised in cases of a
other indications for impaired liver function (hypoalbuminaemia,
secondary involvement of the liver due to a non-hepatic primary
low urea concentrations) are present.
disease, administration of glucocorticoids or anti-epileptic drugs.
There are a number of factors that can result in lowered BA serum
1.1.3 Post-hepatic hyperbilirubinemia
concentrations. Some examples are anorexia lasting more than
In post-hepatic icterus, intraluminal or extraluminal problems
1-2 days (fasting BA value possibly lowered); delayed emptying
result in a mechanical obstruction to the excretion of bilirubin.
of the stomach; changes in intestinal transit time; malabsorption,
Examples of this are pancreatitis, neoplasia (bile ducts, pancreas,
severe disease; and resection of the ileum.
duodenum), cholelithiasis, rupture of the gall bladder or a bile
duct with biliary peritonitis.
Determination of BA concentrations is routinely conducted as a
screening test for liver function in small animal veterinary practices, as the collection of samples from the patients and laboratory
determination are both straightforward. Maximum information is
obtained through determination of a 12-hour fasting and a 2-hour
postprandial value. Suitable food for the determination of the
postprandial value in dogs and cats is canned food with a moderate fat content: two teaspoons for animals of below 5 kg or two
dessertspoons of food for heavier animals.
False positive results can occur. Postprandial values should still
be determined if preprandial values have been determined and
these are normal. Higher fasting values than postprandial BA
values are occasionally found. The causes of this are interdigestive gall bladder contractions during fasting prior to the conduc-
tion of the test, as well as individual variation in emptying of the
2 Cytology and histology of liver samples
stomach, response to cholecystokinin secretion, or intestinal
transit times.
Differentiation between different liver diseases is usually not possible based on the clinical symptoms and changes in laboratory
Determination of BA concentrations is not indicated in icteric
values alone. Further information should be gained by taking
patients with hepatobiliary disease as it does not provide any
liver samples for cytological and histological examinations. Such
additional information. Severe haemolysis or severe lipaemia may
sampling is not only indicated to reach an accurate diagnosis,
result in false lowered results.
but also to obtain clues on the response to treatment and disease
Please note that Maltese dogs are distinctive in that they can
progression.
exhibit raised postprandial values without suffering from hepatobiliary disease.
The investigation of material that has been obtained using fine
needle aspiration (cytology) is certainly less informative than ma-
Two qualifying points must be considered in the interpretation
terial obtained through a biopsy (histology). However, fine needle
of BA values. Firstly, different hepatobiliary diseases cannot be
aspiration also has advantages such as less danger of haemor-
distinguished from one another based on BA determinations.
rhage, and it is easier to perform. In addition, especially in cases
Secondly, there is a very poor correlation between the severity of
of diffuse diseases, fine needle aspiration can result in diagnosis,
histological lesions or the degree of a PSS, and the magnitude of
e.g. for lymphosarcomas, mast cell tumours, hepatic lipidosis,
increase in BA values. The only reliable indicator for clinical remis-
corticosteroid- induced changes and amyloidosis. It also enables
sion is a return to normal values, following several BA determina-
diagnosis of a hepatocellular carcinoma.
tions conducted in a patient in order to assess disease progression or therapeutic response.
A liver biopsy is taken using ultrasound monitoring or within the
framework of a laparotomy to collect samples. Animals with
1.3 Ammonia
hepatopathies exhibit a tendency towards coagulopathies and
For the purposes of detoxification, the ammonia (NH3) produced
coagulation testing is therefore recommended prior to the proce-
in protein metabolism is transformed into urea (urea cycle) in the
dure.
mitochondria of the liver cells. Raised ammonia concentrations
Parenteral administration of vitamin K1 can be used prophylacti-
can occur in cases of hepatic insufficiency, when the liver can no
cally 24 hours before the biopsy is taken. A plasma transfusion
longer detoxify the ammonia, or when the portal blood is not flow-
may help if a biopsy must be taken even though a coagulopathy
ing through the liver (PSS). The significance of ammonia determi-
is present. The interpretation of the histological findings can be-
nation is similar to that of bile acid determination. It has recently
come complicated if the sample is too small or not representative
been demonstrated that fasting ammonia values are slightly more
of the lesion.
sensitive and, above all, more specific than bile acid values for
the diagnosis of a congenital or acquired PSS.
