Clinical pathology & Cytology Part 2

Why can you see elevations in liver enzymes in diabetes?

Because insulin deficiency in diabetes mellitus leads to abnormal glucose and lipid metabolism —> fatty infiltation/hepatic lipidosis

Why can infection and dental disease cause elevations in liver parameters?

The liver contains Kupffer cells which are involved in the immune response and filter toxins and bacteria. These cells react to infections, sepsis and endotoxaemia originating at any extra-hepatic site. Dental disease can cause mild to moderately elevated ALT and ALP levels.

What haematological changes are seen in PSS?

• Mild anaemia

• Microcytic red cells with or without anaemia due to iron deficiency

What biochemical changes are see in PSS?

Normal or mild increase in ALP and ALT

Normal bilirubin

Low albumin

Low cholesterol

Low potassium

Low urea

Elevated BAs and ammonia, esp post prandially

Low glucose

What urinary changes can be seen in dogs with PSS?

Presence of ammonium biurate crystals due to increased ammonia and reduced uric acid to allantoin by hepatic uricase

What biochemical changes are usually seen in acute hepatitis?

• Rapid marked increase in ALT, less marked increase in ALP and GGT

• Elevated BAs and bilirubin (depending on the severity of damage)

• Albumin usually normal

What biochemical changes are seen in chronic hepatitis?

• Moderate increase in all enzymes, but level may fluctuate

• Low albumin and urea late in the disease

• ± elevated globulins

• Elevated BAs

• ± elevated bilirubin

• Low cholesterol

• Prolonged coag times

What biochemical changes are seen in cirrhosis of the liver?

• Enzymes can be elevated but they can be normal due to reduced hepatic mass

• Elevated BAs

• Elevated bilirubin

• Prolonged coag times

What biochemical changes can be seen in a bile duct obstruction?

• Marked increase in ALP and GGT

• Mild to moderate elevation in ALT and AST

• Elevated bilirubin

• Markedly elevated bile acids

What biochemical changes are seen with Cushing’s or glucocorticoids?

• Marked rise in ALP

• Normal to mid increase in ALT

• Normal to mid increase in BAs

• Normal bilirubin

• ± hyperglycaemia and hypercholesterolaemia

• ± Low urea

Why is an inflammatory sediment sometimes not present in a dog with Cushing’s disease?

Due to the anti-inflammatory effects of cortisol

What liver enzyme changes are seen in nodular hyperplasia and idiopathic vacuolar hepatopathy?

Marked increase in ALP with mild increases in other enzymes.

What lab findings are associated with cholangitis in cats?

• Elevations of all liver enzymes

• Bilirubin and bile acids are usually elevated

• With the lymphocytic plasmacytic form, all liver enzymes may be normal early on in the course of the disease

• Globulins may be increased ± a lymphocytosis

• May see a protein rich ascitic fluid

What biochemical changes are most commonly seen in hepatic lipidosis in cats?

Mild to marked increase in ALP with much smaller or often no increase in GGT

Increased ALT, AST and elevated bilirubin

The liver is the source of all albumin and most globulin except for what type of globulin?

Gamma globulins

What percentage of liver must remain in order to produce albumin?

33%

Aside from a reduction in functional liver mass, why else can albumin be low in liver disease?

May be mildly reduced as part of the acute phase inflammatory response. There is increased globulins and a reciprocal down regulation of albumin.

Why are globulins commonly increased in liver diseases?

• Inflammation

• Acute phase response

• Decreased clearance of antigen by Kupffer cells resulting in a systemic immune response.

• FIP

• Lymphocytic plasmacytic cholangitis

What are possible causes of a low albumin?

• Reduced production

• Liver disease

• Negative acute phase response inflammation - usually see inflammatory leukogram ± increased globulins

• Downregulation in production secondary to increased globulins eg in myeloma

• Malnutrition

• Increased loss

• GI loss/PLE

• Renal loss/PLE

• External blood loss

• Third space loss eg with septic peritonitis

• Haemorrhage

• Dilutional

• Excessive IVFT

• Fluid retention eg with CHF

• Age related (young puppies and kittens)

List the causes of low globulins

Increased loss

GI loss/PLE

External blood loss

3rd space loss eg with septic peritonitis

Haemorrhage

Decreased production

End stage liver disease

Immunodeficiency

Dilutional effect

Excess IVFT

Fluid retention eg with CHF

Age related - young puppies and kittens

List possible causes of hyperproteinaemia

• Dehydration

  • Increases both albumin and globulin

  • Normal AG ratio

  • Elevated HCT

  • Pre-renal azotaemia

• Increased globulins, usually immunoglobulins, or less commonly due to increased acute phase proteins

• Increased albumin eg with hepatocellular carcinoma or occasionally in 早晨shing’s

How can you evaluate hyperglobulinaemia?

