SA clinical pathology Flashcards
What liver features can be tested for by clinical pathology?
Hepatocellular injury -> leakage of enzymes
Cholestasis (reduced/blocked bile excretion) -> release of enzymes induced by retained bile
Hepatocellular function - decreased production or catabolism of substances
Hepatic portal circulation - decreased extraction of substances absorbed from GIT
Where are liver enzymes found normally?
ALP and GGT - on hepatocyte cell membranes
ALT, AST and SDH - in hepatocyte cytosol (and AST in mitochondria)
Which liver enzymes are there and what are they specific for?
ALT - largely liver specific (but also muscle), small animals
AST and LDH - liver and muscle
SDH and GLDH - liver specific in all species, used in large animals, SDH unstable
ALP and GGT - used in large animals as indicators of liver damage
What does it mean if there are increased liver ‘leakage’ enzymes in the blood?
Indicates hepatocellular damage
Myocyte damage can cause mild increase of AST, LDH +/- ALT (check CK)
Artefact (haemolysis) can increase AST and LDH (check serum/plasma quality)
Magnitude of increase correlates with degree of hepatocellular damage but not with reversibility of injury, prognosis or hepatic function
Short half lives: days in dogs, hours in cats (so even small increases may be significant in cats)
Which ‘cholestatic’ enzymes are there, used in small animals? What else can be used so assess if cholestasis?
ALP - good se for dogs but poor se for cats, scottish terriers have higher activities GGT - more specific Bilirubin Bile acids - more sensitive Cholesterol
What forms of ALP are there?
2 isoenzymes: intestinal and non tissue specific (I-ALP not generally detected in plasma as short half life so lost in GI tract)
Measurable isoforms:
- Liver-ALP (L-ALP) - serum half life of approx 70h in dog and 6h in cat (so insensitive in cats)
- Bone-ALP (B-ALP) - usually causes only mild increases, negative prognostic marker in osteosarcoma
- C-ALP - unique to dogs, induced by corticosteroids, product of I-ALP gene but produced in hepatocytes
What happens to senescent red cells to produce bilirubin (extravascular haemolysis)?
Breakdown of haem to bilirubin in macrophages of reticula-endothelial system (tissue macrophages, spleen, liver)
Unconjugated bilirubin is transported in blood via albumin to the liver
Taken up by facilitated diffusion by liver and conjugated with glucoronic acid
Conjugated bilirubin actively secreted into bile then into intestine
In intestine glucorionic acid is removed by bacteria and bilirubin is converted to urobilinogen
Some of urobilinogen is reabsorbed from gut and enters portal blood
Some of this participates in the enterohepatic urobilinogen cycel
Remainder of urobilinogen is transported in blood to kidney, converted to yellow urobilin and excreted
Types of hyperbilirubinaemia?
Prehepatic - secondary to haemolytic, check for anaemia
Hepatic - can be due to decreased bilirubin uptake, conjugation and excretion (so hepatocyte dysfunction and intrahepatic cholestasis)
Post-hepatic - secondary to obstruction of extra hepatic bile duct, serum cholesterol often high, ultrasound useful
How is bilirubin measured?
Total bilirubin Conjugated bilirubin (=direct bilirubin) Unconjugated = Til-DirBil (=indirect bilirubin)
Clinical signs of hyperbilirubinaemia?
Jaundice persists long after liver function turned to normal due to delta-bilirubin bound to albumin
If jaundice is due to delta-bilirubin there will be no bilirubinuria
Liver functions?
Detoxification
Synthesis of cholesterol, bile acids, plasma proteins, clotting factors
Breakdown of RBCs
Metabolism of carbohydrates, lipids and amino acids
Removal of bacteria
Storage of glycogen, iron, copper, vitamins
What markers are there of decreased hepatocellular function?
Decreased uptake and excretion of bilirubin and bile acids - increased (unconjugated) bilirubin and bile acids
Decreased conversion of ammonia to urea - increased ammonia, decreased urea
Decreased synthesis of metabolites - decreased albumin, cholesterol coagulation factors and inhibitors, glucose (but hypoalbuminaemia, hypoglycaemia, hypocholesterolaemia are insensitive markers for decreased liver function)
Decreased synthesis of coagulation proteins - decreased fibrinogen, increased PT and PTT
Decreased immunologic function - decreased clearance of toxins and antigens -> systemic stimulation -> increased Igs
What markers are there of alteration of hepatic blood flow (PSS)?
