Pathology Flashcards
what do you describe in a post mortem
organ
location
distribution
size
shape
demarcation
contour
texture
smell
how would you describe location in a post mortem
which organ
which lobe/part
aspect (dorsal, ventral, medial ect. esspecially important for intestine- changes differential- serosial mucosial)
aaymetric/ symetric
how would ypu discribe distribution of a leasion in a post mortem
diffuse (genrelised)- whole organ
lobular/ regionally diffuse- one lobe
focal/ localized- one discrete spot
genrelised multifocal- multiple discrete spots
localised multifocal- multiple discrete spots in one specific loction
zonal
why is distribution important
it can tell whether a tumour is primary or secondary (e.g a secondaey tumour is more licly to be generalised multifocal as it preads through the bloodstream
demarcation
Clearly demarcated versus blendswith surrounding tissue
contour
Flat versus raised versusdepressed
what could a red colour in a post mortem indicate
Dark red, red-black, pink
Blood – hyperaemia, congestion, haemorrhage, erythrocyte lysis, haemoglobin imbibition
what could a red/black colour in a post mortem indicate
Haemorrhage and necrosis (acute) (often go hand in hand)
what could a yellow colour in a post mortem indicate
icterus
what could a blue/green/black colour in a post mortem indicate
psuedomelanosis (post mortem change)
what could a brown/black colour in a post mortem indicate
melanosis/melanoma (
what could a white/grey/cream colour in a post mortem indicate
necrosis, fibrosis (chronic)
what could texture tell you in a post mortem
firm- firm intervertibral disks can indicate intervertabrale disk disease
friable- can indicate that tisues are breaking down ( could be pathology (amyloidosis) or post mortem effect)
gritty- can result in bloodstream when euthanised with barbituates
incidentaloma
pathology that did not lead to the death of the animal
what are the components of the morphological diagnosis
distribution
severity
timescale
organ or tissue
patholgical process
(a summary of the lesions and an interpreta- tion of the likely disease processes)
what would be the morphological diagnosis of very large mesenteric lymphnoes form a dead foal that are puss filled when insised
Diffuse, severe, suppurative, mesenteric lymphadenitis
supprative indicates bacteria- Rhodococcus equi
what would be the morphological diagnosis of generalised dark lung lesions in a pig
multifocal, mild, suppurative pneumonia
Mycoplasma
hyopneumoniae
what would be the morphological diagnosis of multiple spread out, fiberous lesions on a cow liver
Multifocal, chronic, marked, pyogranulomatous hepatitis
microbaterium bovus- tb
pathognomonic
specifically characteristic or indicative of a particular disease or condition.
what should be taken into acount when deciding whether an animal died suddenly or not
History-Is it sudden or unexpected, both or neither?
Any previous history of illness?
Suspicion of neglect?
Gross examination- Body condition, fat stores (serious atrophy of fat) and muscle coverage
Food in stomach?
Injuries, haemorrhage
Histopathology- Serous atrophy of fat
Heart
what are the body sytems can cause sudden death
nervous
cardiovascular- gdv in gastrointestinal system effects this
respiritory
describe how the nervous system can be involved in sudden death
pathology can be very subtle or very obvious
neurogenic shock
Trauma
Hypoxia/anoxia
Oedema
Toxins
Seizures
neurogenic shock
any factor that stimulates parasympathetic activity or inhibits sympathetic activity of vascular smooth muscle and can cause neurogenic shock, this reults in widespread and massive vasodilation
How can the autonomic system be damaged?
Trauma
Hypoxia/anoxia
Oedema
Toxins
Seizures- can be pathologically subtle. cardiac arythmia, respiritory disfunction
what would we examine the brain for in a case of sudden death to prove it was the cause
History- Trauma, Seizures
Gross examination- Skull fractures, Areas of haemorrhage in the brain
Microscopic examination- Unless obvious trauma/haemorrhage, is difficult
Hypoxic events and seizures will typically leave no trace visible by histopathology- Diagnosis of exclusion
any trauma abouve t5 can result in
damage to the autonomic nervous system and sudden death
the adrenal medulla is primerally responsible fro producing
catecholamines- tese hormones are released into the body in response to physical or emotional stress. The main types of catecholamines are dopamine, norepinephrine, and epinephrine. Epinephrine is also known as adrenaline.
agonal changes
Agonal changes take place immediately before death and are due to circulatory failure. The most common change of which to beware in this category is hypostatic congestion of the lower lung, which may be confused with pneu- monia.
what bosy system is the most common cause of sudden death in dogs and cats
cardiovascular
Pathophysiology of death due to cardiac failure
Structural
Electrical
Ischaemic
Pathophysiology of death due to vascular failure
Ischaemic
Shock
causes of sudden death due to the cardio vascular system
Pathophysiology of death due to cardiac failure-Structural
Electrical- diagnosis of exclusion
Ischaemic - a condition in which the blood flow (and thus oxygen) is restricted or reduced in a part of the body- may see hypoxia if animal lives for 6 hours after
Pathophysiology of death due to vascular failure- Ischaemic- infarction, thrombus,
Shock
describe the pathology of vessels when investigating sudden death due to the cardiovasular system
Pathology of the vessels- Evidence of haemorrhage?
Evidence of disease of organs involved in fluid and electrolyte haemostasis? -GI, Urinary, Endocrine (addisons disease?)
Open pulmonary arteries and aortic bifurcation
Take sections of kidney for histeopath - End arteries therefore good place to look for small thromboemboli
waht would be examined in the heart when inspecting sudden death cuased by the cardiovascular system
Pericardial effusion?
Weight
Measurements
Gross lesions
Sections for histopathology
Sudden death - respiratory
“Respiratory failure occurs when there is inadequate exchange of O2and CO2to meet the needs of metabolism, which leads to hypoxaemia, with or without hypercarbia”
Respiratory failure can be divided into:
Type I respiratory failure, in which processes that impair oxygen transfer in the lung cause hypoxaemia (acute or hypoxaemic respiratory failure)
Type II respiratory failure, in which inadequate ventilation leads to retention of CO2 , with hypercarbia and hypoxaemia
‘Mixed’ respiratory failure, in which there is a combination of type I and type II respiratory failure (acute-on-chronic respiratory failure).
The most common cause of death due to respiratory failure in dogs is reported to be accidental asphyxiation due to choking on food material
Acute respiratory distress syndrome- Secondary to inflammation/infections elsewhere in the body, Often pancreatitis, Sudden but likely expected and/or dog already hospitalised
BOAS- Brachycephalic Obstructive Airway Syndrome
Heat stroke
Peri-anaesthetic
Type I respiratory failure
in which processes that impair oxygen transfer in the lung cause hypoxaemia (acute or hypoxaemic respiratory failure)
low presure- high altitude
hypoventilation under anasthesia
ventilation diffusion imparment
perfuion mismathc
right to left shunt
Type II respiratory failure
in which inadequate ventilation leads to retention of CO2 , with hypercarbia and hypoxaemia
abnomalities of cantral respiritory drive
neuromuscular dysfunction
abnormalities of chest wall
abnormalities of airway
abnormalities of lungs
‘Mixed’ respiratory failure
in which there is a combination of type I and type II respiratory failure (acute-on-chronic respiratory failure).
BOAS
“Brachycephalic obstructive airway syndrome (BOAS) is a term used to describe a raft of upper respiratory tract problems that occur in brachycephalic breeds. Clinical signs vary in severity and include stertor (noise), stridor ( increased effort), exercise intolerance, collapse and cyanosis (blue colouring). Affected dogs are also predisposed to syncope, gastro-oesophageal reflux, vomiting and regurgitation.”
pulmonary haemorrhage in horses
cause eof repiritory sudden death
Pulmonary haemorrhage is a common clinical condition and PMfindingin equine athletes.
Exercise-associated fatal pulmonary haemorrhage (EAFPH)-
describe fatal pulmonary haemorrhages in racehorses
Fatal pulmonary haemorrhage is one of the most frequent causes of sudden death in racehorses, and such lethal pulmonary bleeding has been reported long before the acronym EAFPH was coined
The occurrence of acute cardiac failure or spastic contraction of pulmonary postcapillary sphincters have been listed as possible pathogenetic mechanisms for the occurrence of EAFPH, but this has not been proven
Exercise-induced pulmonary haemorrhage (EIPH)-
The term was first used in1981 to describe epistaxis of pulmonary origin, especially after exercise.
EIPH is believed to be an important cause of reduced athletic performance, especially in cases with severe bleeding, however its role in sudden death is complicated
Exercise-associated fatal pulmonary haemorrhage (EAFPH)
A term first reported in 2015, used to describe fatal pulmonary haemorrhages in racehorses
Fatal pulmonary haemorrhage is one of the most frequent causes of sudden death in racehorses, and such lethal pulmonary bleeding has been reported long before the acronym EAFPH was coined
The occurrence of acute cardiac failure or spastic contraction of pulmonary postcapillary sphincters have been listed as possible pathogenetic mechanisms for the occurrence of EAFPH, but this has not been proven
post mortem findings will show widespread blue/black discolouration on the lungs distinguishabke by its spread from the more schronic case seen in EIPH
Exercise-induced pulmonary haemorrhage (EIPH)
The term was first used in1981 to describe epistaxis of pulmonary origin, especially after exercise.
EIPH is believed to be an important cause of reduced athletic performance, especially in cases with severe bleeding, however its role in sudden death is complicated
post mortem findings of the lungs in which this occurs may show blue black areas on the doral aspect of the lungs
will have haemociderophages- macrophages that have taken up blood
suggests chronic condition
what is the aeitological agent of haemorrhagic pneumonia in kenneled dogs
Aetiology = Streptococcus equi subsp. Zooepidemicus
haemorrhagic pneumonia in kenneled dogs
Aetiology = Streptococcus equi subsp. Zooepidemicus
Pathogenesis is unclear however stress and concurrent viral infections may play a role
Typical gross finding is that of a thoracic cavity filled with blood and histopathology of the lungs reveals a necrotizing suppurative bronchopneumonia: suprative- lots of neutrophils, necrotising- necrosis of the alveolar walls
appart from Streptococcus equi subsp. Zooepidemicus, what may cause haemorrhagic pneumonia in dogs
Extraintestinal E. coli will also cause a haemorrhagic pneumonia
what two aeitiological agents may cause haemorrahgic pneumonia in dogs
Extraintestinal E. coli
Streptococcus equi subsp. Zooepidemicus
what things should you consider in Sudden death in ruminants
Several factors make the definition of ‘sudden death’ in farm animals problematic.
The most commonly accepted definition is death that occurs since the last inspection (ie, within 12 to 24 hours).
However, it can be challenging for the farmer to detect early clinical signs of disease in farm animals due to- Extensive husbandry
Infrequent inspection
Difficulty in recognition of individual animals difficult.
Animals often scavenged
Trauma- Dog attack
Electrocution- Often asked to pronounce on suspected lightning strike due to its common inclusion in insurance policies.
Circumstantial evidence, such as nearby trees, water or wire fencing, plus knowledge of a recent storm, is vital to diagnosis.
Multiple animals dead by metal railings if due to faulty electrical equipment
Death is usually instantaneous with few distinguishing features
There may be singe marks on the skin, a rapid onset and disappearance of rigor mortis and rapid decomposition of the carcase with distension of the rumen.
only consistant finding in electrocution cases is quick onset of rigor mortis in combination with it quickly fading
Acute infection- Anthrax
Clostridial diseases
Anthrax
Anthrax is a cause of outbreaks of sudden death, particularly in ruminants, however can affect other grazing animals and humans.
Clinical signs in animals that do not die suddenly include high fever, tachycardia and tachypnoea, and congested and terminally cyanotic mucosae with haemorrhage.
Animals that survive longer than a day may have dysentery, abortion, oedematous swellings of the perineum, throat and abdominal wall, and blood-stained milk.
The characteristic sign in pigs is swelling of the pharyngeal region and neck.
Anthrax is notifiable and zoonotic, so if you suspect it you must call APHA
Triage all callsbefore accepting dead ruminants for PM
Due to the high fever, animals decompose rapidly resulting in typical saw-horse bloated appearance
DO NOT open up the animal, it is spread by spores which can become aerosolised
If the animal has been accidentally opened up, immediate gross post mortem findings include blood filled cavities and marked splenomegaly- not many other things in cattle cause this
often in sudden death the spleen contracts due to shock unless barbituates are involved
Diagnosed in field by taking a blood smear from the ear
Rod-shaped bacteria with capsule
Sudden death in ruminants – clostridia
Whist the GIT is not an organ that can kill you quickly, clostridial organisms found in the GIT can under the right circumstances produce toxins that affect predominantly affect the CVS and neurological systems
Pathophysiology:
Clostridium perfringens is a gram positive anaerobic bacillus
C. perfringens types B and D produce epsilon toxin (ETX) resulting in enterotoxemia
ETX is a pore forming toxin with particular affinity for vascular endothelial cells
This changes the permeability of the small and large intestine
The other target organs of the toxin include the kidneys, lungs and brain
toxins cause disease
Clostridium perfringens epsilon toxin: the third most potent bacterial toxin known
C. perfringens is a commensal however certain situations can result in overgrowth
Typically fat lambs or animals that have broken into feed containers or gone onto lush grazing
Waning of maternal immunity
Antibiotic use in rodents – C. difficile.
watery bloody diherea
fluid filled intestins
toxins go to kidney lung and brain
effect cardiovascular and neurological systems
Gross pathology
Fluid accumulation, mucosal haemorrhaging and haemorrhagic content in the small intestine and large intestine- may occur in pericardial sack
Also seen in all species with clostridial enteritis
Pigs may have emphysematous wall
Widespread haemorrhages
Hydropericardium, hydrothorax and pulmonary oedema
“Pulpy” (soft) kidneys
Brain
General brain swelling leads to flattening of the gyri and cerebellar coning in some cases. cerebella coning- The herniation of the cerebellar tonsils through the forament magnum
Symmetrical focal malacia (softening) - perhaps due to glutamate as this part of brain is heaily relinant on it and it is an exitable chemicale
looks brown grey
Diagnosis- Culture of intestinal content not useful as is a commensal
Ileal content- Look for the toxin, often breaks down before it is found though ( it is “Labile”)
Glucosuria is typical but not pathognomonic as indicates systemic toxaemia
Histopathology- Brain, white matter
protein-rich perivascular oedema (microangiopathy)- this is due to the pore forming toxin
malacia
softening
Sudden death in pigs – salt poisoning
happens when pigs engorge with water- maybe sue to not having acces for a while
show muscle fasciculation, blindness, seizures or were down, non-responsive.
No significant gross pathological lesions are evident in the brain
eosinophils may be seen
ALBUMIN
protien
Produced in the liver at a constant rate
Major contributor to plasma oncotic pressure
Carries ion molecules (calcium, magnesium)
oncotic pressure
Oncotic pressure, or colloid osmotic-pressure, is a form of osmotic pressure induced by the proteins, notably albumin, in a blood vessel’s plasma (blood/liquid) that causes a pull on fluid back into the capillary.
protiens contribute to this
GLOBULINS
Subdivided into: Alpha, beta, and gamma globulins
Includes: Inflammatory cytokines, immunoglobulins
Produced by many different cell types. Major contributors:
Liver: Acute phase proteins (increased during inflammation), coagulation proteins (clotting factors, anticoagulants)
Lymphocytes: Immunoglobulins
Hyperproteinaemia- High albumin
dehydration
Hyperproteinaemia- High globulins
dehydration, inflammation, neoplasia
Neoplasia refers to lymphoma & myeloma, which produce monoclonal immunoglobulins
Detection of monoclonal immunoglobulins can be done via serum protein electrophoresis
Hypoproteinaemia
Hypoproteinaemia can be categorised as follows:
Selective hypoproteinaemia
Hypoalbuminaemia
Hypoglobulinaemia
Total proteins can be WRI
Panhypoproteinaemia
Both albumin and globulins are below WRI
Selective hypoproteinaemia
Hypoalbuminaemia
Hypoglobulinaemia
Total proteins can be WRI (reference interval) - total protien is normal
albumin and globulins should be looked at seperatly
Panhypoproteinaemia
Both albumin and globulins are below WRI (reference interval
When both albumin and globulin are lost together.
Two main categories:
Protein-losing enteropathy (common)- Lymphoma
IBD
Lymphangiectasia
Parasitism
Protein-losing dermatopathy (rare → severe burns)-
Selective hypoproteinaemia: Hypoalbuminaemia
Can be caused by:
Reduced production
Liver disease
Inflammation- negative acute phase protien so a drop in albumin is a normal response to inflamation
Losses:
Renal disease → protein-losing nephropathy (PLN)- kidneys shouldnt loose protien, damage to the glomerulus can cause this to happen. it tend to be albumin that is lossed first due to it being more passible
Movement from blood into body cavitary effusion
Selective hypoproteinaemia: Hypoglobulinaemia
Rare! Encompasses so many proteins, unusual to lose so much of one that it has a significant impact on total globulins.
Check dehydration is not masking concurrent hypoalbuminaemia
Consider double checking with a reference laboratory
Main differential: Immunodeficiency resulting in severe reductions immunoglobulin production (e.g. Severe combined immunodeficiency)
what are enzymes used to check for
Generally used to check for cellular injury.
Commonly measured enzymes can be broadly categorised into those from:
Liver
Biliary tract
Muscle
Pancreas
Some enzymes are produced by multiple tissues → need to look at panels to work out which tissue is affected. ast is prosuced byt the liver and the muscle
There can be multiple different versions of one enzyme: “isoenzyme”
isoenzyme
when there are multiple different versions of one enzyme
Interpreting enzyme changes- mild
1-2 x upper end of reference interval
Exceptions given to when the half life is very small for that species, and for specific enzymes such as GGT.
Interpreting enzyme changes- moderate
2-4 x upper end of reference interval
Exceptions given to when the half life is very small for that species, and for specific enzymes such as GGT.
interpreting enzyme changes- marked
5+ x upper end of reference interval
Exceptions given to when the half life is very small for that species, and for specific enzymes such as GGT.
SDH
(sorbitol dehydrogenase)
not very stable
GLDH better option
produced in liver
Generally only used in large animals
ALT
produced in Liver, muscle
more sensitive marer for liver than muscle
Not useful in large animals
Cat ALT has much shorter half life compared to dogs → smaller elevations are more clinically significant
GLDH
Liver marker
More stable than SDH
AST
equal markers for Liver, muscle
long half life
ALP
Biliary (within the liver), bone, intestines, steroid- diffrent isoenzyme
Steroid isoenzyme only in dogs- important for cushings
growing animals produce more alp due to bone growth but this can also happen with bone cancer
colicing horses have increse
only specilised equipmetn can tell siffrence between isoenzymes
Cat ALP has much shorter half life compared to dogs → smaller elevations are more clinically significant
Canine steroid ALP isoenzyme is elevated with both drugs (corticosteroids, phenobarbital), chronic stress, and hyperadrenocorticism
GGT
very specifically Biliary
Small increases significant- mild, moderate and mared scale less usefull here
Colostrum is high in GGT → increases in calves
Can be used to check for passive transfer
Also elevated in foals but not due to colostrum
in horses:
Increase in GGT associated with chronic biliary stasis
Good indicator of chronic liver disease
Persistent increases above 400 IU/L associated with poorer prognosis
good sensitivity, low specificity in horses
Other hepatobiliary markers
Bile acids
Bilirubin
Cholesterol
Albumin
Glucose
Coagulation factors
Bile acid cycle:
Produced by hepatocytes and excreted into the bile
Degradation occurs in the gut, then the transformed bile acids are reabsorbed
Transported to hepatocytes via the hepatic portal vein
Hepatocytes uptake the transformed bile acids for reprocessing
Bile acid stimulation test
Tests the ability of the liver to re-uptake bile acids from the portal vein.
Patient is sampled after being starved for 8 hrs so no more bile acids are released, then resampled after being fed.