Using serum protein electrophoresis

How does serum protein electrophoresis work?

• Serum is placed on a cellulose acetate gel in an electrical field

• The individual protein fractions migrate towards the anode at difference speeds depending on their size and charge and then are stained and scanned by a densitometer.

Electrophoretic traces from normal animals produce a tall, narrow albumin spike at one end and how many globulin fractions?

Three

What are the three globulin fractions that electrophretic traces are split into?

a globulins, b globulins and y globulins

Canine and feline a globulins and b globulins are further subdivided into a1, a2, B1 and B2 subfractions. What does a1 include?

a1-lipoprotein (HDL) and minor acute phase proteins such as a1-antitrypsin and a1 acid glycoprotein

Canine and feline a globulins and b globulins are further subdivided into a1, a2, B1 and B2 subfractions. What does a2 include?

Haptoglobulin, caeruloplasmin, a2-macroglobulin and the lipoproteins VLDLs and LDLs

Canine and feline a globulins and b globulins are further subdivided into a1, a2, B1 and B2 subfractions. What do B1 and B2 include?

Complement components, transferring, lipoproteins, IgM and IgA

What do y globulins contain?

IgA and IgG

Why is serum the preferred sample for electrophoresis?

As fibrinogen in plasma masks some of the other proteins

Increases in B and or Y globulins are generally due to increases in what?

Immunoglobulins

What is a polyclonal gammopathy?

This refers to an increase in several different types of globulins and is seen with chronic inflammation/infection eg pyometra, skin disease, viral/fungal/protozoal infections. It can also be due to chronic liver disease and immune-mediated disease.

Give examples of what can cause a marked polyclonal gammopathy

In cats:

• FIP

• Stomatitis/gingivitis

• Lymphocytic cholangiohepatitis

In dogs:

• Ehrlichia

• Leishmania

What is a monoclonal gammopathy?

Monoclonal gammopathy is the excessive synthesis of a single immunoglobulin by a single clone of B cells. These commonly consist of IgG which migrates to the gamma region. Less commonly, they consist of IgA (in B or Y region) or IgM which is in the B region

What are the causes of a monoclonal gammopathy?

• Multiple myeloma

• Lymphoma

• Lymphoid leukaemia

• Lymphoplasmacytic lymphoma - gives rise to the production of IgM - since this is the largest immunoglobulin, it is more likely to cause hyperviscosity

What is the reference change value?

A formula used to assess the true difference between two results

What is dispertion?

How much the true result may vary from the measured result

Why do you need to see a very large change in serial SDMA measurements to be sure the difference is significant?

Because the dispersion and RCV are much higher

What is urea?

Urea is the major nitrogenous waste product in mammals

Describe how urea is formed

Proteins are hydrolysed in the intestines to amino acids

Some of the amino acids are degraded by bacteria in the gut to produce ammonia

Ammonia ia absorbed by the enteric circulation to the hepatic portal vein to the liver

It is taken up by hepatocytes and detoxified into urea, which is excreted by the kidneys

Urea is freely filtered at the glomerulus, but up to what percentage is re-absorbed and where?

40% and mainly in the proximal tubule

What increases the amount of urea reabsorbed?

The GFR, so it increases in dehydration or with reduced renal blood flow of any cause

What 3 things does urea concentration depend on?

• Rate of production (liver fx, rate of protein breakdown)

• Rate of tubular absorption

• Rate of excretion

What are urea measurements influenced by?

Many non-renal variables such as:

• Fasting

• Dietary protein content

• GI haemorrhage

• Liver function

• Diuresis

• Hyperthyroidism

What can cause low urea levels?

• Severe liver disease/PSS

• Low protein intake in diet

• Diseases causing a marked polyuria eg Cushing’s or DI

What can cause elevated urea levels?

• Intestinal haemorrhage

• High protein diet/recent meal

• Increased catabolism of body tissues eg fever, starvation, sepsis, massive muscle trauma, c’steroid therapy

• Drop in GFR - either pre-renal, renal or post renal azotaemia

The production of creatinine depends on what?

Muscle mass and lean body mass

What is lean body mass affected by?

Age, gender, breed (large > small)

Puppies often have creatinine at the low end or below the reference interval, it increases up until what age?

1 year

What can cause an increase in creatinine?

• Strenuous exercise such as sprints

• Muscle trauma

• After feeding 9 peaking at 4-6h when it may be 20umol/l higher, before falling to baseline by 12h

What can cause a decrease in creatinine?