Even if the histological investigation of a liver sample does not always guarantee an aetiological diagnosis, as a rule, the following
There are two disadvantages of ammonia analysis compared
statements are possible:
with the bile acid analysis. Firstly, there are the more complicated
handling requirements for the sample – analysis is usually only
1. Disease category: inflammatory/necrotic, neoplastic,
practicable in the practice environment. The reader is advised
vacuolated and vascular
to take particular care to follow the relevant instructions for the
2. Extent of the disease: mild/moderate/severe
analyser. Secondly, the conduct of an ammonium chloride toler-
3. Chronicity of the lesion: acute versus chronic
ance test in animals suffering from encephalopathy can result in a
deterioration in the neurological symptoms.
Chronic inflammatory lesions pose a particular challenge to
pathologists, as the liver reacts in a similar histological fashion to
different types of chronic insult (whether due to toxins, infection
or immune stimulation). Degenerative changes to the liver cells,
inflammatory infiltrates, fibrosis and necrosis are possible manifestations of an inflammatory event. The inflammatory cell type
should be noted when assessing inflammatory changes: Neutrophilic granulocytes (“acute”) are often present at the start of an
inflammation, with the addition later on of lymphocytes and macrophages. The presence of fibrosis is an indicator for a “chronic”
event. The degree of fibrosis is correlated with survival time.
For example, bridging fibrosis (connective tissue connections
In summary, it should be noted that in spite of the limitations of
between portal triads or portal triads and the central vein) indicate
cytological and histological investigations of liver samples, these
a poor prognosis. Eosinophilic granulocytes often indicate an
can serve a very important role in the treatment of patients with a
allergic or parasitic event.
hepatopathy.
Although histological analysis does not always permit a definitive
aetiological diagnosis, it generally does provide indicators for a
possible aetiology. For example, centri-lobular lesions occur in
passive congestion of the liver, while infections like salmonellosis
or toxoplasmosis result in (multi-) focal lesions. Vacuolated le-
Author:
sions indicate the presence of lipid or glycogen accumulation and
PSS also exhibits specific histological characteristics.
Further investigations can be instigated if required. For example,
specific staining can be an aid in the identification of certain infectious pathogens or permit estimates of copper content in a liver
Dr. med. vet. Cécile Rohrer Kaiser
sample. In addition, samples can be obtained for aerobic and
Dipl. ACVIM (Internal Medicine) and
ECVIM-CA (Internal Medicine)
anaerobic bacterial cultures.
LIVER DIAGNOSIS IN HORSES
The liver is one of the main metabolic organs and, as such, is positioned between the digestive tract and the systemic
circulation. The majority of compounds absorbed from the gastrointestinal tract reach the liver directly through the portal
circulation. The liver is the regulatory site of carbohydrate, protein and lipid metabolism.
In addition, the liver is an organ responsible for excretion (bile for
Clinical symptoms
the digestion of fats), a storage organ (glycogen, vitamins, trace
The clinical symptoms of liver disease are often unspecific or may
elements), an organ where synthesis occurs (albumin, fibrinogen,
be completely absent. Signs include lethargy, appetite disor-
prothrombin), and it participates in immune regulation (Kupffer’s
ders, weight loss, lowered performance, dull coat, dermatoses,
cells).
neurological symptoms, icterus, photosensitisation, abdominal
pain and coagulation disorders. Behavioural disorders are to be
Liver function is only impaired once more than 80% of the liver
regarded as typical symptoms for advanced liver disease, caused
has been damaged. However, the liver does possess a unique
by the malfunction of ammonia detoxification by the damaged
capacity for maintaining its specific functions and simultaneously
liver (hepatoencephalopathy).
repairing and regenerating its own tissue.
The severity of the clinical symptoms and the course of a liver disAetiology
ease can vary substantially depending on the distribution pattern,
Liver disease is relatively common in horses, but usually
location and extent of damage to the liver. Basically, a distinction
progresses without any clear clinical symptoms. Liver disease
must be made between reversible diseases (e.g., swelling, fatty
often occurs secondarily, i.e. due to other diseases (viral, bacte-
degeneration) and irreversible damage (necrosis), which may
rial, parasitic infectious disease, internal disease, fatty liver). Such
both be focal (abscess, neoplasia) and/or zonal (centrilobular) in
diseases can, however, also be caused directly by contaminated
their extent. An acute generalized hepatitis causes a loss in func-
feed (mycotoxins) or toxic plants. A full case history is therefore of
tion that is usually associated with enlargement of the liver.
great importance with regard to treatment.
Clinical symptoms only appear when more than 80% of the liver
has been damaged in chronic generalised fibrosis (in the final
stages of cirrhosis). In this case, the liver is reduced in size.