Increases are supportive of either:
Reduced hepatocellular function (NB: does not necessarily indicate failure)
Portosystemic shunt (blood bypasses liver)
Cholestasis (don’t bother running this test if bilirubin is increased!) → bile acids are usually high before and after stimulation testing
Only measure single BA assay in horses
BA stimulation test not applicable for continual grazers
Will increase with prolonged fasting
Highly specific for liver disease
Good prognostic indicator
Normal <15mmol/l
Persistent increase >50mmol/l = Poor prognosis
Bilirubin
Two main types of bilirubin:
Unconjugated bilirubin
Made during breakdown of heme (from dead RBCs)
Insoluble; transported bound to albumin
Conjugated bilirubin → negligible levels in health
Has been processed by the liver and conjugated with glucuronide
Water soluble; majority is transported free
Delta bilirubin = conjugated bilirubin that is bound to albumin (tiny amount)
Analysers can give three different types of bilirubin measurement:
Total bilirubin- Total bilirubin = direct bilirubin + indirect bilirubin
Direct bilirubin → measured value- Total conjugated bilirubin
Indirect bilirubin → calculated value (total bilirubin - direct bilirubin)- Total unconjugated bilirubin
Most analysers give only total bilirubin
Causes of bilirubin increases can generally be broken down into:
Pre-hepatic = excessive breakdown of heme or inhibition of bilirubin uptake by hepatocytes
Haemolysis, fasting (horses, cattle)
Unconjugated bilirubin increases, can eventually lead to both being increased
Hepatic = reduced ability to conjugate bilirubin
Toxic insult, Leptospirosis (dogs, cattle)
Both conjugated and unconjugated fractions increased
Post-hepatic
Gallstones, mucocoele, pancreatitis (cats)
Unconjugated bilirubin increases first, then both increase as the system “backs up”
Pre-hepatic bilirubin increase
excessive breakdown of heme or inhibition of bilirubin uptake by hepatocytes
Haemolysis, fasting (horses, cattle)- cows and horses get a normal ncrease of this
Unconjugated bilirubin increases, can eventually lead to both being increased
Hepatic billirubin increase
reduced ability to conjugate bilirubin- stays in liver cells
Toxic insult, Leptospirosis (dogs, cattle)
Both conjugated and unconjugated fractions increased
Post-hepatic billirubin inxrease
problem with flow of bile
Gallstones, mucocoele, pancreatitis (cats)
Unconjugated bilirubin increases first, then both increase as the system “backs up”
Cholesterol
Produced in the liver but other sources include:
Uptake from food via lymphatics- Usually triglycerides increase also
Release from adipose tissue during negative energy balance- Usually triglycerides increase also
Increases can be particularly high if animal is overweight
Present within the bile in high concentrations
cholesterol Increases due to:
Cholestasis- Look for concurrent increases in bilirubin, GGT, ALP
Starvation/anorexia- Usually triglycerides increase also
Recent meal- Usually triglycerides increase also
Nephrotic syndrome- hepatocytes stimulated to make more cholesterol. not common
choleserol Decreases due to:
Reduced intestinal absorption
GI disease, hypoadrenocorticism
decrease in albumin and glucose
If the liver is end-stage, then these can drop due to reduced production/storage.
causes of hypoglycaemia include:
Diabetic ketoacidosis
Starvation (puppies, working dogs)
Insulinoma → pancreatic neoplasm which produces insulin
Artefact → use fluoride oxalate tube
Coagulation factors
Liver synthesizes coagulation factors
Liver failure → prolonged coagulation times
Muscular enzymes
CK
AST
ALT
CK
Muscle marker
Short half life
CK vs AST timeline
Half life of CK much shorter than AST.
Ratios of these enzymes can vary depending on time point.
NB: Owner handling and recent exercise
Pancreas: Pancreatic lipase
Used to diagnose pancreatitis but can also go up when GFR is reduced.
LIPASE IS ALSO EXCRETED BY THE KIDNEYS, so kidney damage can also effect this
Measured by a multitude of methods:
DGGR lipase- Not fully sensitive or specific, but good screening test
Can increase in dogs with hyperadrenocorticism
Drugs can increase DGGR lipase: corticosteroids, heparin
Specific pancreatic lipase immunoreactivity (cPLI, fPLI)- More specific and sensitive than DGGR lipase
SNAP pancreatic lipase immunoreactivity - Qualitative test for a quick “yes” or “no”
As a general rule, positive = positive, negative = maybe
Pancreas: amylase
Used to diagnose pancreatitis but can also go up when GFR is reduced.
Poorly sensitive in cats
lipase is generally better
Pancreas: TLI
Usually used to diagnose EPI (exocrine pancreatic insuficiency) (whereby levels are decreased)
Can go up with pancreatitis or with incomplete starvation
nonspecific for pancreatitis
tissue structure of renal pysiology
Glomerulus
Renal tubules- Proximal tubule
Loop of Henlé
Distal convoluted tubule
Collecting duct
Renal physiology: Glomerulus
Electrolytes filtered out.
Proteins should remain in blood.- Small amount of protein present in canine urine
Location of juxtaglomerular apparatus (RAAS)
Renal physiology: Proximal tubule
Resorb most electrolytes
Activate Vitamin D- calceum homeostasus
Renal physiology: Loop of Henlé
Absorption of H2O in the descending limb.
Absorption of NaCl in the ascending limb.
Creates the medullary concentration gradient required to concentrate urine in the collecting duct.
Renal physiology: Distal convoluted tubule & collecting duct
Small amounts of electrolytes resorbed in DCT
Collecting duct reabsorbs H2O → concentrated urine
Glomerular filtration rate
Speed at which fluid is filtered out of the blood into the Bowman’s capsule
Controlled by:
Hydrostatic pressure- The rate at which blood enters the glomerular capillaries
The rate at which blood leaves the glomerular capillaries
The rate at which filtered fluid moves through the renal tubules
Oncotic pressure- Amount of albumin within the peripheral blood
Alterations in hydrostatic pressure - renal in-flow
Increased in-flow: High cardiac output
High blood pressure → idiopathic, hyperthyroidism
Decreased in-flow: Low cardiac output → heart failure, shock
Water loss (decreased hydrostatic pressure) → dehydration
less inflow means thing sflow through slower and filtration slows down
Compensation occurs via dilation/constriction of efferent vessel
Compensation limited
Reduced flow through tubules:
Injury to glomerulus
Injury to tubules
Urinary obstruction → urolithiasis
Increased flow through tubules
Excretion of osmoactive substances
Glucose → diabetes
Mannitol → therapy
Diuretics
Loss of medullary tonicity - Psychogenic polydipsia (drinking an exessive amount) or diabetes insipidus → loss of electrolytes = “medullary washout”
Liver failure → loss of urea production
Renal biomarkers
Urea
Creatinine
SDMA
Others used in literature but not routinely in clinical practice:
Clearance of inulin or iohexol
Neutrophil gelatinase-associated lipocalin (NGAL)
Retinol binding protein
Creatinine
Released by muscles at a constant rate
Excreted entirely by kidneys - no reuptake
Concentration in blood dependent on:
Production- Higher with heavy muscle mass → greyhounds
Lower with muscle wasting → young and elderly patients
Rate of excretion (i.e. GFR)- main reason to look at creatinine
kidneys are resiliat- Requires damage to 75% of nephrons for increase so not sensative
Urea
Produced by the liver during protein metabolism
Excreted by the kidneys - small amount of reuptake
Provides the concentration gradient for loop of Henle
raw diet may increase abouve reference interval
GI bleeding may cause it but must be severe and is usually not main reason
Concentration dependent on:
Production- Reduced with liver failure
Increased with high protein diet
Increased with GI bleeding —> Controversy
Rate of excretion (i.e. GFR)- Requires damage to 75% of nephrons
SDMA (symmetric dimethylarginine)
not dependent on muscle mass like creatanine
Released by all nucleated cells at a constant rate
Excreted entirely by kidneys - no reuptake
Concentration in blood dependent on:
Rate of excretion (i.e. GFR)
Requires damage to 25% of nephrons unlike urea
NB: Greyhounds have naturally high SDMA
Other experimental markers for kidneys
Clearance of inulin or iohexol- Used routinely in human medicine
Neutrophil gelatinase-associated lipocalin (NGAL)- Released by injured renal tissues
Measured as urinary NGAL:Creatinine ratio
High sensitivity, specificity unclear (also released by neutrophils)
Retinol binding protein (RBP)- Made by liver and freely filtered
Measured as urinary RBP:Creatinine ratio
Azotaemia
Increase in urea, creatinine, and/or SDMA
Classification:
Pre-renal → renal blood supply, increased urea production
Renal → problem with the kidney itself
Post-renal → obstruction of urine outflow
Azotaemia- Pre-renal
how good is renal blood supply, increased urea production
diet?
Azotaemia- renal
problem with the kidney itself
Azotaemia- Post-renal
obstruction of urine outflow
Azotaemia - urea and creatinine
Require a loss of 75% of functioning nephrons before increases are detected.
For every doubling above reference interval, GFR reduces by 50%.
IRIS scheme
Used to evaluate the degree of renal injury via staging
Useful in guiding therapy
Schemes available for both acute and chronic renal disease
Acute Kidney Iinflation vs Chronic Kidney Disease
Step 1: Signalment
Age
Breed
Step 2: History
Onset of PUPD or anuria
Collapse?
Known toxin ingestion
Skin lesions (Alabama rot)
Step 3: Biochemistry / CBC
Severity of the azotaemia
Multiple organs affected
Anaemia
mild to moderate azitaemia- chronic
marked- acute
other organs effected? liver effected could indicate toxin
end stage disease can lead to non regenerative aneamia
Uraemia
build up of ureamic acids- bosy goes into acidosis
Clinical syndrome:
Lethargy/depression
Mucosal ulceration → oral, gastric
Vomiting/diarrhoea
Respiratory signs → uraemic pneumonitis, metastatic calcification
Hypertension → can lead to hypertrophic cardiomyopathy
Hypokalaemic myopathy (cats) → plantigrade stance, cervical ventroflexion
Hyperkalaemic bradycardia → acute kidney injury & urinary obstruction
Anaemia → non-regenerative
renl problems- Electrolyte disturbances
Sodium- Drops
Chloride- Usually as per sodium, but can increase independently depending on the cause of the injury
E.g. Fanconi’s syndrome
Potassium- AKI or urinary obstruction: Increases in all species → can be severe
CKD:
Dogs & horses: Increases
Cats: Decreases → may need supplementation
Calcium- Variable
AKI: Increases
CKD: Increases at first, then drops during end-stage failure
Urinary obstruction: Drops - but we don’t know why!
Phosphate- Increases, cant be excreted
Magnesium- Increases, cnat be excreted
Renal handling of major electrolytes
Aim is to conserve water by retaining sodium
Creates a concentration gradient to reabsorb H2O
Chloride generally moves with sodium → NaCl
Potassium secreted in exchange for sodium
Sodium as an electrolyte
Most abundant electrolyte
Important factor in maintaining tonicity/osmotic pressure
Increases due to:
Free water loss → diabetes insipidus
Water deprivation
Too much- Salt poisoning
Hyperaldosteronism- aldosterone is th ehormone that encorages the swithcing of sodium and potassium n the renal tubules
Decreases due to losses from:
GI tract → diarrhoea, vomiting
Kidneys → medullary washout, diabetes mellitus (increases gfr) , hypoadrenocorticism (less aldosterone)
Cavitary effusions
Perspiration → horses
electrolytes- Chloride
Second most abundant electrolyte
Increases and decreases are usually in association with sodium
Instances where you lose more chloride that sodium:
Vomiting
Pyloric outflow obstruction → GDV, LDA, foreign body
Instances where you lose more sodium than chloride:
Proximal small intestinal diarrhoea → pancreatitis
Renal tubular injury → Fanconi’s syndrome
Hypoadrenocorticism- not enough ardosterone
Corrected chloride
If NaCl is lost or gained, then sodium and chloride should increase or decrease in equal proportions. In order to work out if chloride levels are in proportion with sodium, a corrected chloride can be calculated.
Corrected chloride = (Na / [centre of Na reference interval]) * Cl
If the corrected chloride is WRI, then chloride is proportionate to sodium
electrolytes- Potassium
Important regulator of pH and cell electrochemical gradient- Used by cells to exchange for H+ when blood pH is altered
Exchanged for Na+ to maintain electrochemical gradient- Higher amounts of K+ inside cells vs outside
Decreases are due to:
Alkalosis
Decreased intake → anorexia (very common in horses)
Perspiration → horses
Increases are due to:
Acidosis
Reduced renal excretion → CKD, AKI, urinary obstruction, hypoadrenocorticism
Artefact (common) → haemolysis, delayed serum separation
Acidosis
Blood pH is tightly regulated ~7.4
Drop in blood pH = acidosis → increased H+
alkalosis
Blood pH is tightly regulated ~7.4
Climb in blood pH = alkalosis → decreased H+
metabolic changes in blood ph
acids or bases building up in tissues/blood
respiritory changes in blood ph
affected by ability to exhale CO2
Metabolic acidosis
Occurs when acids start building up in the tissues/blood or when bases are lost
Metabolic acidosis causes:
If you lose Na in excess of Cl (i.e. loss of NaHCO3)
H2CO3 + NaCl → NaHCO3 + HCl ( retention of hydrochloric acid)
NaHCO3 is excreted in:
Pancreatic/biliary secretions → reabsorbed in small intestines in health
Kidneys → reabsorbed again within tubules in health
HCl gets left behind if you have:
SMALL INTESTINAL DIARRHOEA
Specific types of RENAL TUBULAR INJURY (e.g. Fanconi’s Syndrome)
HYPOADRENOCORTICISM (affects ion exchange in kidney)
Metabolic acidosis causes:
If you retain acids → measurable with Anion Gap
Ketones → DIABETES MELLITUS
Lactate → INJURED OR HYPOXIC TISSUES
Uraemic acids → RENAL INJURY
Metabolic alkalosis
Occurs when acid is lost
Metabolic alkalosis causes:
If you lose HCl- VOMITING → gastric secretions high in HCl
TWISTED STOMACH (GDV) or DISPLACED ABOMASUM → HCl secreted into stomach but cannot enter small intestine to be resorbed → “lost” in the stomach
Pyloric outflow obstruction (GASTRIC FOREIGN BODY) → as above
GASTROINTESTINAL STASIS → as above
Respiratory acidosis
Occurs when CO2 is not exhaled sufficiently
CO2 is an acid → decreased exchange → build up of CO2 → acidosis
Respiratory acidosis causes:
Respiratory tract obstruction
Pulmonary fibrosis
Pulmonary thromboembolism
Pulmonary neoplasia
Pneumonia
Anything that reduces O2/CO2 exchange…
Respiratory alkalosis
Occurs when CO2 is exhaled excessively
Respiratory alkalosis causes:
Tachypnoea
CO2 is an acid → increased exhalation → alkalosis
Mixed acid/base disorders
Where you have more than one acid/base disorder occurring concurrently:
Renal failure with vomiting: Renal failure = metabolic acidosis
Vomiting = metabolic alkalosis
Diabetic ketoacidosis and pancreatitis:
Ketoacidosis = metabolic acidosis
Pancreatitis = metabolic acidosis +/- metabolic alkalosis (if there is vomiting)
Septic abdomen (lactic acidosis) and hyperventilation:
Sepsis = metabolic acidosis
Hyperventilation = respiratory alkalosis
Vomiting causing aspiration pneumonia:
Vomiting = metabolic alkalosis
Pneumonia = respiratory acidosis
Potassium - acidosis
Acidosis = excess H+
H+ taken into tissue and exchanged for K+ → rise in blood K+
Potassium - alkalosis
Alkalosis = H+ deficit
H+ taken out tissue and exchanged for K+ → drop in blood K+
balence of calceum and phosphorous
Balanced controlled by PTH and Vitamin D
Vitamin D less important in horses
Excreted by the kidneys and absorbed by the intestines
UV light for vitamin D production not applicable in veterinary species
Dietary intake and balance important, especially in horses
Total calcium
total calcium bound to albumin and uraemic acids
Total can increase or decrease with fluctuations in these negatively charged molecules → especially albumin
low albumin should cause low calcium- if not check unbounded calcium
Free (aka ionised) calcium
unbound calcium
Levels very tightly controlled by PTH, vitamin D and calcitonin
differentials of Hypercalcaemia-
HARD IONS G
Hyperparathyroidism → decreased excretion and increased bone resorption
Addison’s disease → decreased excretion
Renal disease → decreased excretion
D-hypervitaminosis → psoriasis cream, rodenticide poisoning
Idiopathic → most common cause in cats
Osteolytic → osteosarcoma
Neoplastic → PTHrp
Spurious → artefact, analyser error
Granulomatous disease → macrophages produce vitamin D
differentials of hypocalcaemia
Nutritional- Insufficient dietary intake
Excessive phosphorus intake
Hypomagnesaemia
Renal- Chronic: insufficient Vitamin D production
Not relevant in horses
Acute: reduced tubular reabsorption
Urinary tract obstruction: unknown
Pregnancy/lactation
Pancreatic pathology- EPI: reduced vit D absorption
Acute pancreatitis: unknown
Drugs/toxins- Ethylene glycol
Furosemide
Tissue injury- Massive necrosis (e.g. in tumours)
Rhabdomyolysis, polysaccharide storage myopathy
Rumen overload: unknown
phosphorus increase
Decreased excretion- Renal injury (but not in horses!)
Release from injured cells- Massive necrosis (e.g. in tumours)
Rhabdomyolysis, polysaccharide storage myopathy
Artefact with haemolysis or sample storage
Excessive vitamin D
decreased phosphorous
Increased excretion- Hyperparathyroidism
Fanconi’s syndrome (dogs)
Renal failure in horses
Reduced intake
Hypovitaminosis D
changes in Magnesium level
Only rarely measured in practice.
Has bound and unbound fractions like calcium.
Most important aberrations:
Increases with renal disease (reduced excretion)
Decreases due to dietary deficiencies (aka staggers)
Limitations of population-based reference intervals
Some analytes are very specific to that individual- Intra-individual variation is small but group variation is wide
E.g. creatinine
Analyser precision and accuracy may not be perfect
All analysers should be precise around decision thresholds
A small degree of inaccuracy allowed → varies between analyte
Analysers should be checked regularly → QC very important!
Not all assays are perfectly linear
Once a threshold is reached, higher or lower measurements can become less accurate and precise
Sample haemolysis/lipaemia/icterus can interfere with some assays
Seasonal/diurnal variation- Reference intervals may vary depending on the season or time of day
Can be very important when monitoring medication
Variations with breed and age- Greyhounds have many breed-specific variations
Some analytes change as patients age
Use of generic intervals for exotic species- “Reptile” intervals for lizards and snakes
“Avian” intervals for chickens and parrots
Seasonal ACTH variations in horses
ACTH levels used to help diagnose PPID
ACTH levels peak naturally in Autumn
Different reference ranges required in Autumn
canine t4 varies with
age
sensitivity
the ability of a test to detect sick patients
the ability of a test to exclude a disease when you get a negative result
specificity
the ability of a test to exclude a disease when you get a negative result
when you get a positive reusult you know that the disese is present
Decision thresholds
Some diseases use decision thresholds to rule in/out disease. Very common when diagnosing endocrine disease.
Examples:
Basal cortisol → hypoadrenocorticism
Total T4 → hypothyroidism/hyperthyroidism
ACTH → PPID in horses
IGF-1 → acromegaly in cats
Fructosamine → diabetes mellitus
Can vary depending on severity of the illness being diagnose-
Infectious disease assays usually very sensitive, but may be a trade off for low specificity
Screening tests often less specific
Confirmatory tests often highly specific
ROC curve analysis most common method- Enables selection of cut-off with highest sensitivity and specificity
checklist for trustworthy results
Is my analyser QC and calibration up to date?
Do I know the grey zone for the analyte?
Electrolytes have very small grey zone
Hormones typically have ~20% variability in assay precision
How reliable is my decision threshold?
Check recent papers or textbooks for sensitivity and specificities
Is the sample haemolysed/lipaemic/icteric?- Check reagent inserts to work out if that analyte is affected
Has my sample been taken/handled properly?- Artefacts can occur if not stored properly, Serum should be separated/spun soon after collection, Gel in serum tubes can interfere with some tests e.g. progesterone
Is this test fully sensitive/specific?- Especially important with positive/negative results
Sensitivity and specificity highly important
Consider further tests if:
Result does not fit clinical picture
E.g. FIV positive antibody test in a young indoor cat
Sensitivity or specificity are not sufficient for a confident diagnosis
E.g. Patient tests positive highly sensitive but poorly specific test
Quantitative result required for confirmation or monitoring purposes
E.g. SNAP cPLI/fPLI tests vs quantitative lipase measurement
Summarise the differences in reasons for performing a post mortem on a farm animal compared to a small domestic animal (dog/cat)
Farm animal:
mostly herd health and disease control reasons: can do a thorough search for all kinds of disease that is just not possible on live animals
domestic animals:
in cases of sudden, uneplained death.
to confirm/refine a clinical diagnosis, to obtain samples for further tests (microbial culture, histopathology), to assess the effect of treatment(s), to collect evidence in forensic cases, or to prevent further illnesses and deaths within in-contact animals (for example, within litters of young puppies or kittens).