Decreased uptake and excretion of bile acids - increased bile acids (bilirubin not usually increased as major problem is the bile acids re-uptake from blood)
Decreased conversion of ammonia to urea - increased ammonia
Decreased immunologic function - decreased clearance of toxins and antigens -> systemic stimulation -> increased Igs
Where is ammonia produced and what happens to it?
Produced in GIT by protein digestion or bacteria metabolism
Enters liver via portal vein
Uptaken by hepatocytes to synthesise urea, amino acids, proteins
Urea diffuses to sinusoidal blood or bile canaliculi and is excreted through kidneys or intestine
Things to note when measuring blood ammonia?
Relatively insensitive
Only significant if raised (hepatic encephalopathy)
Not stable in vitro so should be measured immediately after blood sampled
Elevated in:
- congenital and acquired porto-systemic shunts and liver failure
Where are bile salts produced and what happens to them?
Produced by hepatocytes
Released into biliary system and then intestine - allow fat absorption and digestion
>90% then reabsorbed from ileum, enter portal vein, return to liver, re-circulate
Small amounts lost in faeces, replaced by liver
What can increased bile acids in the blood mean?
Reduced uptake/excretion by hepatocytes - reduced hepatocellular mass, impaired hepatocyte function
Disruption of enterohepatic circulation - portosystemic shunts, cholestasis/bile obstruction
No point measuring BA if bilirubin already increased (BA more sensitive)
Interpretation of fasting SBA?
> 25-30mmol/L are abnormal and indicate hepatobiliary pathology - can’t differentiate between cholestasis and liver failure
<25-30mmol/L cannot completely exclude portosystemic shunt, perform bile acid stimulation test (BAST) if still suspecting hepatic pathology
Interpretation of post prandial SBA? How to do it?
BA stimulation test/dynamic BA Take resting sample Fatty meal Post-prandial sample 2h after feeding >25-30mmol/L is abnormal and indicates hepatobiliary pathology - can't differentiate between cholestasis and liver failure
What extra hepatic diseases can cause elevation in liver enzymes?
Hypoxia GI and pancreatic disease Endocrine diseases (fat or glycogen accumulation) Sepsis = 'reactive hepatopathies'
How to determine if raised liver enzymes is due to ‘reactive hepatopathy’ due to extra hepatic disease?
Most have normal bile acids
Other markers of liver function will be normal - unless affected by primary disease
Bile acids may be markedly elevated in sepsis/SIRS/endotoxaemic shock
What haematology may be seen in liver disease?
Microcytosis - portosystemic shunts or severe liver insufficiency, likely due to altered iron transport or metabolism
Ovalocytes (elliptocytes) are frequently seen in cats with hepatic lipidosis
Acanthocytes - lipid disorders, disruption of normal vasculature (e.g. hepatic haemangiosarcoma)
Urinalysis in liver disease?
Often unremarkable but may see:
- isosthenuria or inappropriately low USG
- bilirubinuria (more than 2+ in dogs, any in cats)
- ammonium bitrate crystals or uroliths (40-70% of patients with portosystemic shunts)
Functions of the pancreas?
Exocrine acinar cells (98%) - secrete enzymes involves in initial digestion of food
- proteases (trypsin, chymotrypsin, elastase)
- lipases
- amylases
- high conc of bicarbonate in secretions
- aids B12 and zinc absorption
- antibacterial activity
- intestinal mucosal modulation
- proteases stored as inactive zymogens
Endocrine islet cells (2%) - secrete insulin and glucagon
Tests for exocrine pancreas integrity?
Amylase and lipase tests - identify injury to pancreatic cells (increases most often due to pancreatitis), pancreatitis should be based on a combo of these tests with compatible clinical and imaging signs (abdominal ultrasound)
DGGR - proven to correlate well with more specific tests and clinical pancreatitis, cut offs for pancreatitis diagnosis:
- >34 U/L in cats
- >216 U/L in dogs
Canine pancreatic lipase (cPL) - considered more specific and more sensitive than lipase and amylase, snap test available
TLI (trypsin like immunoreactivity) - used in dogs, cats, horses (species specific assays), detects trypsinogen, trypsin and trypsin bound to protease inhibitors, use in animals with clinical signs of maldigestion/malabsorption, high se and sp for exocrine pancreatic insufficiency, less useful fro pancreatitis, serum TLI < 2.5mg/L in dogs, < 8mg/L in cats
Amylase - function, half life, species used in?