Diagnosis of liver disease
in young animals due to their physiology, specifically the more
The permeability of the cell membrane is impaired in many
active bone metabolism in animals during growth.
pathological processes. Enzymes that are mainly intracellular can
therefore be released into the blood plasma, where they can be
AST (aspartate aminotransferase) / GOT (glutamate-oxalacetate
measured.
transaminase) occurs in the mitochondria and in the cytoplasm
of liver cells, but also in muscle cells and is therefore not liver-
Enzymes specific to the liver in horses
specific. Particularly high levels are observed during the course of
GGT is located in membrane structures, mainly of the biliary
advanced myopathies (sporadic exertional rhabdomyolysis).
system.
Its half-life is approximately 3 days. It is released at an early stage
Assessment of liver function
in liver disease and is often the only parameter that is raised in
Bile acids are synthesised from cholesterol in the hepatocytes.
chronic metabolic disorders affecting the liver. GGT can also
These are continually released into the duodenum (approx.
continue to rise for 1-2 weeks after the cause of the increase
3l/100kg BW) in horses, where they enable the digestion of fats.
in levels has been treated. During convalescence, it may also
They are then partially reabsorbed in the enterohepatic cycle. In
increase if the horse is under pressure, so can be used to monitor
liver disease, the bile acids can no longer be adequately elimi-
the workload.
nated and therefore they accumulate. The finding of raised levels
of bile acids is therefore useful to assess liver function, but does
GLDH (glutamate dehydrogenase) is an enzyme that is bound to
not indicate the type of liver disease.
the mitochondria of the liver cells. Most of its activity is centrilobular, i.e. it reacts highly sensitively to secondary hepatopathies
Ammonia is neurotoxic and is produced in the intestines dur-
(cholestasis, hypoxaemia). An increase to more than 3 times the
ing protein digestion. It enters the liver through the portal vein,
normal value indicates the presence of an acute hepatopathy with
where it is transformed into urea and is then excreted through
liver cell necrosis. Milder increases are seen during infections,
the kidneys. This capacity for conversion to urea is restricted in
fever or the administration of drugs. Its half-life is approximately
functional disorders of the liver and blood ammonia concentra-
3 days.
tions rise as a result.
CNS function can be impaired as ammonia is neurotoxic (hepa-
Other enzymes
toencephalopathy). Determinations should only be made on
ALKP (alkaline phosphatase) is an enzyme bound to the mi-
blood that has been centrifuged immediately after collection and
tochondrial membrane and occurs in many organs (bile duct
on frozen EDTA plasma, as ammonia is highly unstable in blood
epithelia, osteoblasts).
samples.
Increases in enzyme levels are observed in cholestasis, but also
after the administration of drugs (corticosteroids). ALKP is higher
Bilirubin is a breakdown product of haemoglobin that is insoluble in water and is transported to the liver bound to albumin.
Once in the liver, it is conjugated with glucuronic acid in the hepatocytes and secreted into the intestines in a water-soluble form via
the bile. From the intestines, it is either excreted in the faeces or
reabsorbed (enterohepatic circulation). Bilirubin is not a sensitive indicator for liver disease, as it is not only raised in cases of
hepatopathies, but also due to haemolysis (babesiosis, infectious
anaemia, neonatal icterus), anorexia (inanition icterus) and colic.
Persistent hyperbilirubinaemia has been described in otherwise
healthy horses (Gilbert’s syndrome, Crigler-Najjar syndrome).
Plasma proteins are mainly synthesised in the liver (albumin
is synthesised in the liver only), with the exception of the immunoglobulins. Synthesis of these proteins is limited in most severe
and/or chronic liver diseases.
Measuring the concentrations of these proteins can assess the
liver’s capacity for synthesis.
Albumin concentrations are of little significance for diagnosis
as synthesis is reduced only in late on in the course of liver disease.Furthermore, albumin concentrations may also be lowered
Diagnostic
Update
Authors:
in nephropathies, enteropathies, body cavity effusions and
malnutrition.
There may be reduced synthesis of coagulation factors in
severe liver disorders.
Further investigations
Andrea Hille
Following on from the assessment of clinical symptoms and
laboratory tests, ultrasound examinations and a liver
Expert Advisor on Horses
biopsy
can
Dr. Susi Zintner
Horse Veterinary Specialist,
Key Account Manager, Medical Advisor
provide further information on the severity of the disease and its
prognosis. The aetiology often remains unclear even after using
these techniques. It is always recommended to check the coagulation factors prior to conducting a liver biopsy.
Diagnostic options for liver diseases
IDEXX Reference Laboratory
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(
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IDEXX Reference Laboratory Wetherby
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Tel: 01937 544000, Fax: 01937 544001
[email protected]
www.idexx.eu/united-kingdom/
UK033-1008