List the potential zoonotic pathogens that pose a risk to the pathologist when performing a post mortem on a farm animal
E.coli VTEC 0175
anthrax
TB
Camplyobateriosis
Avian influenza
salmonella
cryptosporisiosis
leptosporosis
orf
ovine chlamysiosis
psittacosis
q fever
ring worm
zoonotic diphtheria
agonal changes caused by barbituate
The spleen is particularly susceptible to extreme congestion relating to barbiturate euthanasia
Crystal deposition on the endocardium is another common barbiturate associated change.
which post mortem changes can help with assessing time of death
rigor mortis, livor mortis, and algor mortis
Algor Mortis- the cooling of the body after death
Livor Mortis
The purple-red discoloration of the soft tissues due to postmortem gravity-dependent pooling of blood is livor mortis. Livor mortis may be observed either externally in the skin and mucous membranes or internally in the abdominal or thoracic viscera, most notably the lung, and typically develops within 30 minutes to 2 hours after death in humans.22 Livor mortis must be distinguished from hemorrhage. In livor mortis, the pooling of blood is entirely within dilated vascular channels, whereas hemorrhage is the escape of blood from the blood vessels and into the connective tissues or internal or external spaces. Therefore, hemorrhage within the soft tissues will not blanch when subjected to digital pressure. Livor mortis, however, depending on its stage of development may blanch when subjected to digital pressure.
Rigor Mortis
Immediately after death, a series of biochemical reactions occurs within the skeletal and cardiac muscle fibers throughout the body. Specifically, adenosine triphosphate (ATP), the molecular source of energy for muscular contraction, continues to be consumed by the muscle cells, resulting in cross-bridge formation between myosin and actin fibers. However, the regeneration of new ATP ceases upon death. Because ATP is required for the decoupling of actin and myosin fibers and the resultant relaxation of the muscle, relaxation can no longer occur after the limited supply of ATP is exhausted. As a result, the muscle fibers remain in a state of permanent contraction, unable to relax because of the lack of additional ATP.
Algor Mortis-
the cooling of the body after death
Livor Mortis
The purple-red discoloration of the soft tissues due to postmortem gravity-dependent pooling of blood is livor mortis. Livor mortis may be observed either externally in the skin and mucous membranes or internally in the abdominal or thoracic viscera, most notably the lung, and typically develops within 30 minutes to 2 hours after death in humans.22 Livor mortis must be distinguished from hemorrhage. In livor mortis, the pooling of blood is entirely within dilated vascular channels, whereas hemorrhage is the escape of blood from the blood vessels and into the connective tissues or internal or external spaces. Therefore, hemorrhage within the soft tissues will not blanch when subjected to digital pressure. Livor mortis, however, depending on its stage of development may blanch when subjected to digital pressure.
Rigor Mortis
Immediately after death, a series of biochemical reactions occurs within the skeletal and cardiac muscle fibers throughout the body. Specifically, adenosine triphosphate (ATP), the molecular source of energy for muscular contraction, continues to be consumed by the muscle cells, resulting in cross-bridge formation between myosin and actin fibers. However, the regeneration of new ATP ceases upon death. Because ATP is required for the decoupling of actin and myosin fibers and the resultant relaxation of the muscle, relaxation can no longer occur after the limited supply of ATP is exhausted. As a result, the muscle fibers remain in a state of permanent contraction, unable to relax because of the lack of additional ATP.
Summarise the procedure for post mortem examination of zoo animals
Adequate facilities must be available either at the zoo or within a reasonable distance
for the post-mortem examination of all species held at the zoo
Animals that die at the zoo should be examined post-mortem in accordance with
veterinary advice. Where appropriate, samples for diagnosis or health monitoring should
be taken for laboratory examination
Following post-mortem examinations conducted on the zoo premises, carcasses and
organs should be disposed of swiftly and in accordance with the Animals By-Products- if possible carcasses of interesting or importan tspecies should be offered to a recognised scientidic institution
(Enforcement) (England) Regulations 2011
List the potential zoonotic pathogens that pose a risk to the pathologist when performing a post mortem on an avian species
psittacosis
avian influenza
campylobacteriosis
salmonella
toxoplasmosis
west nile virus
List the available fixatives for preserving tissues for histopathology
Immersion fixation involves the use of fixative solutions.
Formaldehyde (10% neutral buffered formalin) is by far the most popular fixative used in histology since it penetrates the tissue well and creates crosslinks without affecting the sample tissue’s antigenicity. While it is relatively slow to fix, it is highly recommended for immunohistochemical techniques
Glutaraldehyde fixes quickly and provides great cytoplasmic and nuclear detail, but it penetrates poorly and deforms the alpha helix structure in proteins. As such, it is good for electron microscopy but not so for immunohistochemical staining.
Paraformaldehyde (PFA) is an effective fixative that reacts with primary amines found in the protein to form crosslinks (“methylene bridges”). PFA works great in stabilizing proteins and preserving morphology, but it fixes very slowly (more than 24 hours for smaller tissues and up to a few weeks for larger tissues) and may mask antigenic sites.
Perfusion fixation can best be described as fixation through blood flow. The fixative is injected into the heart and spreads through the entire body. Since the tissue doesn’t die until it is fixed, you can get a sample with perfect morphology. Unfortunately, the subject dies during the process.
Outline how formalin fixation preserves tissues
Formalin (a solution of formaldehyde in water) preserves proteins and cellular organelles in a stepwise process. It penetrates tissues quickly then binds to lysine, tyrosine, asparagine, tryptophan, histidine, arginine, cysteine, and glutamine in all of the proteins present in a specimen. It is the reaction between formalin and uncharged reactive amino groups that leads to the formation of cross-links.
Describe the risk posed by the use of formalin as a fixative and how this is mitigated
Formalin fixation is not selective, so cross-linking of target proteins with unrelated proteins can reduce immunoreactivity with target-specific antibodies. In addition, cross-linking can lead to significant changes in the three-dimensional conformation of proteins which may also reduce immunoreactivity. Fortunately, antigen retrieval methods, such as heat-based or enzymatic procedures, can reverse the undesirable changes caused by fixation
why would we sample blood from exotic species
Health screening – to prove animal is healthy (pre GA/mixing/transfer)
To uncover subclinical disease- Many exotics v good at hiding clinical signs, Some species unable to do thorough clinical examination eg. Tortoise (unable to palpate)
To make a diagnosis
To confirm a diagnosis
To guide further clinical investigation/next steps- Focussed testing important
To help determine prognosis
To help monitor progress/changes
Because we can??!
what considerations must be made for reference ranges of exotic species
Consider reference ranges very critically:
Different populations- diffrent genders may greatly vary unlike domestic species
Clinical context
Management context
Lifestage/signalment
Care with extrapolation between species
Consider panels offered and if appropriate for exotic species
Often very non specific clinical signs - Prey species so mask clinical signs
Often seeing advanced disease process in these animals due to masking signs- Rapid diagnostics often important
Need to narrow down differential diagnoses
considerations when prepping an exotic species for blood collection
Warm reptiles
Fasting?- Crop emptying/species variation
Post meal effects- Hypoglycaemia risks when starving these animals
Prepare in advance- Consider which parameters are priority
Skin preparation
Clipping/plucking?- minimally as possible
Cleaning- reptiles grimy
Care with spirit/alcohol- cools reptiles
Suitable equipment gotten before animal is anethsitised- Needle/syringe
Microhaematocrit tubes
Small sample tubes
0.3ml tubes/1.1ml tubes
considerations for blood sample collection in exotics
Venipuncture techniques
Appropriate restraint- Manual/chemical
do we wan t a pre-anesthetic sample
Anaesthesia/Sedation/Conscious- Stress of GA vs handling
Other procedures
Safety of isoflurane vs stress
Experience/confidence level of operator- ga better for lessed experience
stress and GA Can impact on bloods (eg pH/electrolytes from GA/CK from conscious handling)
Appropriate anticoagulant
Heparinised syringes- reduces risk of loosing blood form already small sample. rabbits and rodents also hsve very rapid clotting times
EDTA – effective in some species but some birds & reptiles this will lyse red blood cells
Lithium Heparin – usually appropriate anticoagulant, appropriate size, reduces need to take more samples
Lipaemia
Lipemia is a turbidity of the sample caused by accumulation of lipoprotein particles
Changes can be pathological or physiological
May affect analysis/parameters
Lymph dilution
lymps vessel or sinus has been sampled through and so tissue fluid is in sample
some exotics can only have small blodsamples taken from them. what can be done with this?
Blood smear – 1 drop required- Huge amount of information
PCV – can use microhaemtocrit tubes (40-75ul tubes)- Total Solids from spun section, Assessment of buffy coat
Critical care parameters – 0.1-0.5ml blood- Electrolytes, pH, Glucose etc
Use lithium heparin and don’t spin- Can then use for haematology & biochemistry
what can imapct blood samples from exotic species
Anticoagulants- Haemolysis protein, electrolytes, cell counts, enzymes
Fasting- Ferrets, other small mammals – see lower levels
Post meal- Elevations in glucose, total protein, uric acid
Restraint -Elevations in creatinine kinase, AST
Anaesthesia- Changes in electrolytes, creatinine kinase, . Consider preanaesthetic blood sampling if apprpriate
how much blood can be sampled from a healthy individual (exotics)
Maximum of 10% of total blood volume may be sampled from a healthy individual.
We need to know the blood volume per kg of body weight
Only 5% should be sampled in sick/debilitated animals
Must consider blood loss through haematoma formation
Most patients are compromised
Avian Blood Sampling Volumes
Blood volume calculated as 10% bodyweight- Eg. 10ml circulating blood in 100g bird
Blood volume that can safely be taken in birds around 1% BW- Eg. 1ml sample collection in 100g bird, 3.5ml from 350g bird
Examples:
Canary – 0.2ml
Cockatiel – 1ml
African Grey Parrot – 5ml
Harris Hawk – 10ml
Mute Swan – 100ml
Consider post sampling haemorrhage
Reptilian Blood Sampling Volumes
Lower blood volume than equivalent sized mammal/bird
Blood volume calculated as 4-8% bodyweight- Eg. 4-8ml circulating blood in 100g reptile
Sample volume that can safely be taken around 10% of Blood volume
This equates to approximately 0.5% bodyweight
Eg. 0.5ml from 100g snake, 3.5ml from 700g lizard
exotic Mammalian Blood Sampling Volumes
Blood volume calculated as 6-8% bodyweight- Eg. 6-8ml circulating blood in a 100g mammal
Blood volume that can safely be taken in mammals around 1-2% of BW
Examples:
Dwarf Rabbit – 5ml
Standard Rabbit– 20ml
Giant breed Rabbit – 36ml
Ferret – Jill 5ml Hob 10ml
Guinea Pig – 8ml
Rat – 2-3ml
Mouse – 0.2-0.3ml
Hedgehog 2-6ml
venipuncture sites in exotic mammels-
Cranial Vena Cava- Blind technique
Anaesthesia (or moribund animals)
Landmarks- clavical
use short needle to avoid heart, slightly longer with ferret
Ferret, Guinea pig, Hedgehog
Jugular Vein
Conformation may limit- short neck in guinie pigs
Stress
Respiratory compromise- rabbits breathing compromised in this position- glottis disloged from soft pallet
Cranial pressure can be increased by occluding the jugular
Rabbits, Ferrets, Hedgehogs , Other rodents
Cephalic-
As per dogs/cats
Conformation may limit
Gentle restraint
Small blood samples
Rabbits, Ferrets, Guinea Pigs and other hystricomorphs, Hedgehogs , Other rodents
Lateral Saphenous-
Ideal in rabbits
Often smaller blood samples
Lateral hind leg – cranial to hock
Prone to collapse
Lateral & Dorsal Tail Veins
Rats & Mice
Vasodilation helpful- warm tail in waterbath, anethetic agent, tourocate
Avoid in Gerbils (tail slip)
Marginal Ear Vein
Rabbits, some deer
Small Volumes
Vasodilation helpful- warm ear with hand
Head shaking can->vein laceration- EMLA/Local anaesthetic
venipuncture sites in birds
Basilic/Wing/Brachial/Ulnar Vein-
ventral aspect of elbow
Bend the needle- not much sorft tissue
Very fragile vein prone to haemorrhage- Can bruise easily
Good for: most bird spp. esp if under GA
Less easy in: very small or wriggly patients
Medial Tarsal Vein-
not GA needed
Good for: most bird spp. esp waterfowl, gulls, Less easy in: Raptors (talons), corvids(scaly legs), v small passerines (small)
Right Jugular Vein-
Not all birds have a left jugular or is v. small
Jugular apterium- one of the bare spaces between the feathered areas on the body of a bird.
More difficult for left handed people
Good for: Small passerines/psittacines/ raptors/ corvidsNot in: Pigeons (have no jugular vein or apterium, cervical plexux instead), Waterfowl ( no jugular apterium), Gulls
sites of venipuncture in Chelonians
Jugular vein-
Regarded as gold standard- low risk of lymp dilution
Dorsal branch runs in line with ear (auricular) scale
Ventral branch runs below ear scale
Both v superficial
Handling key – can be very stubborn
Pressure after sampling very important- Can hit carotid artery- Can get haematomas due to clotting defects common in reptiles
Subcarapacial sinus-
Blind technique- Good for larger samples
Higher risk of lymphodilution c/f jugular
Bend the needle
Can use even in drawn in/small animals
Dorsal tail vein-
Dorsal midline
Palpate spine
Smaller volumes
Higher risk of lymphodilution c/f jugular
Brachial plexus-
Useful in larger tortoises
Leg drawn out
Can feel tendon behind carpal joint
venipuncture sites in squamates
Ventral Tail Vein-
Care if species which autotomizes-
Avoid hemipenes- go a quarter of the way down the tail
Midline (like a cow!)
Hit spine and draw back
Reasonable sample sizes
Common in lizards
Only in large snakes
Care if species which autotomizes
Midline (like a cow!)
¼-1/3 way down to avoid hemipenes
Hit spine and draw back
Reasonable sample sizes
Cardiocentesis-
Safer than it sounds
Some clinicians prefer to sedate
Locate using doppler
Stabilise heart
Enter from caudal direction
Advance the needle into the ventricle
Allow to fill with the cardiac output
biochemicla markers of inflamation in exotics
Protein electrophoresis
Albumin:Globulin ratio
Acute phase proteins (Early stages of use in exotics)
Total Protein
Not very sensitive as doesn’t differentiate between different proteins
Includes albumin and globulins
↑ Haemoconcentration, Inflammation, vitellogenesis, lipaemia
↓Malnutrition/absorption, parasitism, hepatopathy, nephropathy, enteropathy, skin losses, lymph dilution
Albumin in exotics
Difficult to assay accurately in practice- Low levels compared to domestic patients, Pre-albumin can affect analysis of albumin
Protein Electrophoresis most accurate way to assay proteins- interpretation needs to be species specific expert
Avian/Reptilian
↑ Haemoconcentration, vitellogenesis, follicular stasis
↓Malnutrition/absorption, parasitism, hepatopathy, nephropathy, enteropathy, skin losses
Globulins in exotics
Avian
Alpha globulins (acute phase proteins)
↑ Acute inflammation, reproductively active female (vitellogenesis)
↓hepatic disease but rarely seen
Beta globulins (acute phase proteins & immunoglobulins)
↑ Acute inflammation, Acute or chronic disease response eg Aspergillosis, psittacosis
Gamma globulin (immunoglobulins)
↑ Chronic inflammation, Acute or chronic disease response eg Aspergillosis, psittacosis
Mammals
Rabbits with E cuniculi often have higher gamma globulins
Decreased Albumin:Globulin Ratio
Alanine Transferase (ALT) & Alkaline Phosphatase (ALP) in exotics
ALT
Widely used as marker of hepatocellular injury in domestic patients
Not released in liver damage in exotic species so of little use
ALP
No use in relation to cholestasis
Purely a marker of bone damage and turnover, elevated in young animals & MBD
Aspartate aminotransferase (AST) in exotics
Elevated in liver and muscle damage
Must assess alongside Creatine Kinase for muscle damage and GLDH for liver damage
↑Hepatocellular damage, tissue damage
Creatine Kinase (CK) in exotics
Avian
Can be elevated from difficult venipuncture of muscle damage/necrosis
↑tissue damage (can see large magnitude increases), IM injections, catabolic states
Short half life (16 hours)
if very high pay attention
Reptilian
Present in skeletal and heart muscle and less in kidneys
↑ with muscle damage and post traumatic venipuncture
Mammalian
In skeletal, smooth & cardiac muscle
↑ with any disease process associated with muscle
Glutamate Dehydrogenase (GLDH) in exotics
Very short half life
specific to liver damage but not sensitive to it
Avian
Specific for hepatocellular damage but not very sensitive
↑Hepatocellular damage
Reptilian
Useful indicator of hepatocellular damage but limited evidence
Mammalian
Not widely used
Gamma-glutamyl transferase (GGT) in exotics
Marker of cholestasis in domestic patients AND exotics
Also present in renal tissue in exotics- Excreted into urine so can’t be picked up on blood biochemistry
Avian
Specific for hepatocellular damage but not very sensitive
↑Biliary Stasis (Similar to ALT in mammals)
Bile acids in exotics
Mammals
Circadian rhythm
In rabbits caecotrophy makes fasted sample collection challenging so hard to tell if elevated levels relevant
Only useful if consistently raised
Hepatic coccidiosis in rabbits
Avian
Reliable indicator of liver function
Beware post prandial increases up to 4.5 x normal range
Some species lack gall bladder
↓ common in birds with microhepatica, poor feathering, overgrown malformed beak
↑Hepatic insufficiency
Reptiles
May be useful – major bile acid varies across reptilian taxa
Much reduced post prandial elevations c/f birds (max 1.7 x range)
Bile Pigments in exotics
Bilirubin
Some birds produce this and see inconsistent elevations with liver disease
Some birds cannot produce bilirubin as most don’t have biliverdin reductase
Generally don’t see icterus in avian patients
Biliverdin
Biliverdin primary bile pigment/end product of haemoglobin in reptiles
Reptiles lack biliverdin reductase enzyme to produce bilirubin from biliverdin
No commercial assay- so detect with Biliverdinuria-green urin
Glucose in exotics
Handheld vs laboratory assays
Human glucometers tend to underestimate) vs Veterinary glucometers oversestimate
Mammals
Herbivores vs carnivores
Rabbits – useful prognostic indicator
↑ Stress, pain, food intake, severity of disease, GI obstruction vs stasis
Ferrets (Guinea Pigs)- ↓ Insulinomas, septicaemia, severe liver disease, starvation- sick ferrets should have glucose checked
Reptiles
Very variable with spp, nutrition, environment, season
↑ with stress, high temps, pancreatic neoplasia and rarely DM
↓ anorexia, malnutrition, severe hepatic dysfunction, sepsis
Lactate in exotics
Produced during times of tissue hypoperfusion
Serial monitoring rather than single value for porgnosis
↑ shock, low cardiac output, acute liver failure, sepsis, seizures
Mammals
Ranges not well defined – rabbits may have higher lactate values than other mammals
Reptiles
Is a marker of anaerobic metabolism, can be used to assess physiological stress
Birds
Capture myopathy
Uric Acid in exotics
Is useful marker to detect renal insufficiency
Not v sensitive s doesn’t elevate quickly
Avian
Main indicator of renal function
↑ Renal disease, postprandial, esp in carnivores, dehydration, egg production, gout
↓Malnutrition/absorption, liver disease
Reptiles
Also primary end product of protein metabolism
Dependent on reptile’s natural environment
Terrestrial reptiles are uricotelic, Aquatic usually ureotelic
Mammals
Not useful – mammals excrete the water soluble nitrogenous waste product urea
Urea in exotics
Mammals
Main marker of renal function alongside creatinine
↑dehydration, renal dysfunction (NB once 50-75% nephrons affected)
Rabbits
Diurnal fluctuation (related to caecotroph ingestion)
Peaks in late afternoon/evening
Avian
Limited value
↑ 4-5 x with dehydration, not sensitive for detecting renal insufficiency
Reptiles
Less useful parameter except for in tortoises
↑dehydration as urea can be resorbed across the bladder to reduce water losos
Creatinine in exotics
Levels low consistently in birds and reptiles
Don’t change with renal insufficiency
Calcium
Total Calcium
Ionised (c. 45%) – metabolically active (can diffuse into cells)
Bound to Albumin (c. 40%) Being transported – not metabolically active
Associated with other ions eg Lactate and Phosphate 10-15% - biologically inert
Essential to measure Ionised calcium
Ca:P ratio important
In egg laying females
See huge elevation in Calcium to produce egg shell
This is predominantly Bound and not metabolically active
Mammals
↑ neoplasia, renal failure, impaired excretion, Ca-rich diets (rabbits/G pigs)
Hypocalcaemia
Again total Ca can be misleading if iCa low but total Ca normal
Mechanisms of cell injury
Anything that causes cell injury disrupts cellular homeostasis.