Catalyses hydrolysis of complex starches Short half-life (hours) Salivary and intestinal not found in serum More useful in dogs than other species Can increase due to decreased GFR
Lipase - function, half life etc?
Catalyses hydrolysis of triglycerides
Very short half-life (2 hours)
Mostly from pancreas
Can increase (mildly) due to decreased GFR
Different tests have different positive and negative predictive value
Diagnosis of pancreatitis?
Elevation in lipase (and amylase) - suggestive but other causes possible, higher increase=higher likelihood, degree of increase doesn’t = severity of disease
Need compatible clinical signs
Additional tests - PLI (specific pancreatic lipase), ultrasound, fluid analysis, biopsy
Suggested criteria for diagnosis of acute pancreatitis in dogs?
Absence of surgical disease on abdominal radiographs or analysis of abdominal fluid
And abdominal ultrasound with evidence of primary pancretitis
And one or more of the following:
- Spec-cPL >400ug/L
- Positive SNAP-cPL
- Gross lipaemia
- Serum PE-1 >17.24 ng/ml
- Total lipase >3x the upper reference interval
Indications for cytology?
Lesion (nodule, mass, plaque) palpable externally or seen on imaging Organomegaly Cavitary effusion Cancer staging PUO
Problems reducing cytology sample quality?
Ruptured cells - incorrect sampling or smearing technique
Inadequate staining
Identifying cell types on cytology?
Inflammatory cells - neutrophilic, eosinophilic, lymphocytic, histiocytic, mixed
Tissue cells - expected from organ aspirated, abnormal cells
Normal cytology of skin and subutaneous tissue?
Few keratinocytes
Scant fat
Rare sebocytes
How to assess neoplastic cells and malignancy from cytology?
Cell arrangement
- discrete or cohesive
- any cytoarchitecture
Cell shape
- round, polygonal, spindle
Criteria of malignancy
- hypercellularity (in mesenchymal tumours)
- pleomorphism (anisocytosis, anisokaryosis)
- high/variable N:C ratio
- multinucleation
- karyomegaly
- mitoses (especially if atypical)
- nuclear moulding (rapid cell growth)
- large, angular, or variably sized nucleoli
Minimum of 3 criteria to be classified as malignant (nuclear are stronger) - adapt depending on specific organ/cell type, allow some with inflammation and in histiocytes
If absent criteria of malignancy -> benign or well differentiated malignant
How do round cell tumours, epithelial tumours and mesenchymal tumours typically present on cytology?
Round cell tumour - ‘sea’ of round, discrete cells
Epithelial tumour - ‘islands’ of cohesive polygonal cells
Mesenchymal tumour - spindle cells with indistinct edges embedded in extracellular ‘matrix’
What round cell tumours are there?
Histiocytoma Plasma cell tumour Mast cell tumour Lymphoma Transmissible venereal tumour (not in UK)
Histiocytoma - Typical presentation? Cytology?
Dome shaped, alopecia, may be ulcerated
Tend to regress in a few weeks
Often (but not only) in young dogs
Cytology - light blue cytoplasm fading into the background, small lymphocytes often present and may predominate at later stages, sometimes difficult to differentiate from plasma cell tumour and lymphoma
Histiocytic sarcoma - cytology?
Arises from interstitial dendritic cells
Usually marked cell pleomorphism with karyomegaluy and multinucleation
Localised or disseminated
Lymphoma - cytology?
Round cells with high nucleus to cytoplasm ratio
Large blasts (larger than a neutrophil)
Monomorphic population
Mast cell tumour - cytology?
Magenta granules in the cytoplasm - sometimes don’t stain well with Diff Quik (send some unstained slides to lab)
Poor granulation can be due to poor differentiation (usually means a more aggressive tumour)
Plasmacytoma - cytology?
Cutaneous plasmacytomas are usually benign
Can be well differentiated to pleomorphic (most commonly)
Deep blue cytoplasm, perinuclear halo, eusinophilic borders
Round, eccentric nucleus
Types of keratinising lesions?
Epidermal/follicular cysts - cysts lined by normal keratinised epithelium, keratin accumulates in centre
Hair follicle tumours - many types, mostly benign, often cytologically identical to cysts, sometimes basaxoid cells
Trichoblastoma - what is it? cytology?
Benign skin tumour
Scant amount of cytoplasm
Uniform nuclei
Sebaceous adenoma - cytology?
Raised cauliflower alopecic lesions
Clusters of cohesive heavily vacuolated cells
Cannot differentiate from hyperplasia on cytology
Hepatoid gland tumour - where? cytology?