Cachexia

Why is creatinine a more accurate indicator of GFR than urea?

Because it is freely filtered at the glomerulus but it isn’t re-absorbed.

Why is there such a wide reference range for creatinine?

Because it is influenced by muscle mass and size of dog

Creatinine has high individuality - what does this mean?

That the normal physiological variation of creatinine is fairly small. A small change from the HSP may be clinically significant even if the value may still be well within the reference interval.

How is SDMA produced?

It is a byproduct of protein methylation produced by all nucleated cells and is released into the circulation following protein degradation.

What is SDMA not affected by?

• Lean body mass - therefore age/sex or food

What should you do in order to assess SDMA?

Sequentially check the SDMA

If the values are significantly different, then the reference change value should be used

What diseases can cause an increase in SDMA?

• Hyperthyroidism

• Malignant neoplasia due to increased protein metabolism

What does the kidney’s ability to concentrate urine depend on?

• The presence of healthy tubules

• Release of ADH and the response to ADH

• Presence of a hypertonic medulla

Reduced release of ADH (pituitary dependent DI) and primary nephrogenic DI are rare, but acquired resistance to ADH is common and may result from?

• HAC

• HyperT

• Inflammatory toxaemic states eg pyometra, pyelonephritis

• Liver failure

• Renal failure

• Hypokalaemia

• Hypercalcaemia

Loss of medullary hypertonicity can result from what?

hAC

Low urea

List the factors that affect renal biomarker lab findings?

• Pathology > declining renal function

• Analyser variation

• Biological variation

• Individuality

Dispersion is a combination of what variations?

Analyser and biological and it is how much the true result may vary from the measured result

What is the reference change value?

A formula used to determine if 2 sequential lab results are significantly different from one another, and the difference is not due to a combination of analytical and biological variation

After what percentage of nephrons lost does the SG fall into the isosthenuric range?

75%

Dogs and cats with what kidney condition sometimes retain concentrating ability even once they have developed azotaemia?

Primary glomerular disease

How can you distinguish acute kidney injury and severe CKD?

• AKI has a short history, whilst CKD has previous hx of PUPD

• AKI generally in good BCS, possibly large kidneys where CKD has small kidneys/poor condition

• CKD - moderate to severe non-regenerative anaemia

• HyperK common in AKI, not usually until terminal CKD, although may occasionally develop in dogs with CKD that are eating renal diets esp if concurrently tx with ACE inhibitors or ARB blockers

• AKI - usually low urine output, CKD increased until terminal stages

• Phosphate high in both

• Urinalysis - may see active sediment in AKI

• Marked electrolyte disturbances in AKI

• Marked signs for degree of azotaemia in AKI

Why is proteinuria a significant finding in kidney disease?

As it is a potential target for therapy

It is associated with a worse prognosis

The origin of protein in patients with CKD can be glomerular or?

Tubular

Why does a uroabdomen result in azotaemia?

Because urea and creatinine are passively absorbed into the plasma

Because urea it a small molecule, the level in the free fluid equilibrates with the plasma level fairly quickly in a uroabdomen. What happens to creatinine?

Creatinine, a larger molecule, remains higher in the fluid than in the plasma

What electrolyte abnormalities can also be seen in a uroabdomen?

A hyperkalaemia and hyponatraemia. Because urine contains so little sodium, this moves in the opposite direction from plasma into fluid along a concentration gradient.

The response to hyperkalaemia and hyponatraemia in the blood in a uroabdomen increases the release of aldosterone. What effect does this have?

This drives excretion of potassium into the urine and hence into the abdominal fluid, so the potassium concentration in the fluid remains higher than in the serum.

What symptoms are associated with severe hyponatraemia and why do they happen?

Neurological signs eg ataxia, seizures and coma. As a result of brain oedema which happens if hyponatraemia develops rapidly

List causes of hyponatraemia

• Excess sodium loss > vomiting and diarrhoea (GI loss), hypoadrenocorticism (excess renal loss), frus/thiazide diuretics, loss into an effusion due to peritonitis, pancreatitis, uroabdomen, pleural effusion, end stage kidney disease

• Water retention > CHF, hepatic failure, nephrotic syndrome

• Shift of water into ECF > DM/DKA hyperglycaemia causes an increase in serum osmolality which draws water out of cells into the extracellular compartment, thus diluting the serum sodium

• Reduced sodium intake

By what mechanisms can hypernatraemia result from?