Cells can be injured by myriad causes, both from intrinsic and extrinsic sources.
Damage occurs to cells through one or a combination of of four basic mechanisms:
Adenosine triphosphate (ATP) depletion
Permeabilization of cell membranes
Disruption of biochemical pathways, especially those of protein synthesis
DNA damage
Damage occurs to cells through one or a combination of of four basic mechanisms:
Adenosine triphosphate (ATP) depletion
Permeabilization of cell membranes
Disruption of biochemical pathways, especially those of protein synthesis
DNA damage
causes of atp depletion
Hypoxia/anoxia- oxygen essentail to make
Specific toxins
causes of Permeabilization of cell membranes
Hypoxia/anoxia
Reactive oxygen species (free radicals)
causes of Disruption of biochemical pathways
Hypoxia/anoxia
Reactive oxygen species (free radicals)
causes of DNA damage
Endogenous causes
Reactive oxygen species (free radicals)
Replication errors
Exogenous causes
Ultraviolet light
X-rays and gamma rays
Certain plant toxins
Viruses
Morphology of cell injury: The common appearance of an injured cell is-
Due to
Failure of ATP-dependent enzymes and pumps/channels
Increased membrane permeability
Acute cell swelling is also known as hydropic degeneration
Particularly in the liver (hepatocytes) and kidney(renal tubular epithelial cells
In other cell types such as the skin (keratinocytes), cell swelling from influx of water is called ballooning degeneration.
Can be reversible if inciting factor/agent is gone
Three key histopathological changes in acute
early and reversible cell injury visible with light microscopy are:
Cell swelling
Cytoplasmic vacuolation
Hypereosinophilia
Organelle changes, membrane blebs and myelin figures are typically only seen using ultrastructural examination (transmission electron microscopy)
cell injury: Fatty change
Physiology of lipid metabolism
Lipid is delivered to the hepatocyte from dietary sources or body fat stores in the form of free fatty acids (FFAs).
A small amount of FFAs are also synthesized in the hepatocyte itself from acetate.
Some of the FFAs are utilized for the synthesis of cholesterol and phospholipids, and some may be oxidized to ketone bodies (1).
Most of the intracellular FFAs are esterified to triglycerides (2).
Once triglycerides are produced, they must be complexed to a lipid acceptor protein (or apoprotein) prior to export from the cell (3) as lipoproteins.
This requires protein and energy
Triglycerides may accumulate if the balance between the synthesis of triglycerides and their utilization or mobilization is deranged. When intracellular triglycerides accumulate, a fatty liver results.
Hypoxia
Protein synthesis- Aflatoxin, Carbon tetrachloride
Outcomes of cell injury
Four possible outcomes
Repair
Adapt- chronic low grade injury
Senescence- dna damage
Death
Dysplasia -> dna damage- neoplasia
Unsurprisingly, mitochondria, which are the organelles most susceptible to injury, are thought to direct many of the processes of cellular adaptation, senescence, and programmed death.
The point at which reversible cell injury becomes irreversible is debateable, but likely is dependent on calcium haemostasis within the cell
cellular adaptation
In the face of chronic sub-lethal injury (or stressor), cells can adapt involving the following mechanisms:
Hypertrophy
an increase in cell size by virtue of an increase in number and size of organelles
Hyperplasia
an increase in cell number that only those cells capable of mitosis can undergo
Atrophy
a decrease in cell size by virtue of a decrease in number and size of organelles
Metaplasia
a change from one differentiated cell type to another of the same germ layer (e.g., from ciliated epithelium to stratified squamous epithelium in the respiratory tract)
Dysplasia
abnormal differentiation with features of cellular atypia
These are physiologically normal responses, however can result in their own pathologies.
Hypertrophy in cell adaptation
an increase in cell size by virtue of an increase in number and size of organelles
Cellular hypertrophy is the process by which postmitotic cells, such as cardiac or skeletal myocytes, can grow.
Physiological response to increased workload and/or hypoxia.
In the heart, in the short term this is compensatory resulting in increased capacity.
In the long term, accompanying changes, can lead to decompensation of the affected organ.
The classic example in veterinary medicine is hypertrophic cardiomyopathy (HCM) in cats.
The complex pathophysiology of HCM is still unclear.
Reversible if due to treatable disease
hyperthyroidism
Do not confuse hypertrophy with hyperplasia
Hyperplasia
an increase in cell number that only those cells capable of mitosis can undergo
Physiological response to increased, often hormonal, stimulus
Puberty and pregnancy
Thyroid hyperplasia - Goitre
Maternal iodine deficiency will result in hyperplasia (and hypertrophy) of thyroid follicular epithelial cells in ruminants
Rarely see it as bad as the image to the right, so should always weight and store thyroid glands (fixed and frozen) in cases of perinatal/neonatal death in ruminants
Non-neoplastic enlargement of a tissue.
Hyperplasia is often the result of hormonal influences (e.g. benign prostatic hyperplasia, perianal gland hyperplasia), tissue injury (e.g. regenerative nodules in the liver, granulation tissue with fibroplasia) or antigenic stimulation (lymphoid hyperplasia).
Aspiration of hyperplastic lesions:
Higher than expected cellularity
Cells may display some mild criteria of malignancy
Mildly increased N:C ratio
Darker blue cytoplasm
More prominent nucleoli
Finer chromatin than normal
Atrophy
a decrease in cell size by virtue of a decrease in number and size of organelles
Physiological response to lack of use/innervation/stimulation
Classic example is the small liver of the puppy with a portosystemic shunt
Metaplasia
a change from one differentiated cell type to another of the same germ layer (e.g., from ciliated epithelium to stratified squamous epithelium in the respiratory tract)
Typically from a specialised and/or fragile type (ciliated or simple cuboidal) to a less specialised, more protective type (eg: stratified squamous or goblet cell)
Vitamin A deficiency in parrots results in squamous metaplasia of respiratory and upper GIT epithelium
Poorly understood mechanism
Results in hyperkeratosis of oral cavity, conjunctiva, nasal lacrimal duct, upper alimentary tract, and respiratory tract
Ducts of glands can become blocked by keratin, often resulting in huge keratin granulomas and rhinoliths
Dysplasia
abnormal differentiation with features of cellular atypia
When applied to epithelium, dysplasia implies an increase in the number of poorly differentiated or immature cells
Can be a precursor to neoplasia
Microscopic features:
Variation in size (anisocytosis)
Variation in shape (poikilocytosis)
Hyperchromatic nuclei
Large nuclei (karyomegaly)
Increased size or number of nucleoli
Mitotic figures
Making the call between dysplasia and neoplasia is thus difficult
A continuum
Classic example is actinic keratosis on the pinna of white cats due to UV light
A carcinoma will breach the basement membrane
Dysplasia = disordered growth
Common in epithelial tissue secondary to inflammation or irritation.
Loss of uniformity of the individual cells
Disordered architectural arrangement of the cells.
Atypical cytologic features:
Nuclear to cytoplasmic asynchrony
Increased cytoplasmic basophilia
Anisokaryosis and anisocytosis.
Dysplasia can be cytologically difficult to distinguish from neoplasia as dysplastic lesions often contain more criteria of malignancy than strictly hyperplastic lesions.
labile cells
cell with rapid turnover
cells that continuously multiply and divide throughout life. This continual division of labile cells allows them to reproduce new stem cells and replace functional cells that are lost in the body.
quiesecent cells
a cellular state in which a cell remains out of the cell cycle but retains the capacity to divide. The unique ability of adult stem cells to maintain quiescence is crucial for life-long tissue homeostasis and regenerative capacity
liver ect
terminally differentaited cells
neurons
in the course of acquiring specialized functions, has irreversibly lost its ability to proliferate
stem cells
an undifferentiated cell of a multicellular organism which is capable of giving rise to indefinitely more cells of the same type, and from which certain other kinds of cell arise by differentiation
Senescence
Senescent cells are somatic cells that stop dividing- previously laibile or quessent cells can become senescent
Senescent cells remain metabolically active
Signals that induce senescence are typically due to DNA damage and/or tumour suppressor genes
mediated by p53-21 or p16
p53 tells damaged cells to stop
causes:
geonimoc instability
telomere attrition- aging causes these to get shorter
epigenetic alteration
loss of proteostasis
deregulated nutrient sensing
mitochondrial dysfunction
cellular senesence
stem cel exhastion
altered intracelleular communication
describert the Morphological appearance of cell ageing
Long-lived post mitotic cells (terminally diferentiated)- Neurones and muscle- will see Lipofuscin
Senescent cells- Heterochromatin (dense and innactive, non replicating chromatin) accumulations
seen by Transmission electron microscopy
Not clinically relevant
Biochemical markers of senescence- Lack proliferation markers
Senescence-associated secretory phenotype (SASP)
Research orientated
Lipofuscin in the context of cell aging
Wear-and-tear pigment
Normal accumulation over time of lipoprotein in secondary lysosomes
May accumulate excessively in certain circumstances- Phalaris poisoning in ruminants- the pigment wouth be seen in cytoplasm of neurons
Intracellular, golden-brown, globular
Ceroid- Very similar to lipofuscin but only accumulates in disease states- rare to see
Brown gut in dogs with vitamin E deficiency
Intracellular and extracellular
Ceroid
Very similar to lipofuscin but only accumulates in disease states- rare to see
Brown gut in dogs with vitamin E deficiency
Intracellular and extracellular
Lysosomes
membrane-bound cell organelle that contains digestive enzymes. Lysosomes are involved with various cell processes. They break down excess or worn-out cell parts. They may be used to destroy invading viruses and bacteria.
Intracellular
Membrane-bound vesicles
Contain enzymes
Genetic disorders- lysosomal storage disorders- effects the brain- quite rare
two types of intracellular pigment
exgenous- carbon, tattoo ink-can be seen in post mortem of lympnodes- vaccine adjuvant carotenoids, tetracyclins-
endogenous- heamatogenous, non-heamatogenous
Endogenous pigmentation – non-haematogenous
Lipofuscin and ceroid
Melanin- Incidental colouration: Leptomeninges, Pig lungs - melanosis- distingushed form melanoma by flat symetrical appearence
Hyperpigmentation- Endocrine skin disease (unclear why)
Melanoma
Hypopigmentation- Vitiligo- immune sytstem attacks melanin, Melanin incontinence: Some skin diseases of dermo-epidermal junction- could be indication od disease in dermis(pemphigus)
Endogenous pigmentation - haematogenous
from Blood-
Haemoglobin
Haemosiderin- brown pigment form broken down blood in macrophages- heamociderophages- stain blue. happens with heamorage. can indicate heart failure when found in the lungs
Haematoidi
Porphyria- Heme synthesis disorder
Deposition of porphyrin pigments in tissues
can turn bone pinker and makes them florese
cattle teeth may turn brown pink
Parastitic haematin- Liver fluke eat blood and pass it out as parasitic haemotin- seen in liver
Methaemoglobinaemina- Heme iron is oxidized from the ferrous (2+ ) to the ferric (3+ ) form
Methaemoglobin is constantly being formed, but it is reduced to haemoglobin by the methaemoglobin reductase pathway
Marked oxidant exposure may promote the formation of methaemoglobin.
Oxidative agents include gallotannin metabolites- Red Maple/Acercan induce this in horses
Associated with Heinz bodies (see haematology lectures next week)
May also see haemolytic crisis
The blood and mucous membranes may appear brown when >10% of the total hemoglobin has been converted to methemoglobin
Rare genetic disease in humans
causes brown pigmant in skin
Carbon monoxide
CO bind haemoglobin, forming carboxyhaemoglobin
Much stronger binding than oxygen and slow to revert
Cherry red mucous membranes, muscle and brain
Bile and breakdown products- juandice
Endogenous pigmentation – post mortem
Haemoglobin imbibition- freeze thaw artifact, animal may have had masave haemorage then not pm-ed for a number of days
Psuedomelanosis
blue green streaking of tissue and facea
Action of hydrogen sulphide (bacteria) on haemoglobin changes into FeS (ferris sulphide)
Exogenous pigmentation - carotenoids
Carotenoids are found in leafy vegetables and in horses and Jersey cattle impart a yellow colour to adipose tissue and lipid-laden organs.
dont confuse with yellow fat disease
Calcification
Pathological calcification = deposition of calcium salts in soft tissues - typically as phosphates and carbonates
Metastatic calcifi
cation is due to increased circulating calcium levels
Dystrophic calcification is secondary to necrosis
Liquefactive/lytic necrosis
Cells are lysed, and the necrotic tissue is converted to a fluid phase
Caused by:
Bacteria
Fungi- cryptococcus neoformans in the brain
Most common in the CNS- cant really get any other type of necrosis
Due to hypoxia
Large amount of cells and cell membranes with little connective tissue so nithing really holds it in
Gross-
Soft, viscous focus, often with cavity containing creamy-yellow material (pus).
Histo:
Cell debris, eosinophilic fluid
Very messy- hard to tell what struture is being observed
Coagulative necrosis
Tissue architecture/basic outline of necrotic cells is preserved. Suggestive of:
hypoxic injury
bacterial toxins
chemical toxins
Gross- Often well demarcated
Especially if due to infarction
Rim of inflammation
Histo-
Overall tissue architecture preserved, often with retention of basement membranes
Necrotic cells display typical histologic evidence of necrosis
Inflammation
Early attempts at healing- Especially in kidney if basement membrane intact
Caseous necrosis
Cheese-like
A chronic coagulation necrosis- Typically due to body’s inability to remove the agent
- intracellular bacteria
Common in birds and reptiles - Reduced amounts of myeloperoxidase in heterophils (neutrophils).
Gross:
Friable, granular, white appearance
Typically encapsulated
Abscess/granuloma/pyogranuloma
Histo:
Loss of architecture- mush of calcified tissue
Central accumulation of remnants of lysed leukocytes
May have border of granulomatous inflammation and outer fibrous tissue (‘granuloma’)
Often dystrophic calcification centrally.
A classic bacterial example is
Corynebacterium pseudotuberculosis
types of gangrenous necrosis
wet/moist, gaesious, dry
Gangrenous necrosis
Three types
Wet/moist:
Area of necrotic tissue further degraded by liquefactive action of saprophytic bacteria.
Death of animal may occur due to toxaemia/ sloughing of tissue
Gross: soft, moist, red-brown to black. +/- gas, putrid odor (hydrogen sulfide).
Gaseous:
Bacteria proliferate and produce toxins in necrotic tissue.
Usually anaerobic bacteria eg. Clostridium perfringens/ septicum.
Bacteria introduced by penetrating wounds, necrotic tissue becomes anaerobic, bacterial growth and toxin production.
Gross : dark red-black, gas bubbles, fluid and haemorrhagic exudate.
Dry:
Coagulation necrosis secondary to infarction followed by mummification (dehydration).
Usually lower portion of extremity (tail, ears, udder).
Ingested toxins- ergot, fescue, and frostbite. Peripheral arteriolar constriction and damage to capillaries. Thrombosis and infarction (also direct freezing injury and ice crystal formation in frostbite).
Necrotic tissues depleted of water eg. by low humidity, resulting in mummification.
NO bacteria proliferation.
Gross: dry, shrivelled, brown-black. May slough.
wet/moist gangrenous necrosis
Area of necrotic tissue further degraded by liquefactive action of saprophytic bacteria.
Death of animal may occur due to toxaemia/ sloughing of tissue
Gross: soft, moist, red-brown to black. +/- gas, putrid odor (hydrogen sulfide).
gaseous gangrenous necrosis
Bacteria proliferate and produce toxins in necrotic tissue.
Usually anaerobic bacteria eg. Clostridium perfringens/ septicum.
Bacteria introduced by penetrating wounds, necrotic tissue becomes anaerobic, bacterial growth and toxin production.
Gross : dark red-black, gas bubbles, fluid and haemorrhagic exudate.
can occur post death
dry gangerenous necrosis
Coagulation necrosis secondary to infarction followed by mummification (dehydration)- trapped animals in dry enviroments may display this
Usually lower portion of extremity (tail, ears, udder).
Ingested toxins- ergot, fescue,
frostbite- Peripheral arteriolar constriction and damage to capillaries. Thrombosis and infarction (also direct freezing injury and ice crystal formation in frostbite).
Necrotic tissues depleted of water eg. by low humidity, resulting in mummification.
NO bacteria proliferation.
Gross: dry, shrivelled, brown-black. May slough.
Fat necrosis
Nutritional, enzymatic, traumatic, and idiopathic.
Focal areas of fat destruction.
Nutritional fat necrosis
Also known as steatitis or yellow fat disease
Diet high in unsaturated fatty acids and low in vitamin E or other antioxidants
ROS production and lipid peroxidation.
Enzymatic necrosis of fat
Pancreatitis
Release of activated pancreatic lipases which liquefy adipocytes
Fatty acids combine with calcium to form chalky white areas (saponification)
Faint outlines of cells with basophilic calcium deposits and inflammation
Traumatic necrosis of fat
Crushing
Idiopathic fat necrosis
Necrosis of abdominal fat of Jersey/ Guernsey breeds
Large masses of necrotic fat in mesentery, omentum and retroperitoneum.
May cause intestinal stricture/stenosis.
Gross:
Firm, white, chalky.
Histo:
Necrotic fat not lost in processing.
Eosinophilic to basophilic (if FFA’s react with Ca2+ to form soap) necrotic adipocytes.
Nutritional fat necrosis
Also known as steatitis or yellow fat disease- dont confuse with normal yellowing in horses
Diet high in unsaturated fatty acids and low in vitamin E or other antioxidants- ROS production and lipid peroxidation.
Enzymatic necrosis of fat
Pancreatitis
Release of activated pancreatic lipases which liquefy adipocytes
Fatty acids combine with calcium to form chalky white areas (saponification)
Faint outlines of cells with basophilic calcium deposits and inflammation
hiseologically will show as spaces for fat surrounded by inflamation and heamorage. soponificaition will leave blue material
Traumatic necrosis of fat
Crushing
Idiopathic fat necrosis
Necrosis of abdominal fat of Jersey/ Guernsey breeds-
Large masses of necrotic fat in mesentery, omentum and retroperitoneum.
May cause intestinal stricture/stenosis.
Gross:
Firm, white, chalky.
Histo:
Necrotic fat not lost in processing.
Eosinophilic to basophilic (if FFA’s react with Ca2+ to form soap) necrotic adipocytes.
Fibrinoid necrosis
Blood vessel wall necrosis- associated with inflammation = vasculitis
Antigen and antibody complexes deposition in arterial walls and fibrin leakage.
Histo:
Bright pink, amorphous hyaline (glassy)
Thrombosis- inflamation
hypertoropic epithelil cells in lumen
Haematopoeisis
Definition - The formation of the cellular components of blood
Location - In the mature animal this occurs primarily in the bone marrow
Maturation, activation and some proliferation of lymphoid cells occurs in thymus, spleen and LNs
Liver and spleen can also be recruited to produce blood cells – “extramedullary haematopoeisis”
Erythropoeisis
the production of red blood cells.
Tissue hypoxia detected in peritubular interstitial cells kidney
These cells produce erythropoietin (EPO)
EPO binds to BLU-E/CFU-E/rubriblasts stimulating signalling cascade
Results in differentiation and survival of erythroid progenitors
Leading to increased production of mature RBCs
Other growth factors involved include IL-3, GM-CSF and Thrombopoeitin (TPO)
Hormones that promote erythropoiesis in combination with EPO – thyroid hormone, androgens, glucocorticoid
this means significant kidney disease can result in anemia- chronic renal disease
Myelopoeisis
the production of leukocytes in blood, such as MONOCYTES and GRANULOCYTES. This process also produces precursor cells for MACROPHAGE and DENDRITIC CELLS found in the lymphoid tissue.