Usually benign
Perianal or along thighs
Cutaneous
Clusters of ‘hepatic’ cells - similar to hepatocytes (large polygonal shaped cells, abundant pink-blue granular cytoplasm, prominent single nucleolus)
Surrounded by few ‘reserve’ cells - smaller and higher N:C ratio and oval nuclei
Squamous cell carcinoma - cytology?
Polygonal cells with variable tendency to cohesion
Typically marked pleomorphism
Often secondary neutrophilic inflammation
Anal sac adenocarcinoma - cytology?
Classically ‘naked nuclei’ appearance
Often cells form rosettes and ‘rows’
Sheets of bland looking cells but high metastatic potential!
Most common soft tissue sarcomas in dogs and cats?
Dogs: - perivascular and nerve sheath tumour - fibrosarcoma Cats: - fibrosarcoma/injection site sarcoma
What tumours are ‘confusing’ as not easily classified as round, epithelial or spindle?
Endocrine and neuroendocrine tumours
Histiocytic sarcoma, chondrosarcoma, osteosarcoma - may be round or spindle
Melanoma:
- well differentiated melanocytes are stellate
- undifferentiated melanocytes may be fixate, round, cuboidal with some tendency to cohesion
Are melanomas usually benign or malignant?
Usually benign and well differentiated in haired skin
Often malignant in nail beds, oral cavity
Lipoma - cytology?
Can’t differentiate from ‘normal’ subcutaneous adipose tissue
Where are erythropoietin and renin synthesised and where is vitamin A activated?
Erythropoietin: peritubular interstitial cells
Renin: juxtaglomerular cells
Vitamin D activation: proximal tubular cells
What electrolytes/substances and conserved and excreted by a healthy kidney?
Conserve:
- water
- amino acids/proteins
- glucose
- bicarbonate
- Na+ and Cl
- Mg2+ and Ca2+
Excrete:
- urea
- creatinine
- K+
- H+
- PO4
- ketones and lactate
- bilirubin
- haemoglobin and myoglobin
What is not filtered by glomerlar filtration and what is it restricted by?
Cells, proteins larger than albumin and most lipoproteins
Restricted by:
- size
- charge - albumin is size of pores but negatively charged
Which tests of renal function are there? What are they all useful to assess?
Biochemistry: nitrogenous waste excretion (urea and creatinine), degradation of lipase and amylase, electrolyte balance
Urinalysis
Haematology: erythropoietin secretion
Blood gas analysis: acid base balance (pH, BE, HCO3)
How can GFR be measured?
Directly with plasma clearance of creatinine or iohexol - impractical
Indirectly - urea and creatinine
SDMA
How much urea and creatinine is normally reabsorbed in the kidney?
Urea: up to 40%
Creatinine: not reabsorbed, a bit secreted
Causes for increased blood urea?
Decreased GFR
Mild increases:
- upper GI haemorrhage: protein catabolism, hypovolaemia/dehydration
- high protein diet
- recent meal
- catabolism (fever, starvation, corticosteroids)
Causes for decreased blood urea?
Severe liver disease or portosystemic shunt Low protein diet Aggressive fluid therapy PUPD - cause and effect Young animals Deficiency of enzymes in urea cycle
Causes of increased and decreased blood creatinine?
Increase: - decreased GFR - high muscle mass (mild increase) - high dietary protein - non creatinine chromagens Decrease: - reduced muscle mass - usually not clinically significant
Define azotaemia and uraemia?
Azotaemia = increased creatinine and/or urea Uraemia = complex of clinical signs seen in patients with renal disease: V+, D+, anorexia, weight loss, anaemia, ulcerative stomatitis, muscle tremors, convulsions, coma
All uraemic patients are azotaemic but not vice versa
Causes of pre-renal azotaemia? Clinical signs/pathology/how can you tell it is prerenal?
Dehydration or decreased CO (due to decreased blood flow to kidneys):
- clinical evidence of dehydration and/or hypovolaemia
- maximally concentrated USG (>1.030 in dogs, >1.035 in cats)
- urea often increased more than creatinine
- should respond to fluid therapy
Increased ammonia load (high protein diet, or GI haemorrhage)
- increased urea but not creatinine
- maximally concentrated USG
- signs of iron deficiency possible
Causes of renal azotaemia? Clinical signs/pathology/how can you tell it is prerenal?