Free water loss

Fluid loss in which the water loss exceeds the sodium loss (hypotonic water loss)

Hypernatraemia occurs when the patient fails to drink enough water to replace the increased loss (eg, water deprived or too sick to drink sufficiently)

Excessive sodium intake

Give examples of when free water loss can occur

• Diabetes insipidus

• Heat stroke, fever, burns

• Reduced water intake in sick/comatose animal or animal deprived of water

Give examples of when hypotonic water loss can occur

GI: vomiting and diarrhoea

DM results in osmotic diuresis (can result in hyper and hyponatraemia)

What can cause excessive resorption of sodium?

Hyperaldosteronism

What mechanisms can cause alterations to potassium?

• Altered intake

• Altered potassium loss through the kidney or GIT

• Movement of potassium from plasma to cells or vice versa

Explain how blood pH affects K+?

In acidosis, hydrogen ions build up and potassium moves out of cells and swaps places with the hydrogen to combat acidosis = hyperkalaemia

List reasons why you might not believe a high K+ level?

Can be spurious due to delayed serum separation where potassium is released from cells. This can be especially marked if there is a leukocytosis or thrombocytosis, in clotted/serum samples, or if there is a marked reticulocytosis and reticulocytes contain higher levels of K+ than mature RBC

Akitas Shibas and other Japanese breeds have high levels of K+ in their red cells so haemolysis in this breed will result in marked hyperkalaemia.

Contamination of the sample with EDTA

True hyperkalaemia is most commonly due to a reduced urinary excretion of potassium. Give examples

• Hypoadrenocorticism

• UO

• Ruptured bladder/uroabdomen

• Anuric/oliguric severe acute renal failure

Why do you see hyponatraemia in blood in a uroabdomen?

Because sodium diffuses from blood into fluid

What are some other less common causes of hyperkalaemia?

• Severe metabolic acidosis due to loss of bicarbonate

• Pseudohyperkalaemia - where there is a marked leukocytosis or thrombocytosis in clotted samples

• Marked hyperglycaemia due to solute dragging K+ out of cells

• Extensive crush/re-perfusion injuries due to movemnt of K+ out of damaged cells

• Prolonged use of K+ sparing diuretic spironolactone

• Rpt drainage of effusions

• Hypoaldosteronism (with normal cortisol production)

What acid base abnormality is commonly seen in hAC?

Metabolic acidosis

What other conditions can cause pseudohypoadrenocorticism?

Severe diarrhoea due to Trichuris vulpis, Salmonella and other infections

What are the most common causes of hypokalaemia?

Vomiting and diarrhoea

Renal wasting

Fluid therapy with K+ depleted fluids

Insulin administration in the initial treatment of DM

Give examples of less common causes of hypokalaemia

Diuretics eg frus, thiazides

Excess mineralocorticoid therapy

Primary hyperaldosteronism

Cushing’s

Young Burmese cats

Metabolic alkalosis

Hyperthyroidism

At what level of potassium do clinical signs of hypokalaemia develop?

<3?

What clinical signs can be seen in hypokalaemia?

Reduced GI mobility = ileus, constipation

Skeletal muscle weakness

Marked = severe muscle weakness and persistent ventroflexion of the neck, a crouched posture, stilted gait and muscular pain

What is the typical cause of hyperaldosteronism?

An aldosterone secreting adrenal tumour (adrenoma, adenocarcinoma)or unilateral or bilateral adrenal gland hyperplasia

What changes are typically associated with hyperaldosteronism?

• Low K+

• High Na

• Metabolic alkalosis (as aldosterone promotes acid secretion)

• CK elevated

• Hypophosphataemia

• Hypomagnesaemia

Why can hypertension be an indicator of hyperaldosteronism?

Because sodium retention leads to hypernatraemia and increased water resorption in the kidney which can lead to hypertension

Why can a secondary myopathy be seen in cases of hyperaldosteronism?

Because rhabdomyolysis can develop in cases with severe hypokalaemia (mechanism unknown)

How can you make a diagnosis of hyperaldosteronism?

• Moderate to marked hypokalaemia

• Mild hypernatraemia

• Adrenal mass, elevated aldosterone and ideally a low renin.

Why is renin important or useful when making a diagnosis of hyperaldosteronism?

Because it is used to distinguish primary hyperaldosteronism from a response to activation of the RAAS system due to hypovolaemia

In what other conditions can aldosterone concentrations be increased in?

In cats with heart disease and kidney disease. Magnitude is usually <1000pmol/l

What is Burmese Hypokalaemia AKA familial episodic hypokalaemic polymyopathy?

An inherited autosomal recessive disorder that presents within the first year of life. The underlying mchanism is likely to be a potassium wasting nephropathy

Give causes of a low Na:K ratio

• hAC

• Severe diarrhoea

• Urinary tract rupture, UO

• DKA

• Pleural and peritoneal effusion, especially due to repeat drainage