Stimulated by factors such as IL-3, GM-CSF and G-CSF
Act on CMPs/GMPs to differentiate into myeloblasts
Production massively upregulated in response to inflammatory stimulus (eg macrophage inflammatory protein-2, IL-8, C5a
Eosinophil differentiation – TH2 lymphocytes involved by producing Il-5
Basophil differentiation – IL-3 and others involved
Thrombopoeisis
the formation of platelets in the Bone marrow
Stimulated by thrombopoietin (TPO) which is produced mainly in liver
TPO stimulates megakaryocyte production and survival from megakaryoblasts
Other factors involved include IL-3, GM-CSF, IL-11 and EPO
animals anemic due to iron deficiency will have increased no. of platlets
animals with liver disese still produce adiquate platlets
Neutrophils
recognised by segmented nucleus
granulocites
Vital part of innate immune system
“First responders” – hallmark of acute inflammation
Methods of attack – phagocytosis, degranulation and extracellular traps (NETs)
Eosinophils
recognised by pink granuals
Involved in response to parasitic infection
?Involved in response to some viral infections- not clasically assositated with this though
Major mediators of allergic responses
Basophils
Play a role in reactions to parasites and allergic reactions
Release histamine (along with mast cells)
Lymphocytes
mononuclear cells. thin rim of cytoplasm. smallest luekocyte
Adaptive immune response
T cells – cell mediated immunity
B cells – humoral (antibody) production
NK cells – innate immune system
Many functions!
Monocytes
can be confused with neutrophils of leukocytes. bigger with more blue colouring an dmore cytoplasm
Orchestrators of the immune response
Phagocytosis and microbicidal
Regulates immune responsevia cytokines
Scavenger role
Secretory
Leave circulation to become tissue macrophages
what can be diagnosed from Blood film examination
Clinically significant thrombocytopenia
Regenerative vs non-regenerative anemia
Spherocytes
Rouleux vs agglutination
Hypochromia (iron deficiency)?
Check machine leukocyte differentials
Left shifts
How to perform a manual platelet count:
Must be performed at x1000 (ie x100 oil lens)
Count number of platelets in 10 x1000 fields of view
Take average and multiply by 15-20
This gives an approximate count x10^9/L
Don’t forget to correlate with clinical signs (eg petechiae)
Remember clinically significant bleeding 2nd to thrombocytopenia unlikely unless count <50x10^9
Rule of thumb – if you can find platelets easily on the film patient unlikely to be bleeding from thrombocytopenia
Try and perform the count in the monolayer – not too thin and not too thick
how to distinguish if the patient’s anemia regenerative or not?
Key to answering this question is to assess for the degree of polychromasia
Polychromatic red cells larger and bluer – retained RNA, released earlier from bone marrow- indicates regenerative anemia
spherocytes
Spherocytes are red blood cells that are sphere-shaped rather than the usual round doughnut shape. Spherocytes are more fragile than normal red cells and their presence is accompanied by anemias of varying severity.
Dogs not cats
Must look in right area of blood film – in monolayer where you can compare to red cells with central pallor
Not in tail of film – everything will look like spherocytes!!
Spherocytes appear smaller, perfectly round and more densely staining
Their presence in significant numbers is suggestive of IMHA
Rouleux vs Agglutination
Agglutination (“bunch of grapes”)- immune mediated anemia
Rouleux (“stack of coins”)- high protien level
Hypochromia
Iron deficiency
Increased central pallour
Pale staining
Frequently fragile red cells (schistocytes, acanthocytes and keratocytes)
Often associated thrombocytosis
What is a left shift and how do I recognise it?
an increase in the number of immature cell types among the blood cells in a sample of blood. Many (perhaps most) clinical mentions of left shift refer to the white blood cell lineage, particularly neutrophil-precursor band cells, thus signifying bandemia.
Release of earlier granulocyte precursors from marrow
Indication of increased neutrophil demand/consumption
Inflammation/infection
“Band” neutrophils
What should I confirm with an external lab for haematology?
Neoplastic cells
Toxic change?
Infectious agents?
Dysplastic change
Laboratory assessment of Erythrocytes:
Complete blood count (CBC)
Blood film examination
Ancillary tests
Measured Red Cell Parameters
Haemoglobin (Hb) (uses a biochemical method)
Red blood cell concentration (RBC/ul)
Mean cell volume (MCV); average size of RBCs
Calculated Red cell parameters: Haematocrit(HCT)
calculatedfrom those measured (HCT = MCV x RBC)
Equivalent of manual PCV
Calculated Red cell parameters: Mean corpuscularhaemoglobin(MCH)
calculated (MCH =Hbx10 / RBC)
Calculated Red cell parameters:Mean corpuscularhaemoglobinconcentration (MCHC)
MCHC=Hb/HCT
analysers in haemotology
Red cell distribution width (RDW) – indication of variation in red cell size- bone marrow is trying to respond to an anema, larger cells are being released
Reticulocyte counts/percentage – measure of regeneration, how many immature cells are being released from bone marrow
Reticulocyte Hb – potential marker for iron deficiency
Plateletcrit (PCT) – equivalent of HCT for platelets
Packed cell volume (PCV)
PCV – percentage of red cells in a volume of blood. Manual technique.
Centrifuged whole blood, red cells read as a % of column.
Buffy coat assessment
Plasma – clear/ straw or pink if haemolysed
Total proteins measurement
Blood film exam of RBCs
Evaluating 3 main things:
Red cell density – does it look anemic?
Red cell regeneration – is the marrow trying to regenerate?
Red cell morphology – are there any clues to the cause of an anemia? (Eg spherocytes, organisms etc)
is the patient anaemic?
Very subjective
Will depend on how the blood film is made
Use as a crude quality control measure – does the red cell density appear compatible with the HCT provided by our analyser?
why is it good to differentiate between regenerative and non regenerative anemia
Regenerative anemia – relatively short differential diagnosis list
Non-regenerative - long differential diagnosis list
Regenerative anemia signs
Anisocytosis
Polychromatophils- when stained with diff quick type stain. Reticulocytes- stained with methalyn blue. same thing. these are immature red celss that still contain ribosomes
mathalyn blue precipitates ribotsomes, making them reticulated. hence reticulocytes
these immature red cells are seen in regenerative anemia as bone marrow is putting them out to regenerate the red blood cells
Nucleated RBC- not cliniclaly significant in birds and reptiles. earlier stage of maturation of ed blood cells. shows bone marrow is trying to regenerate red blood cell count in the case of re generative anemia
Howell-Jolly bodies- Are nuclear remnants. usually would be removed in bone marrow except in this case
Reticulocytes and Polychromatophils
On a Diff – Quik or Giemsa stained smear, young red cells containing reticulin show up as larger, bluer cells: polychromatophils
Stain the same cells with New Methylene Blue, and you can see the reticulin : the cells are then called reticulocytes
THEY ARE THE SAME CELLS
RBCs – Morphological abnormalities to help distiguifh causes of anemia
Most helpful/specific-
Spherocytes
Agglutination
Eccentrocytes
Heinz bodies
Hypochromia
Acanthocytes
Schistocytes
Less helpful/specific-
Echinocytes (crenated cells)
Keratocytes
Ovalocytes
Dacrocytes
Heinz bodies and eccentrocytes
sign of oxidative injury which can cause anemia. Haemoglobin is oxidised and pushed to cell margin, no longer functiona
eccentrocytes - cresent moon shape
heinz bodies- protruding heamoglobin, red nose shape
Paracetamol in cats, onions, some toxins (eg Zinc), diabetic ketoacidosis are some common causes of Heinz bodies/eccentrocytes
Acanthocytes
red cells with spicules (small uneaven projections)
form with alteration of lipid metabolsm which effects the cells membrane
Lipid disorders
Liver disease
Fragmentation injury (eg haemangiosarcoma- neoplams of blood vessels involving liver and spleen)
Schistocytes
Red cell fragments forming secondary to being squeezed through abnormal blood vessels (eg haemangiosarcoma) or being fragile (eg iron deficiency)
Hypochromic RBCs
This usually occurs when there is not enough of the pigment that carries oxygen (hemoglobin) in the red blood cells.
ancillary PCR (EDTA sample) tests are available for:
the haemotropic Mycoplasmas in cats
Babesia species in dogs
Ehrlichia and Anaplasma in dogs
FIV/FeLV
ancillary Serology (serum sample) is available for
Leptospirosis
Panleukopaenia,
canine parvovirus
equine infectious anaemia
Feline coronavirus
Slide agglutination test
To try and distinguish rouleux and true agglutination
The sample is examined microscopically after the addition of saline.
Rouleaux formations disperse, but agglutination persists.
Coomb’s test
Coomb’s reagent (containing antibodies to IgG, Igm and C3) in dilutions is reacted with washed patient red cells to detect those cells opsonised with antibody and complement
Opsonised cells binding antibody agglutinate
Agglutinated red cells fail to settle in round bottom wells
Significantly positive result provides support for immune mediated component to anemia
Faecal occult blood test
Up to 50% blood volume may be lost into the GI tract before it is grossly visible in the faeces.
These tests generally detect peroxidase activity (present in haemoglobin) and may be 50x more sensitive than visual examination.
They are prone to false positives due to meat diets, vitamin C and some vegetables (eg brassicas, cantaloupe melon (true, it’s on Wikipedia!!)).
It is advisable to feed restricted diets for at least 3d prior to the test. Which diets?
Traditionally white meat has been used
Rice / cottage cheese may be better
Commercial dry diets. Variable but if heat treated may be OK. Can always send in sample of dry commercial food at same time as faeces.
Compensatory mechanisms of tissue hypoxia
so a higher proportion of O2, which is carried by the Hb, is available to be released to the tissues.
Increased cardiac output: Serves to increase tissue oxygenation
Increased RBC production: Tissue hypoxia causes the release of the hormone erythropoietin from the kidneys.
Vasoconstriction: Shunting of blood away from tissues with low O2 demand (i.e. skin) to tissues with high O2 n demand (i.e. brain).
Potential clinical signs of anemia
Mucous membrane pallor
Lethargy
Exercise intolerance
Tachycardia
Tachypnoea
Collapse
Icterus
Melaena
Pica
RBC indices
Based on MCV values
Normocytic: erythrocytes of unremarkable size
Microcytic (low MCV): iron deficiency
Macrocytic (high MCV):
Presence of immature RBCs (ie marrow trying to respond)
some poodles,
in some bone marrow disorders
a common artefact in stored/old (usually posted) blood samples (RBCs swell up)
Normochromic
Hypochromic (low MCHC/MCH):
in iron deficiency/ poor iron incorporation (with microcytosis)
Presence of immature RBCs (are not fully haemoglobinised)
Hyperchromic (high MCHC/MCH):
Not physiologically possible
Always artefact (i.e. haemolysis)
regeneration of rbc
Regeneration is the body’s response to a fall in oxygenation
The kidneys respond by releasing erythropoietin (EPO)
This stimulates the bone marrow to increase red cell production
Takes 3-5 days, and younger red cells (polychromatophils/ reticulocytes) increase in circulation
Dogs and cats - expect reticulocyte response – horses retain retics in bone marrow, not seen in circulation; cattle/ sheep only with severe anaemia
Regenerative anaemia
Haemorrhage, haemolysis
Non-regenerative anaemia
Intra- and Extra-marrow diseases
Haemorrhage (external)
Melaena (GI bleeding)
Haematuria (Urinary tract)
Epistaxis (nose bleeds)
Post trauma/ surgery
Marked ectoparasitism
Some endoparasites (e.g. hookworm)
With acute haemorrhage, 3 – 5 days needed for a marrow response – before this, appears non or poorly regenerative : ‘pre-regenerative’
lab results:
Anaemia
initially regenerative BUT
prolonged external haemorrhage»_space;> iron deficiency»_space;> non-regenerative anaemia as iron supplies are exhausted
Proteins
will be normal in the acute stage, then decrease as fluid re-equilibrates
Haemorrhage (internal)
Cause not always obvious-
Bleeding tumours
Trauma
Bleeding disorders
Into tissue, cavities, lungs
Surgery
Respiratory” blood may be coughed, swallowed and appear as melaena
No iron deficiency
RBC breakdown products available for recycling
Proteins normal or raised
types of haemolysis
a cause of regenerative anemia
Extravascular- outside of blood vessels-
More common
Macrophages
Spleen and liver
± Icterus (jaundice)
Intravascular-
Acute, severe
Haemoglobinaemia
Haemoglobinuria
Ghost cells
+/- icterus
boith can occur at once- presentation is very accute
Haemolytic anaemia - Causes
Immune-mediated (common – primary verses secondary)
Infections (less common)
Babesia
Mycoplasma
Inherited RBC metabolic defects (rare)
Toxins
Severe Hypophosphataemia (rare)
Immune-mediated heamolitic anemia
One of the most common causes of anaemia in dogs
One of the most common immune-mediated Dz
Primary IMHA (AIHA)
Most common
Idiopathic
Breed predispositions
Young-middle aged
Female>male
clinical signs-
Lethargy, anorexia
Pale mucous membranes +/- tachycardia, bounding pulses, systolic murmur
±
Tachypnoea
Jaundice
Hepatosplenomegaly
Pyrexia, mild lymphadenopathy
Secondary IMHA
Infections
Neoplasia
Drugs
Systemic extra bone marrow disorders causing anemia
common
. Chronic inflammation
Most common cause
Usually mild/moderate
Fe sequestration and inflammatory mediators shorten erythrocyte survival
Endocrinopathies
Hypothyroidism
Hypoadrenocorticism
Chronic renal failure
Decreased erythropoietin production
Decreased RBC lifespan with uraemic toxins
At risk of GI haemorrhage
Primary intra-bone marrow disorders causing anemia –
less common
. Aplastic anaemia
Damage to stem cells, microenvironment or both
Non-regenerative anaemia, bicytopenia or pancytopenia
Idiopathic
Secondary to
Toxins (i.e. bracken fern)
Drugs (i.e. chemotherapy)
Infections (i.e. Parvo)
Immune mediated mechanisms
- Pure red cell aplasia/PIMA
Selective BM erythroid precursor damage
Non-regenerative anaemia
Immune mediated
Secondary to FeLV - Myelophthisis
Represents a space-occupying lesion in the BM that inhibits or displaces normal haematopoietic cells
NEOPLASIA usually
bone marrow derived but may originate elsewhere
Lymphoma, Metastatic disease
Non-regenerative anaemia, - Myelofibrosis
Proliferation of BM with fibrous elements that inhibits or displaces normal haematopoietic cells
Idiopathic
Secondary to other BM diseases
Often need BM biopsy for histopathology, not aspirate, to diagnose
ERYTHROCYTOSIS
having a high concentration of red blood cells in your blood. This makes the blood thicker and less able to travel through blood vessels and organs. Many of the symptoms of polycythaemia are caused by this sluggish flow of blood
Increased red cell mass, evidenced in increased :
Haemoglobin
Packed cell volume (PCV)
Haematocrit (HCT)
Red blood cell count (RBC)
Physiological-
Breed related
Reference intervals should be different for
Greyhounds vs other dogs
Thoroughbreds vs ponies
Relative
Dehydration (PCV up to 60%)
fluid loss with a stable red cell mass
Clinical signs may be present
Lab test abnormalities
increased serum total proteins
Often but not always increased serum sodium
increased urine specific gravity
PRIMARY(absolute)
Polycythaemia rubra vera
Bone marrow neoplastic dz
Clonal proliferation and maturation of RBCs WITHOUT normal feedback mechanisms
EPO low (in theory!)
neurological signs, seizures, paroxysmal sneezing, cardiopulmonary signs, retinal changes (high blood viscosity)
High PCV!! (>65
SECONDARY(absolute)
Hypoxia (altitude, heart/pulmonary dz)
Bone Marrow responds>EPO»RBC
Solid tumours
Renal carcinoma» EPO»»_space;RBC
WBC morphology
Left shift
Toxic change
Reactive lymphocytes
Atypical cells
Infectious agents
(NB – Analysers will not reliably pick out these things)
WBC Ancillary tests
Acute phase proteins (Dogs CRP, Horses Fibrinogen and SAA)
PCR and serology for infectious agents
Bone marrow aspiration/biopsy
PARR test (lymphoproliferative disease)
Flow cytometry (classification of leukemias)
Anti-neutrophil antibodies (uncommonly tested)
Neutrophilia
a higher neutrophil count in the blood than the normal reference range of absolute neutrophil count.
- TO MEET DEMAND
Infections
Immune mediated diseases
Inflammation
Neoplasia
(COMMON) - INDEPENDENT OF DEMAND
Bone marrow neoplasia = Leukaemia
(UNCOMMON)
- Persistence in circulation
Chronic stress
Glucocorticoids (steroids) - Redistribution (shift from marginating to circulating pool)
Excitement (epinephrine)
Stress(glucocorticoids)
increased blood pressure
Neutropenia: Causes
Increased demand (migration into tissue)
Bacterial sepsis, abscess
Endotoxaemia, tumour necrosis
Redistribution
In response to acute endotoxaemia (shift from circulating to marginating pool)
Decreased production
Bone marrow dz, Drugs
Increased destruction
Immune mediated
Neutrophils- Morphological assessment
Band neutrophils-
released with increased demand
Usually “U” shaped or “S” shaped nucleus with parallel sides ie minimal indentation/ segmentation
Toxic neutrophils-
Maturation defects
Seen with increased demand
Not necessarily sepsis, can happen with sterile demand
Pyothorax, pancreatitis, pyometra etc.
Dohle bodies
Foamy cytoplasm
Bluish cytoplasm
Toxic granules - rare
diagnosing toxicity is objective
LYMPHOCYTOSIS
- Increased production in response to increased demand
Persistent antigenic stimulation (fungal, protozoal, viral), Post vaccination, young animals - Increased production without demand
Lymphoid Leukaemia, Lymphoma - Redistribution
Excitement/acute stress (epinephrine response) (physiological; as for neutrophils)
inhibition of recirculation, release from the thoracic duct
Hypoadrenocorticism (10-20% of cases)
LYMPHOPENIA
Loss of lymphocytes
Loss of chylous fluid (rich in lymphocytes)
Protein losing enteropathy, chylothorax
Decreased production
Viral infections, lympholytic drugs (for chemotherapy)
Redistribution
Chronic stress, Glucocorticoids (steroids)
Trapped in lymph nodes
move from circulation into bone marrow and tissues
lymphocytolysis
MONOCYTOSIS
- Increased production by the bone marrow to meet demand
Infections, immune-mediated dz, inflammation, necrosis, sepsis, neoplasia - Increased production by the bone marrow without demand
Leukaemia (myelomonocytic) - Redistribution
Chronic stress, Glucocorticoids (steroids)
Move monocytes from the marginating to the circulating pool
EOSINOPHILIA
- Increased production in response to increased demand
Parasitic infection,
allergic disease”,
inflammation of mast cell rich tissue (intestines, skin, lungs, uterus) - Increased production without demand
Neoplasia (lymphoma, mast cell tumour, squamous cell carcinoma)
Hypereosinophilic syndrome
Peripheral eosinophilia & infiltration of organs with eosinophils without obvious cause
Eosinophilic Leukaemia (rare) - Hypoadrenocorticism
(lack of glucocorticoids)
BASOPHILIA
- Increased numbers due to increased demand
Hypersensitivities
drugs, food, insect bites/stings
Parasitism
especially Dirofiliaria,
but also GI parasites, fleas and ticks
Inflammation - Increased numbers without demand
Paraneoplastic (particularly with mast cell tumours)
Basophilic leukaemia (rare)
Stress leukogram
Leukocyte pattern due to the effect of increased cortisol
Classically neutrophilia +/-monocytosis with lymphopenia and eosinopenia
Most commonly seen dogs and cats
May be absent when expected eg Addison’s disease
Feline Haemotrophic Mycoplasmas
Endemic in UK
Feline Haemotrophic Mycoplasmas collectively known as haemoplasmas
Cause of Feline Infectious Anaemia - acute haemolytic anemia
Transmission method not definitively established - suspected fleas or aggressive interactions
higher prevalence in older male cats with out door access
transmission in blood - all blood donors should be screened
M. haemofelis (Mhf)- most pathogenic
mild to severe clinical disease in healthy cats
Candidatus M. haemomimutum (CMhm)- asymptomatic/milder disease
most likely to be clinical in immune compromised patients /concurrent disease (e.g. FeLV, FIV)
Candidatus M. turicensis (CMt)
could cause small reduction in RBC indices, unlikely to develop clinical anemia - unless concurrent disease /immunocompromised
Post infection -anaemia develops in 2-34 days
Bacteremia and anaemia can last for weeks
Cyclic/fluctuating numbers of bacteria
Antibiotic therapy may control acute infection but won’t clear infection
Recovered cats develop carrier status
Low grade intermittent bacteremia with no anaemia or develop milder disease when stressed.