Cause: decreased functional nephrons
Variable USG:
- isosthenuria (1.008-1.012) when >66% of nephrons lost
- suboptimal USG (>1.012 but <1.030 in dogs and <1.035 in cats)
- falls progressively
- cats can lose concentrating ability later than dogs
Causes of post renal azotaemia? Clinical signs/pathology/how can you tell it is prerenal?
Urinary tract rupture or blockage -> failure of urine output
Hyperkalaemia
If ruptured bladder:
- creatinine in abdominal fluid > blood
How good are urea and creatinine as markers of renal function?
Insensitive - only azotaemia when >75% of functional nephrons lost
What is SDMA produced from? Is it filtered by the kidneys? Benefits for kidney disease? When to measure? RI?
Produced from intranuclear demethylation of L-arginine
Freely filtered by glomerulus
More se and sp than creatinine:
- not influenced by muscle mass
- increases with 40% reduction of GFR
Measure when creatinine is normal or borderline to identify animals in IRIS stage 1/2 with absent or milf clinical signs
RI <14ug/L (+1 in puppies, kittens, greyhounds)
What electrolyte changes are seen with kidney disease?
Hyperphosphataemia:
- small animals have a high phosphate intake
- exception is horses as low diet P so tend to be hypo
Hyper or hypokalaemia:
- increased with acidosis, decreased urine output, marked decreased nephrons, anuria/oligura (AKI), bladder rupture, obstruction
- decreased with increased urinary losses (esp CKD), decreased intracellular stores, GI losses
Hypochloraemia:
- increased renal loss (follows Na)
- GI loss with vomiting
Hyper or hypocalcaemia
- hyper in horses
- hyper in low % of smallies (usually only tCa)
- most commonly hypo in smallies as decreased vit D and alimentation and tissue deposition
What does it mean if the USG is hyposthenuric?
Requires functioning nephrons
Diluted by kidneys
Typically ADH problem
When is a suboptimal USG significant? Cause?
If low USG + dehydration/azotaemia First look for non kidney causes: - lack of aldosterone or ADH - diuretics, glucocorticoids, glycosuria, medullary washout, pyelonephritis - low urea, Na or K, or high Ca If not, renal disease likely
Proteinuria: When is it significant? Causes?
Small amounts up to +1 may be normal More significant if low USG Causes: - glomerular damage (renal) - protein overload to glomerulus (pre-renal) or urinary tract inflammation (post-renal)
Urine protein:creatinine ratio: Why is this useful? Reference range for dogs and cats? What does it suggest?
Measure of proteinuria independent of urine concentration
>0.5 (dogs) >0.4 (cats) = proteinuric
0-2-0.5 - borderline
Elevated ratio in absence of an active sediment or hyperproteinaemia is a strong indicator of renal (often glomerular) disease
Glycosuria: When seen? Significance?
Seen with serum glucose >10mmol/L in dogs and >16.6mmol/L in cats
If serum glucose normal = renal glycosuria (failure of tubular reabsorption)
What is the normal urine pH for dogs and cats? What does it mean if different?
Usually 5.5-7
Low with metabolic acidosis
Can be inappropriately high with metabolic acidosis due to tubular dysfunction
Alkaline due to UTI with urease producing bacteria
What are casts un urine? Significance?
Cylindrical moulds of tubules composed of mucoproteins +/- cells
Occasionally hyaline and granular casts may be normal
Granular, cellular and waxy indicate tubular damage
Crystals in urine: How significant?
Fresh urine: likely developed in vivo
Stored urine: can precipitate post collection
Lots of normal animals have crystalluria
What types of crystals are there in urine? Significance? When formed?
Struvite:
- magnesium ammonium phosphate
- UTI, urolithiasis or normal
- form in alkaline urine and in vitro
Amorphous:
- urate in acidic pH: no clinical significance
- phosphate in alkaline pH, no clinical significance
Calcium oxalate di-hydrate:
- can be normal
- can form in vitro
- can be seen with ethylene glycol toxicity but not pathognomic
Calcium oxalate monohydrate:
- hyperoxaluric disorders or hypercalciuria (e.g. ethylene glycol toxicity or ingestion of oxalate rich foods like peanut butter)
Ammonium bitrate or uric acid:
- portosystemic shunts, liver disease, dalmatians
Cystine:
- associated with metabolic defect in tubular resorption of cystine (bulldogs)
Normal distribution of electrolytes?
ECF: Na, Cl, HCO3
ICF: K, P, proteins