Clinical Signs :
M. haemofelis can cause variable, but often severe anemia
Weakness, lethargy, pale mucous membranes
Intermittent fever
Tachycardia, tachypnea, haemic murmurs, depression, collapse
Clinical Pathology Results:
Regenerative anemia *
Macrocytic and hypochromic anemia
Increased reticulocytes (and may have nucleated red cells)
+/- mild hyperbilirubinemia (haemolysis)
Primarily extravascular haemolysis
Coombs +ve and autoagglutination (production of Ab against
erythrocytes)
* Anemia may not be regenerative
1. Pre-regenerative anemia
Bone marrow response takes 3-5 days to produce peak response
2. Immunocompromised patient/concurrent disease (e.g. FeLV)
Impairs a normal bone marrow response
PCR positive with non-regenerative, needs further investigation.
diagnosis:
Evaluation of fresh blood smear
tiny rod, cocci or rings on the surface of erythrocytes
In aged blood samples (e.g less day old), fall off erythrocytes
low sensitivity test
Need to distinguish between stain precipitate,
Howell Jolly bodies, basophilic stippling
Fastidious and non-culturable
Gold standard is PCR detection in blood (detecting DNA from the bacteria)
May get false negative if low numbers (decline in parasitemia,
chronic carrier phase)
Babesia Canis
Emerging Tick borne disease
Endemic in South and Central Europe
Cases of infected non-travelled dogs, Babesia found in local tick population.
Various tick sp. Dermacentor reticulatus, Riphicephalus)
Restricted geography
West Wales, South Devon, Essex.
Different sp with varying pathogenicity, B. canis of concern in UK
Can have co-infections with other TBD ( Ehrlichia, Borrelia, hepatozoon(imported dogs))
Tick infect blood with sporozoites during feeding
Sporozoites invade RBC, differentiate into merozoites, divide by binary fission, rupture RBC, infect neighbouring RBC
clinical signs-
Variable clinical signs and presentations
Mild to moderate disease and chronic carrier status can develop
Complicated forms of disease
Immunological syndromes
Primary feature is haemolytic anaemia
Haemolytic anemia, mild to severe (often severe with B. canis)
Fever, lethargy, anorexia, jaundice, vomiting, haemoglobin
Regenerative anemia
Extravascular /intravascular destruction
Intravascular destruction leads to haemoglobinuria and haemolysed plasma/serum
Can be associated with development of autoantibodies (immune mediated component)
Can have spherocytes and Coombs + result
Often thrombocytopenia (and neutropenia)
diagnoisis-Blood smear evaluation
Low sensitivity method
Intraerythrocytic
Large paired piriform organisms
Can be singular
Other Babesia sp, can be ring shaped
Canine Hepatozoonosis
Emerging disease (imported dogs)
Rhipicephalus ticks (endemic Mediterranean basin)
Unlikely to become endemic in UK, screen imported dogs.
Ingestion of infected tick, sporozoites penetrate intestinal
epithelium
Disseminate to the haemolymphatic system, undergo replication
Releasing merozoites that infect neutrophils.
clinicla signs-
Clinical signs related to the severity of parasitemia
Low burdens may be asymptomatic/mild
Higher parasite burdens - can have severe clinical signs
Infected neutrophils have reduced bacteriocidal activity - risk of secondary infections
Non-specific clinical signs - fever, lethargy, weight loss, chronic infections
Hyperglobulinemia (from infections/chronic inflammation)
Hypoalbuminemia (negative acute phase protein)
Non-regenerative anaemia (anaemia of inflammatory disease)
Neutrophilia
diagnosis-
Blood smear evaluation
Elliptical to pale blue gamonts
% of infection can vary, may be infected with no peripheral
gamonts, especially in asymptomatic/mild cases
Buffy coat smear
(Similar as above but increases the number of neutrophils
evaluated)
PCR
most sensitive method of detection
Heartworm
Present in Europe/Americas, screen imported dogs
Mosquito vector
Nemotode - Dirofilaria immitis,
Dogs are definitive host (patent infections), cats are non-definitive hosts
Three phases of life cycle:
Microfilaria (blood)
Larve (mosquito and tissue)
Adult worm (pulmonary artery or in heavy burdens right atrium)
clinicla signs in dogs-
mild- aysymptomatoic or cought
moderate- cough, exersise intolerance, abnomal lung sounds
severe- cough, exersise intolerance, abnomal lung and heart sounds, enlarged liver, syncope, ascites, death
caval syndrome- severe acute lethargy weaknesss, hemoglobinema, heamoglobinuria
diagnosis/ detection-
Features that affect detection
Long life cycle - takes up to 7 months post infection before patent adults in PA
Need to test after 7 month last possible exposure
Only mature adults produce MF - occult infection of adults but no MF
Need to know patient is Heartworm free before treating with microfilarial drugs (ivermectin / moxidectin) in case reaction to dying MF
Animals infected with Heartworm require lengthy treatment protocol that will involve 6-8 weeks of STRICT cage restriction. Prevention is better than cure.
Two types of screening tests
1. Detection of antigen
Antigen (protein secreted by the mature adult female)
Specificity good
Sensitivity good and will detect most occult infections but….
Low adult female worm burdens may yield false negatives.
Male only infections will yield false negatives
Consider a negative result ‘not detected’
All positive results MUST to be confirmed prior to treatment- with a different antigen test, MF, cardiac ultrasound/radiographs etc.
2. Microfilarial test (modified Knott Test)
1ml EDTA blood: 9ml 2% formalin
Mix and centrifuge
Remove supernatant, put sediment on slide with coverslip
Evaluate at low power under microscope
Positive tests
Validate positive antigen tests
Indicate dog is a reservoir of infection
Warning that high MF burden may react to microfilaricides
American Heart worm society recommendations
Dogs should be tested with BOTH antigen and Knotts test
Positive antigen tests need confirmation before treatment
Cats are not definitive hosts, greater resistance against
Generally have low worm burdens - males >females (1-2 worms, often single sex)
Seldom circulating microfilaria
Clinical signs - generally associated with respiratory signs but can be asymptomatic
Rarely severe dyspnoea, tachypnoea, collapse
Testing is not reliable in cats
Antigen testing (but a negative does not exclude infection)
+ Radiographs, echocardiography
Bovine Babesiosis
Red Water fever / Piroplasmosis
High prevalence in SW England
Difference sp. but B. divergens and B.major (transmitted by Ixodes ricinus) occurs in UK.
Small Babesia sp.
Merozoites invade the erythrocytes, multiply, burst out destroying erythrocyte
Cause of significant intravascular haemolytic anaemia
Disease may be mild to severe, may be less severe in endemic regions if have immunity
Severe clinical signs relate to the severity of haemolysis (can result in death)
Fever, depression, icterus, anorexia, tachycardia, tachypnea, pale mucous membranes, haemoglobinuria
Other signs could include reduced fertility/production/abortion.
diagnosis-
Clinical signs/pigmenturia/local knowledge/changes in pasture etc
Fresh blood smear evaluation
Single or paired round oval or pear shaped structures
Low sensitivity
PCR
Bovine Anaplasmosis
A. Phagocytophilium - cause of Tick-borne fever
Ixodes ricinus
Obligate intracellular bacteria - unusual residence in neutrophils
Causes disease via immunosuppression
Reduced number of cells
Reduced bacteriocidal function
Affects lymphocyte signalling/function
Infection on its own usually causes self limiting febrile disease
Can also cause abortion, mild drop
Increases susceptibility to other diseases (pasteurellosis, septicaemia)
Causes persistent infection/carrier status -
evades detection (changes surface proteins)
Status can last up to 2 years
Presence of bacteremia at various time points
Peak bacteremia >50% granulocyte infected.
diagnosis-
Blood smear evaluation (but low sensitivity)
Mulberry like micro-colonies (morulae) of coccobacciliary bacteria
Size 0.2-05 um diameter
Within cytoplasmic bound vacuoles
PCR (pan-Piroplasmosis PCR for Babesia and Anaplasma, APHA)
Serology (IFA, ELISA) (not routinely used )
Equine Piroplasmosis
Disease of concern
Protozoa: Theileria equi (formerly Babesia equi) Babesia caballi
Endemic in tropical and subtropical regions
Tick borne disease - multiple tick vectors inc. Dermacentor reticularis
Intra-erythrocytic disease
Replicates in RBC, ruptures cells, merozoites released into the circulation, infects further RBC
clinical signs-
Both sp. induce similar disease, cannot be distinguished clinically
Different forms of disease acute, sub-acute, chronic
Acute
Haemolytic anaemia (varying severity, can be severe)
Icterus
Haemoglobinuria (severe cases)
Thrombocytopenia (destruction or consumption)
Fever, inappetence, oedema (occasionally death)
Chronic/carrier states
Mild variable and non-specific signs
Clinically indistinguishable from chronic inflammatory diseases
Variable anaemia, lethargy, anorexia, weight loss, reduced performance and fertility, may be asymptomatic
Still pose a reservoir for tick borne and iatrogenic transmission
Not reportable or notifiable in UK
No formal UK requirements for pre-import screening
Asymptomatic horse could enter UK and seropositive horses are present in UK (2016 study reported 8% seropositivity of pre-export submissions!)
Exposure to Dermacentor tick - could theoretically establish/transmit disease
Iatrogenic transmission possible
Can treat (imidiocarb) but T. Equi
generally considered life long disease
Serology tests (3 methods of antibody detection commercially available)
cELISA- considered better test for detecting chronic infection
CFT - more likely to detect acute infection
IFT
PCR - false negatives occur
No test is perfect and a combination of serologic tests may be required (especially if suspicion of clinical disease).
USA, Australia and Japan are disease free and require pre-import testing
USDA recognises the cELISA as approved screening test
Camelid Mycoplasma
Candidatus Mycoplasma haemolamae
Transmission unknown but suspected to be ticks?
MANY infections are subclinical
Above study; herd 131 llamas, 29% prevalence (PCR +ve) but no evidence on blood smear,
healthy, non-anaemic population
represents sub-clinical infection
only one case developed clinical signs of anaemia (but had focus of inflammation)
Mycoplasma haemolamae - potential cause of regenerative anaemia
M. haemolamae secondary cause/associated with chronic disease
GIT parasites (Haemonchus) most common cause of anaemia
Higher GIT parasitemia more likely to be severe anaemia
Haemonchus and M. Haemolamae together cause more severe anaemia
diagnosis-
RBC small and elliptoid
Lots of small blue dots on surface of RBC
Identify on blood smear (make fresh as organisms will fall off)
PCR
Positive result interpreted in context of clinical signs
Is there anaemia?
FEC, other disease
cachexia
weakness and wasting of the body due to severe chronic illness
psuedomelanosis
post mortem change
green blue staining by FeS
FeS is formed by H2S from putrefactive bacteria and iron from HB from lysed red blood cells
will progress to black colouration under the right circumstances
chilling a carcuss will often effect the cornea and or lense of the eye. how?
it will increase the opacity
haemoglobin inhibition
the red discolouration of tissue due to haem from lysed erythrocytes
commonly seen as a freeze thaw artifact
can also happen over time, esspecially in organs that contain a lot of blood like the heart
Antemortem
Occurred to the animal before it died
Incidental findings
True lesions
Agonal
Occur at the time of death
Lung and liver congestion
Artefact
Occur as part of killing the animal
Barbiturate crystals in heart
Splenic congestion due to barbiturate euthanasia
Gun shot damage to head
Freeze-thaw/chilling artefact
Post mortem change
Occurs after the animal is dead
Rigor mortis
Algor mortis
Livor mortis/hypostatic congestion
Pseudomelanosis
Putrefaction
Haemoglobin imbibition
Autolysis
blood cyst
Focally raising the contour of the atrial surface of the mitral valve is a single, smooth red, round focus, measuring approximately 3mm in diameter
Interpretation: blood cyst
Seen frequently in young ruminants
Incidental
endocardiosis
Multifocally at the line of closure of the mitral valve the valve is thickened and pale cream with raised coalescent nodules.
Age-related change commonly seen in older dogs
must be interepreted in context
How is this different to mitral valve disease?- vegitative lesions. not smooth.
Bronchial Mineralization
incidental finding
Difficult to see grossly but can sometimes in older dogs’ lungs, feel multifocal firm to gritty small areas within the parenchyma
Age-related incidental calcification
If widespread the main differential would be uraemic mineralisation/calcification due to renal failure of vitamin D poisoning
Paralaphestrongylus tenuis
incidental finding
Description: Multifocally within the meninges and over the surface of the dorsal cerebrum are multiple nematodes
Interpretation: Paralaphestrongylus tenuis
Non-pathogenic parasite of the white tailed deer (USA)
Incidental in definitive host (white tail deer), causes neurologic disease in a number of aberrant ruminant hosts.
Multifocally within the meninges and over the surface of the dorsal cerebrum are multiple nematodes
Paralaphestrongylus tenuis
Non-pathogenic parasite of the white tailed deer (USA), therefor typically incidental in these hosts
Incidental in definitive host, causes neurologic disease in a number of aberrant ruminant, camelid and equine hosts.
Ascarid migration
incidental finding
Description: Multifocally affecting all lobes of the liver are poorly-demarcated, flat, vaguely round, white to pale tan foci
Interpretation: Ascarid migration
Various species
Hepatocellular adenoma/carcinoma
possible incidental finding
Description: Entirely obliterating the left lateral lobe of the lever is a large nodular vaguely round pink to red firm mass
Interpretation/morphological diagnosis: Hepatocellular adenoma/carcinoma
history important
Only an issue if they rupture, can grow quite large in old dogs completely un-noticed
However, a small proportion will produce insulin-like peptides or IGF-2
-> hypoglycaemia
Nodular hyperplasia
posiible incidental finding
Description: Focally expanding the parenchyma and raising the contour of the spleen is a focal, well-demarcated, black to red to pink mass
Interpretation/morphological diagnosis: Nodular hyperplasia
Can grow large, can rupture
There are however lots of malignant neoplasms that can form masses in the spleen-
Haemangiosarcoma
Lymphoma
Histiocytic sarcoma
siderotic plaques
possibel incidental finding
Description: Focally extensively raising the capsule of the lateral aspect of the body of the spleen are raised cream to white to grey gritty multifocal to coalescing (squishing toghtther) plaques
Interpretation: siderotic plaques
Accumulations of debris associated with erythrocyte turnover.
Histopathologically are quite pretty with multiple variations of metabolised haem-
Haemosiderin
Hemotoidin - Gamna-Gandy bodies
Renal cysts
Renal cysts
Can be incidental
Can also be pathological
Quantifying and contextualising is important
can be common finding in slaughter pigs but alco couldy be pkd
bird erythrocytes
Nucleated
Larger than mammals’ smaller than reptiles’
Elliptical cells
Elliptical, central nucleus
Higher oxygen demand so higher than mammals
Normal PCV 35-55%
RBC Life span 28-35 days
Lower red cell mass in juveniles
Few significant gender influences
1-5% Polychromasia
Higher % in juvenile birds
Generally as a result of haemorrhage
Toxicosis another common cause
reptile erythrocytes
Nucleated
Larger cells than birds and mammals
Blunt ended ellipse
Central round/ovoid nucleus with Irregular margin
Lower oxygen demand so lower PCV
Normal PCV 20-40%
RBC life span 600-800 days!
Influence of gender, hibernation, environment – lots of variation
May have mitotic figures in RBCs
May see increased regeneration
With blood loss or without blood loss
mammal erythrocytes
Anucleated (No nucleus)
Anisocytosis/Polychromasia common in some species
Shorter half life c/f dogs and cats
Rodent and rabbit RBCs shorter half life c/f cats/dogs (45-68 days)
PCV varies a little with species (Rabbits 30-50%)
Can see high levels of regeneration
Esp juveniles
Non regenerative anaemia - reptiles
Most common form of anaemia in reptiles
Systemic disease
Starvation
Chronic disease
GI disease
Neoplasia
Suboptimal Husbandry
Non regenerative anaemia - Birds
Systemic disease
Starvation
Chronic disease
Neoplasia
Drugs
Suboptimal Husbandry
Many infectious diseases
- spergillosis, Chlamydiosis, TB, circovirus, chronic bacterial infection
Leukocytes in Exotics
Granulocytes
Lymphocytes
Monoocytes
Azuropohils (snakes)
Heterophils/Neutrophils
Functionally the same
Different staining
Most mammals have neutrophils
Rabbits and some rodents – heterophils
Reptiles, Birds – heterophils
Most common leukocyte to vary in disease
Predominant leukocyte usually- Some species lymphocytic
Rabbits, G. Pigs, mice, rats, gerbils
Heterophil:lymphocyte ratio
Function & Appearance of Heterophils/Neutrophils
Phagocytosis and lysosomal action
Irregularly round cell
Granules
Often rod shaped granules esp in birds
Granules often fused together
Basophilic nucleus
Lobed - 2-3 lobes in birds
Non lobed oval nucleus in reptiles
Very variable appearance in
reptiles
Heterophilia
Stress (transport, restraint, fear, crowding, management, trauma, temperature stress, anaesthesia)
Infectious causes, inflammation
Neoplasia
Necrosis
Differentiate between physiological and pathological heterophilia
Left shift (increased band heterophils)
Less lobed nucleus
More basophilic (bluer), fewer granules
Toxic Changes (main marker in reptiles)
NB reptiles huge variation
Biochemistry changes (Fibrinogen assays, A:G ratios, acute
phase proteins
Pseudoheteropaenia
True Hetero/Neutropaenia
Sepsis
Viral infections
Myelosuppression
Iatrogenic/drugs
Neoplasia)
Combined with left shift in severe consumption in overwhelming infection
NB in Lymphocytic species, heterophil:ymphocyte ratio more reliable
Eosinophils in exotics
Exact function unknown in some species
Loosely foreign antigen
Larger than heterophils with round outline
Red/orange round cytoplasmic granules
Some species have pale blue granules eg AGP/iguanas
Birds – bilobed nucleus
Reptiles – elongated/round nucleus
Generally in fewer numbers
Raptors higher eosinophils <15%
Reptiles variable
Eosinophilia/ Eosinopaenia in exotics
Eosinopaenia
Typically low numbers so eosinopaenia hard to identify/define
Stress
Glucocorticoids
Eosinophilia
Less clearly defined than in dogs/cats where usually parasites
Artefactual
Foreign Antigen
Marked tissue trauma
Parasites
Rarely hypersensitivity
Azurophils
Slightly smaller than the monocyte
Non segmented nucleus
Darker more basophilic cytoplasm than monocyte
Prominent red cytoplasmic granules
Appearance and function more similar to neutrophils
Can be <35%
Increase with acute inflammation or bacterial infection (similar to neutrophils) but do also have heterophils
No real clinical benefit in differentiating from monocytes
Thrombocytes in birds and reptiles
Nucleated cells
Second most numerous cell in peripheral blood
Small, round to oval cells
Round to oval nucleus
High Nucleus:Cytoplasm ratio
May contain eosinophilic granules 1 or 2 in one area of the cytoplasm
Clump or form aggregates in blood films
Avian thrombocytes capable of phagocytosis
Thrombocytopaenia
Decreased BM production
Excessive peripheral utilisation
Chronic inflammatory conditions
Severe septicaemia
Thrombocytosis
Chronic inflammation
Haemoparasites in exotics
Protozoan Haemoparasites
Insect vectors (leeches in aquatic reptiles)
Haemolytic anaemia
Haemoproteus
Plasmodium
Leukocytozoon
Trypanosomes
Microfilaria of filarial nematodes
Avian Biochemistry
Rarely provide definitive diagnosis
Significant variation with species, age, gender, lifestage
Liver and Muscle
AST & Bile Acids most sensitive indicators of liver disease
AST not liver specific – can be elevated with muscle damage, inflammation, sepsis
GLDH most specific marker oof hepatocellular damage
Bile acids reliable indicator of liver function (must collect serum as heparin affects it)
Post prandial bile acid elevation so fasted samples preferredCK elevation with muscle damage so should be used alongside AST and GLDH to differentiate
ALT, ALP & GGT very non specific
Bilirubin concentrations inconsistent in liver disease and across species
Biliverdin primary bile pigment
Renal
Uric acid most reliable test of renal disease (NB affected by many factors)
Dehydration and renal disease can both elevate uric acid
Can be used as prognostic indicator for gout (>600uml can lead to precipitation in joints)
Urea and creatinine not useful for renal disease
Creatinine not synthesised by birds (Creatine instead)
Urea can be useful for assessing hydration status (but 10-15 x increase not uncommon)
Other
Protein levels lower in birds c/f mammals
Glucose levels more stable in birds as not utilised by RBCs but see stress and postprandial↑
BG ranges in birds higher than mamamls
Calcium – consider total and ionised calcium
Egg laying biochemistry
Reptilian Biochemistry
Lymph dilution common and will reduce all biochemical values
Effects of sex, season profound
Liver and muscle
ALT, ALP, LDH non specific
AST found in liver, kidneys & muscle so should be assessed alongside CK and LDH
GGT in liver and kidneys; sensitive for hepatic & renal disease
GDH may be useful indicator of hepatocellular necrosis
Biliverdin is primary bile pigment – no assay commercially available
Bile acids vary across reptilian taxa
Renal
Uric acid main excretory product of protein metabolism – produced in the liver (gout)
Fasted samples required in carnivorous reptiles
Uric acid main excretory product of protein metabolism – produced in the liver
Fasted samples required in carnivorous reptiles – postprandial elevations
Uric acid marker of renal disease, dehydration
Persistent elevations can –> visceral & articular gout
Reduced uric acid levels seen in hepatic disease
Urea and creatinine less useful as low and variable levels
May see elevated urea levels in early rehydration as can be resorbed across bladder wall
Ca:P ratio one of most useful indicators of renal disease
Sodium ↑ with dietary intake and dehydration and ↓ with renal disease, GI losses
Other
Calcium – egg laying females
Exotic Mammal Biochemistry
Variations between carnivores & herbivorous species
Liver & Muscle
ALT elevations seen with hepatocellular damage so can be useful initial screening parameter
AST found min muscle and liver so should be interpreted alongside SK
ALP non specific for liver, elevated in young, growing animals or high bone turnover
Calcium
Rabbits unique amongst mammals in their Ca absorption mechanism
Dietary intake can cause hypercalcaemia
Renal
Urea elevations as per other mammals with dehydration, renal disease, obstruction
Can see hyper or hypcalcaemia
Other
Hepatic lipidosis elevated trigycerides & cholesterol
exotic haemotology: Metabolic bone disease & Hypocalcaemia
Calcium leached out of bones
Calcium and Vit D intake +/- UV exposure
Must measure iCa as well as Total Ca
Hypocalcaemia in African Grey Parrots
Often collapse, seizures. Tremors
iCa diagnostic
Further lectures to cover this in more detail
Egg Laying Biochemistry
Female birds and reptiles
Classic pattern of biochemistry
Constituents of egg and shell mobilised
↑ Total Protein, Globulin, Uric Acid, ALP, Total Calcium
Check ionised calcium
Comparative pathology
Comparing human disease with that of animal models
Animal models can be spontaneous or induced
Spontaneous models in inbred mice are some of the most well known and technically simple
The NOD (non-obese diabetic) mouse was discovered and established in Japan and is model for human type 1 diabetes mellitus.
Induced can be chemical or genetic
Streptozotocin given to rats is toxic to pancreatic beta cells, resulting in type 1 diabetes mellitus
Genetic involves knock-in, knock out or transgenic
mouse knock-in model generated to upregulate HER2, or the mouse orthologue Erbb2, in breast cancer
comparitive pathology Models of atherosclerosis
Refers to a type of arteriosclerosis (narrowing of vessels)- Composed of lipid, fibrous tissue, and calcium deposits in vessel walls
Hugely important in human medicine and one of the major causes of coronary heart diseases (heart attack)
Rare in animals
Dogs
Secondary to hypothyroidism, diabetes mellitus, and dogs with breed-related hyperlipidemia (Miniature Schnauzers)
A recent study identified gonads of dogs as common locations for atherosclerosis, +/- amyloid deposits, associated with age and interstitial cell tumours (Ushio et al, Vet Pathol, 2021)
Rabbits
Chickens
Pigs
Nonhuman primates (NHPs)
Rabbits are the most commonly used research species- Three types of rabbit model are commonly used for the study of human atherosclerosis and lipid metabolism: (Fan et al, Pharmacology & therapeutics,2015)-
Cholesterol-fed rabbits
Watanabe heritable hyperlipidemic rabbits, analogous to human familial hypercholesterolemia due to genetic deficiency of LDL receptors,
Genetically modified (transgenic and knock-out) rabbits
comparitive pathology Hamster models of COVID-19
In contrast to other rodents, hamsters are naturally susceptible to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
As soon as angiotensin converting enzyme-2 (ACE2) was discovered as the principle human cellular receptor for the viral spike (S)-protein, systematic structural protein analyses of mammalian ACE2 homologs predicted that macaques, mink, ferrets, common marmosets, felines, rabbits, hamsters, and few other mammals but not mice would be naturally susceptible to the infection
The Syrian hamster (Mesocricetus auratus) rapidly developed into a popular model. - It recapitulates many characteristic features as seen in patients with a moderate, self-limiting course of the disease such as respiratory tract inflammation and age dependence.
Had already served as a model for SARS-CoV infection in 2005
The Roborovski dwarf hamster (Phodopus roborovskii) more closely mimics the disease in highly susceptible patients with frequent lethal outcome, including devastating diffuse alveolar damage and coagulopathy
comparative pathology Models of Duchenne muscular dystrophy
Duchenne muscular dystrophy (DMD) is a progressive, fatal, X-linked monogenic muscle disorder
Results in progressive muscle wasting. Caused by mutations in DMD, a gene encoding the protein called dystrophin
In order to test treatments for DMD, a range of natural and engineered animal models have been developed, including mice, rats, rabbits, dogs, pigs.- There appears to be an effect of body size in models of DMD, as the severity of the clinical signs increases with increasing body size across species.
All DMD models have different advantages and disadvantages
The best known model is the dystrophic mdx mouse- 2800 papers published using this mouse
Good biochemical model of DMD
But, the mice have only a slightly shortened life span and show no obvious clinical signs of muscular dystrophy
A number of different breeds of dogs presented at veterinary clinics have been diagnosed with dystrophin-deficient muscular dystrophy.- Several of these have been subsequently used to develop dog models of DMD
The most common being a Golden Retriever with Muscular Dystrophy (GRMD)
While these dogs do show clear clinical signs analogous with DMD, there are some significant differences. A number of the GRMD dogs die within the first 6 months of life and there is considerable dog-to-dog variation, which, with the small numbers available for each experimental group, makes it difficult to show clear statistically significant results
The rabbit model is a recent creation using CRISPR/Cas9- Rabbits have the advantage of being easier to breed and less costly than dog or pig models, but have clear clinical signs, in contrast to many mouse models.
outline the drug developmet process
identify the target- understand the disease
validate the target- show that the target is clinically relevant
ADME- Pharmacokinetics of agent
Toxicity- is organ damage caused by agent?
tolerabiluty- advere effects of behavioural agent?
pharmacodynamic effect in target organ- agent engages target and causes clinically measurable effect
Humanized mice
= immunodeficient mice engrafted with functional human cells and tissues
It all started with the SCID mouse- Commonly known as the nude mouse
Athymic due to a loss of function mutation in the PRKDC gene
Resulting in impaired development of T and B lymphocytes which gives rise to severe combined immunodeficiency (SCID).
The most common approach is to engraft human CD34+ hematopoietic stem cells which migrate to the bone marrow of the mouse and differentiate to all lineages of the mature immune system
Some models involve whole body irradiation prior to engraftment
Patient-derived xenografts in humanized mice- Not a new idea injecting human tissue into mice
Orthotopic models involve implanting a human tumour into the corresponding tissue in animal models.- E.g. breast cancer cells directly into mammary tissue in mice
Using a humanized mouse for this more appropriately models the tumour microenvironment, the contribution of the immune system to the eradication of tumour cells, and could predict drug response
Veterinary diagnostics enable us to …
Establish a diagnosis
Provide prognostic information
Guide selection of treatment or control strategies
Monitor effect of treatments
Veterinary molecular diagnostics
Amplify the genome of viruses present in or released from infected cells
DNA of bacteria, fungi or parasites
Polymerase chain reaction (PCR)
denaturing, anealing, extention
Uses oligonucleotide primers to amplify region of interest (gene)- for specific antigen, toxin ect
Cycles of heating and cooling drives each step
Millions of copies can be produced in minutes
Number of copies provides information on presence and/or amount of starting material
PCR - denaturation
High temperature breaks hydrogen bonds holding base pairs together- 94ish
‘Melts’ double-stranded DNA revealing bases in specific order
PCR - annealing
At cooler temperatures, complementary bases can bind- 50ish
Oligonucleotide primers ‘match’ small regions of the target area (gene of interest)
They bind to the matching areas (anneal)
Primers must be designed so that one matches the sense strand and the other matches the antisense strand
PCR – extension/elongation
Temperature raised to approximately 74°C
Synthesis of new complementary DNA strand from 3’ end of primer
Only regions where primers bound will be amplified/copied. So it’s really important that they only match the region we’re interested in
PCR – cycling
In one cycle (denaturation; annealing; extension) we have gone from one copy to two
Cycle is repeated multiple times and product number increases exponentially
Average PCR run is 40 cycles
Semi-quantitative PCR
The amount and size of the PCR product can be visualised using staining and gel electrophoresis
This visually confirms if our pathogen / gene of interest / strain is present
Known as semi-quantitative PCR
Quantitative PCR (qPCR)
the amount of DNA amplified in each cycle are quantified.
increased florecence indicates increased presesnce of target
targrt floreced via cyber green or Taqman
TaqMan more specific- floreces when “knocked off” target stran d of dna by formation on new strand
cQ value
This value tells how many cycles it took to detect a real signal from your samples.
the point in qPCR where a flourecent signal is detectable
Reverse transcriptase PCR (RT-PCR)
Uses reverse-transcriptase enzyme to produce double stranded DNA from RNA
This provides template for normal PCR
Can also be incorporated into qPCR = RT-qPCR
PCR for viral infections
very useful
Serological assays are not always feasible when detecting viral infections so PCR can be used
Lack of species-specific secondary antibodies
Suitable cells for growth and titration are not available
primer needs to be developed
methods for molecular diagnostics of bacteria
Culture- labour intensive, can take long time, contamination of culture common, multiple plates and culture mediums may need to be used
Stain
Test
not useful for, for example:
Helicobacter-
Many different species that can infect veterinary species- which one do u culture? which one is actually casing disease
Individuals can be infected with more than one species at the same time
Fastidious bacteria (difficult to culture)
Helicobacter
Many different species that can infect veterinary species
Individuals can be infected with more than one species at the same time
Fastidious bacteria (difficult to culture)
PCR primers designed to detect a single species
Rapid test
diagnostic methods for Fungal infections
Slow growth in culture
Diagnosed histologically
Can be diagnosed with PCR
PCR can be used if no identifiable fungal species cultured or morphologically identifiable
Pathogen-specific primers
Generic fungal primers (e.g. rRNA)
Genus-specific primers
Benefits of PCR
Specificity is very high- primers can be designed to be incredibly specific
Sensitivity is very high- you cna have very little dna and it will work (some assays are still more sensitive though- giardia assay duse to fecal sample having pcr inhibitors
Rapid turnaround time
Overcomes culturing restrictions
Negatives of PCR
PPV can be low
Not for all assays/samples
Requires specialist equipment
Overcomes culturing restrictions
Positive predictive value (PPV)
The probability that a test positive animal is diseased
Disease agents may colonise healthy animals as well
PCR detects DNA/RNA in live and dead organisms
May be positive even if infection is controlled or cleared
Interpretation of results for a single animal can be difficult
PCR inhibitors
False negatives present a challenge
Often occur due to PCR inhibitors in the sample
Natural inhibitors include:
Bile salts
Polysaccharides in faeces
Haem from blood
Glycogen and fats in tissues
Proteinases in milk
Urea in urine
to combat this there are:
Co-purified with DNA/RNA
Extraction kits designed to remove them
Organism vs antibody detection
Detection of organisms gives most information
Assays not always available or optimal
Antibody detection still commonly used
Combinatory approach can be used
PCR positive result can occur prior to seroconversion – prove infection in acute cases
PCR can be negative later in course of disease
Serum antibodies are detectable
Serology
Detect antibodies or antigen in blood sample
Indirect method
Limitations due to lack of species-specific secondary antibodies
Seropositivity may not indicate acute infection
Antigen:
Specific portion of pathogen
Protein found on surface of pathogen
Non-self
Antibody:
Proteins (immunoglobulins (Ig))
Part of the immune system (self)
Created to bind to specific antigens
Specific antigen binding sites
The most common antibodies have 2 binding sites, some have 4 or 10
Binds to antigen
Neutralises antigen
ELISA - Enzyme Linked Immunosorbent Assays
2 types of ELISA:
Direct test - Antibodies used to test for antigen
Indirect test – Antigens used to test for antibody
Can test for:
Bacteria or bacterial toxins
Viruses
Protozoa
Ab to any of these or Ab to parasites, yeasts,
Direct test ELISA
Antibodies used to test for antigen
Sample proteins immobilised on plate/well
Enzyme labelled antibodies added
Antibodies bind to antigen
Enzyme-specific substrate added
Reaction takes place and produces colour
Colour change (signal intensity) detected
indirect test ELISA
Antigens used to test for antibody
Indirect ELISA
Detection of antibody:
Antigen immobilised on plate/well
Sample is added
Any antibodies present will bind to antigen
Enzyme-labelled secondary antibody added
Substrate added
Reaction takes place and produces colour
Colour change (signal intensity) detected
Sandwich ELISA
Detection of antigen:
Plate/well coated with capture antibodies
Sample is added
Any antigen present will bind to antibody
Direct: enzyme-labelled antibody used
Indirect: Enzyme-labelled secondary antibody added
Substrate added
Reaction takes place and produces colour
Colour change (signal intensity) detected
Competitive ELISA
multiple ways to set up depending on whether the antigen or antibody is of interest
Detection of antigen or antibody:
Sample antigen/antibody competes with reference antigen/antibody
Analyte concentration is indicated by signal interference
Coat plate with reference antigen
Incubate sample (unknown antigen concentration) with limited amount of labelled antibodies
Low antigen conc. in sample = large portion of labelled antibodies have nothing to bind to
Add this mixture to the antigen coated plate
Any free labelled antibodies will bind to reference antigen
Wash plate to remove antibodies bound to sample antigen
Add substrate
Stronger colour = less antigen present in sample
ELISA – important steps
Plate coating: Samples are diluted in buffer, then pipetted into a microwell plate. After incubation, the solution is discarded and plate is washed with a wash buffer. ONLY immobilised antigen/antibody remains
Plate blocking: Blocking buffer is added to the plate. This binds to any remaining protein-binding sites in the coated wells, reducing non-specific binding of antibodies to the plate. Plate then washed again
Antibody incubation: Following incubation, wash away unbound antibodies with a wash buffer.
Detection: The enzymes covalently attached to the antibodies will start producing a coloured reaction product. Stop solution is added to terminate the colour development and the absorbance of each well is read. The signal intensity allows you to determine whether a sample contains the antigen/antibody of interest, and at what concentration. By stopping all wells at the same time (with stop solution) the signal intensity if indicative of the antigen (or antibody) concentration.
signla detection in a Lateral flow immunochromatographic assay
ELISA
Detect the presence of a target substance (antigen) in a liquid sample
‘Signal’ is still a colour change
But does not require specialised equipment (patient-side)
Chromatography paper = capillary action
Antigen binds to antibodies with tag
Flow along test paper
Collect at test line – fixed antibodies that will bind to antigen(Collection = visible line)
Flows across control line – fixed antibodies that bind to tagged antibodies
Detection of signal indicates detection of antigen-antibody complex
Epithelial cells
All endoderm and some ectoderm
Cells form bulk of parenchyma of organ, glands or line organs
Hepatocytes
Skin
GIT
Bladder
Cell-to-cell and cell-to-basement membrane adherence
Mesenchymal cells
Mesoderm
Supporting cells
Fibroblasts -> collagen
Endothelia
Bone
Round cells
Mesoderm
Cells of the haemo-lympho system
Erythrocytes
Leukocytes
Histological appearance of epithelia
Polygonal
Poorly-defined cell borders
Abundant cytoplasm
Polar to central round nuclei
Basement membrane
Histological appearance of mesenchymal cells
Fusiform/spindloid- long, stringy
Poorly-defined cell borders
Variable cytoplasm
Fusiform/spindloid nuclei
Extra cellular matrix
Histological appearance of round cells
Round
Well-defined cell borders
Individualised
Variable cytoplasm
Round to variable nuclei
Immunohistochemistry
Immunodiagnostic
Uses antibodies to detect an “antigen” in a tissue
IHC can be used to differentiate between inflammation and neoplasia
A classic example is inflammatory bowel disease versus lymphoma
In both diseases lymphocytes accumulate in the intestine
If inflammatory this is polyclonal
Multiple lymphocytes replicating
If neoplastic this is monoclonal
One cell becomes neoplastic and replicates
Antibodies made by injecting antigen of interest into an animal
Two types of antibodies
Monoclonal
Antibodies produced by the same clone of plasma B cells
Hybridisation with tumour cells
Higher specificity, lower sensitivity
Polyclonal
Heterogeneous mix of antibodies
Derived from the immune response of multiple B-cells
Each one recognizes a different epitope on the same antigen
Higher sensitivity, lower specificity
cytokeratine lables
Epithelial cells
vimentin lables
Mesenchymal cells
Indications for a kidney biopsy:
Proteinuria
Acute renal failure
Chronic renal failure
Renal azotaemia that cannot be categorized as acute or chronic renal failure
Proteinuria
the presence of abnormal quantities of protein in the urine, which may indicate damage to the kidneys.
Proteinuria is, once a urinary tract infection is ruled out, almost always due to disease of the glomerulus. Whereas low urine specific gravity, which would indicate reduced ability to concentrate urine, is typically due to disorder of the tubule.
Podocytes
The structure of the glomerular capillaries is important in determining the rate and selectivity of glomerular filtration.
The glomerular capillary wall consists of three layers:
the capillary endothelium
the basement membrane
the visceral epithelium
= podocyte
Greek pous, pod- ‘foot’
Immune-mediated glomerulonephritis
Results from the deposition of immune complexes in glomeruli
formation of antibodies against the glomerular basement membrane
activation of inflammatory cascade
Any condition that stimulates the immune system for long periods of time can cause IM-glomerulonephritis
What is cytology?
Microscopic examination of tissue samples spread onto slides
Histology looks at architecture, cytology has no architecture - look at individual cell morphology
Aspirates – solid tissue
Needle only
Intermittent suction
Continuous suction
Impression smears- mucosa, ears, biopsies
Wash / Lavage – trachea, BAL, nasal, prostate
Ultrasound guided – watch out for ultrasound / lubricant gel!
No anaesthetic and low risk of complications
Relatively quick procedure and less invasive
Rapid results – emergency cases, clients waiting, same day intervention
Older patient - removal of a benign tumour?
Grading of tumoursto he lp with surgical or chemo planning
Surgical planning
Staging of tumours
Fluid classifications – Transudate, Modified Transudate, Exudate
Infections (septic abdomen, ear cytology)
Ancillary tests-
PARR analysis and Flow Cytometry
PCR for infectious diseases
How to take a fine needle aspirate for cytology
22-gauge needle
5-10ml syringe
Glass slides
Pencil
place needle without syringe in mass- not in purilent area
rediredct needle a few times
take out needle
put air filled syringe on needle and put needle over slide
squirt cells down onto slide
suction technique-
same but pit syringe on first and apply suction while in mass
may cause haemodilution
the sample must then be spread-
Squash pull / drag method best, send both slides
Fluids-
Blood smear (push / wedge)
Line concentration
Cytospin
Pre-analytical factors for cytology
Submission forms
Formalin
Sample types used for fluid
Lubricant gel
Cellularity / Pre-staining
Preservation
Cell thickness
Greasy slides
Formalin Exposure in cytology
Exposure of unfixed cytologic specimens & air-dried unstained smears to formalin fumes → Interferes with staining
Fluid Specimens
BAL
Pleural or Abdominal fluid
Prostate wash
Urine – sediment vs. cytology- diffretn things!!!!. braf test for transitional cell carcinoma
Bile-most samples you see nothing but cats get bacterial infections
Cerebral Spinal Fluid
One or more direct preparations
Air dried and submitted with fluid
EDTA (cyto) and plain tubes (culture)
Fluid is a very poor preservative - cells deteriorate very quickly in fluid. could be done for FIP
Cellularity
The number of cells in a given tissue sample
How to assess this before sending to the lab?
Pre-staining- can effect labs staining
Assessment of unstained slides = lower the condenser
differentials for a greasy slide after a fine needle aspirate
Lipoma?
Other differentials include:
Steatitis / panniculitis
Perinodal fat
Mast cell tumour
Liposarcoma
Other soft tissue sarcoma
Not representative
Sample preparation – allow to dry and stain flat
Diff Quick – in house stain technique
Diff-Quik stain consists of:
Fixative agent - methanol, blue
Solution I - eosinophilic, orange
Solution II - basophilic, blue
Technique
Dry slides in the air
Dip slides sequentially into each solution 5 times (one second each dip)
After every immersion wait a moment to drain excess.
Rinse with tap water
Allow to dry (do not use heat sources)
Slides must be air-dried before staining – it is important to make sure the slides are very dry before staining, water may still be present when the slide appears dry – so make sure to leave sufficient time for adequate drying.
Heat fixation is not recommended as this may interfere with staining quality and can rupture cells.
When using diff quick dip slides into each solution 5 times for 1 second, followed by a water rinse and dry the slide vertically
Some tips for the ideal stain include:
If the slides are thin or have only a few cells on them - shorten staining times
Thick smears or those with high protein may need longer staining times.
Ensure stain reagents are fresh and well maintained - if your cytology slides have a lot of stain precipitate on them , it is probably time to refresh the stain.
If you get a gold film forming on the top of the stain – this means it is also time to change the reagents
Organisms in the stain may contaminate the cytological slides.
Automatic staining – external lab
may make mast cells look like histeocytoma!!!!!
MGG Quick stain – in house stain technique
new! better than diff quick
May-Grunwald Giemsa Quick stain
Performs similar to the traditional May Grunwald-Giemsa used in labs
But is quick to perform like most rapid stains
Technique
Dry slides in the air
Dip slides sequentially into each solution 5 times (one second each dip)
After every immersion wait a moment to drain excess.
Rinse with tap water
Allow to dry (do not use heat sources)
In lab automatic staining
Modified Wright’s
only material in middle of lide is stained so slides must be preped appropriatly
Modified Wright’s
Get your slides out ready
Ensure you never put any slides you have taken out of the box back in the box – avoids cross contamination
If doing needle only attach the syringe over the slides
Always start with needle only to ensure you don’t get too much blood contamination
Never package with formalin
Always submit more than one slide
Don’t put cytology slides in the fridge- erythrocytes can freeze
Avoid using a hairdryer or flame to dry- cells rupture and fragment
Greasy slides need longer to dry and should be stained flat
Make sure you use fresh stain
How to approach analysing cytology samples
Cellularity and preservation
Haemodilution
Inflammatory or Proliferative
Inflammation – infectious, immune mediated, necrosis
Proliferative type
Epithelial, Mesenchymal, Discrete / Round cell, neuroendocrine
Hyperplastic, dysplastic or neoplastic
Neoplasia – benign or malignant
Definitive diagnosis
what might be found on Haemodiluted cytology slides
Erythrocytes
Platelet Clumps
Nucleated cells in peripheral blood- are they from mass or just from the haemodilution???
neutrophils and monocytes- from blood
macrophage- from tissue
eosinophils and basophils
lymphocytes
Inflammation in cytology
White blood cells-
Neutrophils, macrophages, eosinophils, lymphocytes, mast cells (a couple are OK).
Neutrophils-
Non-degenerate neutrophils – well preserved
Degenerate neutrophils – microorganisms and necrosis.
Pale blue swollen nucleus
Macrophages-
Activated - vacuolated and foamy
Phagocytosis –fresh smear as can do this in vitro
Infectious agents (Slow growing bacteria, Fungi, Parasites
Cell debris
Erythrophagia - previous haemorrhage, breaksdown to haemosiderin and haematoidin
Reactive lymphoid hyperplasia
a benign nodular lesion, histopathologically characterized by marked proliferation of non-neoplastic, polyclonal lymphocytes forming follicles with an active germinal center.
you will see:
Mixed small, intermediate, large lymphocytes
Mott cells
Plasma cells
compare Inflammation and Infection in analysis of cytology samples
Inflammation -
Acute / suppurative - neutrophils
Chronic active / pyogranulomatous – neutrophils and macrophages
Chronic / granulomatous – macrophages (multinucleated), lymphocytes, plasma cells
Specific e.g. eosinophilic
Concurrent tissue reaction – hyperplasia / dysplasia
Infection-
Bacteria – rods / cocci / coccobacilli
Yeasts and fungi
Mycobacterium
Protozoa
Aspiration of hyperplastic lesions may show….
Higher than expected cellularity
Cells may display some mild criteria of malignancy
Mildly increased N:C ratio
Darker blue cytoplasm
More prominent nucleoli
Finer chromatin than normal
Atypical cytologic features of dysplasia:
Nuclear to cytoplasmic asynchrony
Increased cytoplasmic basophilia
Anisokaryosis and anisocytosis.
Dysplasia can be cytologically difficult to distinguish from neoplasia as dysplastic lesions often contain more criteria of malignancy than strictly hyperplastic lesions.
Types of Neoplasia
Epithelial
Mesenchymal
Round Cell
Neuroendocrine
Epithelial neoplasm
Arrangement-
Monolayer sheets, clusters, rows, palisades and acinar.
Cohesive with distinct tight junctions (desmosomes)
Clear lines between cells.
Shape
Columnar, cuboidal or polygonal
Nuclei
Round to oval.
Epithelial cells can look round in fluid or when poorly differentiated.
E.G.: Squamous cell carcinoma, adenoma and adenocarcinoma
Benign: Adenomas
Malignant: Squamous cell carcinomas
Mesenchymal neoplasms
Arrangement
Often see individual cells but occasionally seen in aggregates.
Loosely arranged with extracellular matrix and individualised spindle or stellate cells.
Shape
“Spindle cell” - spindle shaped cells, fusiform or stellate.
Wispy cytoplasmic borders, the borders are not distinct. Cytoplasm can contain vacuoles
Nuclei
Round to elliptical (oval). Cytoplasm can contain vacuoles.
Samples are often poorly cellular in benign lesions, but can be very cellular in malignancy.
Arise from connective tissue e.g.: fibroblasts, osteoblasts, adipocytes, myocytes and vascular lining cells.
E.g Osteosarcoma, Haemangiosarcomas etc.
Benign: Fibromas, lipomas
Malignant: Sarcomas, soft tissue sarcoma, haemangiosarcoma etc.
Lipoma
Greasy slides
Large fat filled adipocytes in aggregates
Often have capillaries going through them
Often benign, can have infiltrative forms
Liposarcoma - malignant version
Round Cell neoplasm
Arrangement-
Discrete and individually arranged
Shape-
Round tumour cells
Well defined / distinct cytoplasmic borders
Nuclei-
Round to oval large nucleus (can be indented)
Exfoliate well
5 main examples:
Mast cell tumour
Lymphoma / leukaemia
Histocytoma / histiocytic sarcoma
Plasma cell tumour / plasmacytoma / multiple myeloma
Transmissible venereal tumour
Mast cell tumour cytology
Round cells
Central round nuclei
Lots of magenta granules in the cytoplasm
Fibroblasts, eosinophils and ribbons of pink matrix
Lymphoma cytology
Monomorphic population of lymphocytes individually arranged
Low N:C ratio
Large cell vs. small cell lymphoma
Lymph nodes, skin, spleen, intestines
Histiocytoma cytology
Rounds cells individually arranged
Light blue cytoplasm, Nuclei round to oval to cleaved
Often accompanied by lymphocytes and plasma cells
Mass in a young dog, likely histiocytoma, although can get them in older dogs
Plasma cell tumour cytology
Rounds cells individually arranged
Looks like well differentiated plasma cells, however the cells are predominantly plasma cells. If this was reactive you would see a mix of lymphocytes and plasma cells
Prominent golgi zone
Multinucleation and Macronuclei even though commonly benign
Transmissible Venereal Tumour cytology
Used to be a rare disease in the UK
Seeing more and more cases
Sexually transmitted tumour is thought to be of histiocytic origin - transfer of intact neoplastic cells
Often around the mouth or genital region, but can be seen in other sites.
Cytoplasm is characteristic: Abundant light blue to grey with moderate to many discrete margined vacuoles
CTVT cells have 59 chromosomes compared with the normal canine karyotype of 78 chromosomes.
PCR now available to help diagnosis
. Neuroendocrine neoplasm
Arrangement-
Free nuclei arranged in small rosettes / sheets
Shape-
No cytoplasm – mostly just bare nuclei
Nuclei
Round
Exfoliate well
Examples: Thyroid tumours, pheochromocytomas
Cytology Description and Identification
Identification:
Nuclear features
Cytoplasmic features
Miscellaneous-
Background – joint fluid, product, mucous, chylous
Haemodilution – inflammatory?
Accompanying cells – contamination or tissue?
Concurrent process – haemorrhage, inflammation, necrosis
Presence / absence – e.g. lymphoid cells
When looking at a slide it is important to be systematic-
Always start at the top left of the slide.
Scan the slide at low power (4x magnification)
Assess the cellularity, background colour and then find the main cellular population
Then get closer at 40-100x
Careful to not get oil on the 40x!
it is important to appreciate certain nuclear features. Bizarre mitotic figures are often very interesting and can really help you determine whether a lesion is neoplastic. In this example here the mitotic figure is very atypical and has become fragmented. You can have multinucleate cells with different sized nuclei within the same cell. Also large prominent nucleoli are a key feature to look out for.
Nuclear features to look for in cytology
Size and shape
Chromatin
Nucleoli
Nuclear membrane
Relationship / nuclear moulding
Mitotic figures
Cytoplasmic features to look for in cytology
Vacuolation
Basophilia
Product
N:C ratio
Nuclear position
Criteria of Malignancy in cytology
Anisocytosis and Anisokaryosis
Increased nuclear to cytoplasmic ratio (not always seen e.g.: lymphocytes)
Nuclear features:
Mitotic figures, how many there are and if they are atypical
Multinucleation
Nuclear moulding
Angular nuclei
Chromatin (instead of being smooth it becomes coarse and clumped)
Nucleoli:
Prominent, multiple, angular, anisonucleoliosis
Macrocells, macrokaryosis and macronucleoli - macro is always bad news!
Necrotic cells in between neoplastic cells. These cells look like smudge cells (been rubbed out with a rubber)
Phagocytosis - cannibalism
Emperipolesis
Crowded cells
Lymphoma subtyping – Cytological criteria
Features of the abnormally represented population:
Small, intermediate or large lymphocytes
Blastic appearance (presence or absence of nucleoli)
Position of the nucleus in the cell, eccentric or central
Shape of the nucleus, round, oval, cleaved, convoluted
Relative volume of cytoplasm
Appearance of cytoplasm, eccentric, concentric, mirror-handle
Presence/absence of golgi zone
Relative numbers of mitotic figures and presence of atypical mitoses
Dimorphic lymphocyte distribution
Presence of ancillary cells, tingible body macrophages, eosinophils
Careful cytological assessment will efficiently diagnose lymphoma and allow estimation of the classification, phenotype and grade
Cells harvested for cytology are not formalinised allowing molecular and immunological investigation of stained slides
The big 5 canine lymphomas have characteristic cytological patterns recognisable to skilled cytologists, allowing a quick and cost effective diagnosis.
On cytology you can see:
Disproportionate representation of a specific lymphocyte population – In general lymph-nodes should be heterogenous, but instead in lymphoma you have a homogenous effacing population
Cytology also allows you to see very fine cellular details of the neoplastic population such as the chromatin pattern and atypical mitotic figures.
Lymphoma subtyping – ancillary tests
PARR analysis
Assessment of clonality (genotype)
Clonality Testing (PCR for antigen receptor rearrangement; PARR) uses end-point PCR to predict whether lymphocytes in a sample are a clonal or polyclonal population
Flow cytometry
Assessment of phenotype
Immunocytochemistry
Assessment of phenotype
things to avoid when taking good histeopathology samples
Don’t crush it- think about handling, dont handle critical areas, can distort tissue- lymphoid tissie and tumour cells vunerable to this
•Don’t cauterise it
•Don’t freeze it
•Don’t scrape mucosal surfaces (e.g. GIT)- rinse under water instead if needed
•Fix it ASAP!
Don’t package cytology and histology together
appropriate containers for histeopathology samples
sample pots- Label your sample pots- important! wha tis the sample!
•Use appropriate containers
•Use correct amount and type of fixative
•10:1 ratio of formalin: tissue
•10% neutral buffered formalin
dont use-
Narrow-necked bottles “pill pots”- sample may swell when fixed
Glass containers- health and saftey
Sharps container- cant open!
Different types of margins
Cross-sectional or radial
•Bread loaf or pie
Cost effective •Easy •Allows for measurement of margins •Assumes centrifugal growth from centre •Only evaluates a portion of marginal tissue
Tangential / shaved / orange peel
•Tumour bed
•Theoretically complete assessment of all marginal tissue •Technically difficult •More slides, more cost •Does NOT allow measurement of margins •Binary answer –excision complete or not
suture tags can be used to comunicate thing to pathologist
you can also ink the sample- green and blue ink best
What is heart disease vs. failure?
Heart disease is any condition affecting the cardiovascular system
- chronic in nature e.g. cardiomyopathy
- acute in nature e.g. myocarditis
- may or may not have clinical signs associated with it e.g. syncope, exercise intolerance
- abnormalities on physical examination usually present e.g. heart murmur
Heart failure is a syndrome where the heart can no longer meet the metabolic demand of the body
- Usually acute onset
- Clinical signs present e.g. exercise intolerance, syncope, lethargy, anorexia etc.
- Physical examination abnormalities present e.g. fluid thrill, dyspnoea, crackles, jugular pulsation etc.
clinical signs of heart disease
Often non-specific/ absent
Exercise intolerance and weakness
Can be syncope/ pre-syncope
Cyanosis
Coughing- left atrium can press on brochi. fluid could fill in lungs,
Paresis-condition in which muscle movement has become weakened or impaired. thrombi can be lodged in aorta. thrombi can aso cause lamness
(acute blindness, neurologic signs)
on physicla examination:
Body condition score
Respiratory rate and effort
Ascites- condition in which fluid collects in spaces within your abdomen
Distended jugular
Pulsating jugular
Peripheral oedema
Cyanosis
Right to left shunting defect from deoxygenated circulation to oxygenated
Central cyanosis
Differential cyanosis
Peripheral cyanosis
Heart murmur (dog vs cats)
Arrhythmia (dogs vs cats)- cats is assosiated with heart disease but more varied differential with dogs
Gallop sound
Cat with cold hindlimbs
Cyanosis
Presence of goitre
Retinal detachment
Some neurological signs
clinical signs of Heart failure
Tachypnoea/ dyspnoea (L CHF usually)
Weight loss (less cats)
Swollen abdomen (R CHF)
Peripheral oedema (R CHF)
Signs as per heart disease
mm often normal!!!
Dyspnoea/ tachypnoea
Crackles in lungs
Ascites (fluid thrill)
Jugular distension
Significant jugular pulsation
Muffled heart or lung sounds
Positive hepatojugular reflux
Pulsus alternans/ paradoxus
Peripheral oedema
Plus usually signs of heart disease
Jugular pulsation
Normally only 1/3 up neck
If to angle of jaw – abnormal
Arrhythmias
Tricuspid regurgitation
Pericardial disease
Jugular distension
Should collapse after pressure released
Elevated systemic venous pressure
R heart failure
Pericardial disease
Obstruction of cranial vena cava
Hypervolaemia (cats)- fluid overload, is a condition where you have too much fluid volume in your body
Thoracic examination- what to lookout for if heart disease is expected
Auscultation-
Heart – both sides thorax, apex and base
Heart murmur
Gallop sounds
Arrhythmia – Atrial fibrillation, AVB…
Heart rate – brady/tachyarrhythmias
All lung fields
Compare sides and dorsal vs. ventral
Muffled
Crackles
Palpation/ percussion/ compression-
Apex beat displaced
Thrill
Percussion for fluid/ soft tissue/ air
Reduced compressability (cats)
gallop sounds on heart auscultaion
Normally heard in dogs and cats-
Occur during systole
S1
‘lub’
Closure of AV valves
S2
‘dub’
Closure of semilunar valves
Not normally heard in dogs and cats-
Gallop sounds (occur in diastole)
S3
Early diastolic filling
Not heard in compliant ventricle
Systolic dysfunction
S4
Atrial contraction
Forceful atrial ejection into a noncompliant ventricle
Hypertrophic/ restrictive cardiomyopathy
Abdominal examination- for heart faliur
Palpation-
Fluid thrill – ascites
Splenomegaly occasionally CHF
Hepatomegaly too
Painful sometimes
Hepatojugular reflux-
Squeeze just behind ribs
Watch jugular vein
If jugular vein distends whilst squeezing – positive response
Positive response suggests high R sided filling pressure
R side can’t accommodate extra blood
Pulsus alternans
Alternates strong with weak
Myocardial failure
Pulsus paradoxus
Decrease pulse quality on inspiration
Pericardial effusion
What is a heart murmur?
Extra sound
Occur during systole (most commonly) – between ‘lub’ and ‘dub’
Or diastole – after dub
Or both (continuous murmur) – all the time
Due to regurgitation of blood e.g. mitral regurgitation
OR turbulent flow across a narrowing e.g. sub-aortic stenosis, VSD
OR reduced viscosity of blood (anaemia)
Blood flows DOWN pressure gradient
High pressure – low pressure
The greater the gradient the faster the flow
Depending also on resistance to flow
Flow ONLY occurs when a pressure difference exists
Can explain many phenomena:
PDA – continuous murmur (L-R shunting)
Murmurs on right often quieter than left
VSD shunts L-R ‘normally’
Reverse shunts tend to be quieter
Descriptive characteristics of heart murmers
Grade
1 – barely audible, need quiet room
2 – audible but quieter than heart sounds
3 – clearly audible and as loud as heart sounds
4 - louder than heart sounds
5 – THRILL (PALPATION) present- buzzing feeling of chest
6 – Audible with stethoscope lifted off chest
Mild/ moderate/ loud/ thrilling (Ljungvall 2014)
Timing
Systolic/ diastolic/ continuous
Can be further classified as holo- and pansystolic
Location
Right/ left
Apex/ base (dogs)
Sternal/ parasternal (cats)
Radiation- does it radiate dorsaly? to the left? ect
Apical systolic murmurs
Mitral regurgitation-
Grade can correlate with severity (MMVD not DCM)
Pansystolic worse (MMVD)
Can be musical/ whooping
Often radiates to right
Tricuspid regurgitation-
Difficult to distinguish from radiating left sided murmurs
Vary with respiration
Tricuspid valve dysplasia
Pulmonary hypertension
Degeneration of valve
Basilar systolic murmurs
Aortic stenosis-
Harsh sounding
Radiate widely to thoracic inlet
Low grade are difficult to distinguish from physiologic/ innocent
(Cats – HOCM dynamic)
Pulmonic stenosis-
Left heart base
Radiate dorsally
Innocent/ functional murmurs
Innocent-
Puppies and kittens
No structural heart disease
Grade 1-3
Systolic
Left heart base
Don’t radiate widely
Functional-
Associated with disease process
Anaemia
Hyperthyroidism
Fever
Hypertension
Pregnancy
No structural heart disease
Grade 1-3
Systolic
Left heart base
Don’t radiate widel
Other systolic murmurs
Ventricular septal defect (VSD)-
Usually left to right
Smaller defect louder murmur
Right sternal border
Increased right side pressure
Quieter
Bi-directional
Right to left
Absent
Atrial septal defect (ASD)-
Murmur not directly related to ASD
Only large defects
Increased blood flow
Relative pulmonic stenosis
Left heart base
Diastolic murmurs
Aortic regurgitation-
Heart base
Quiet
Endocarditis/ congenital
Early closure of MV – Austin flint murmur - severe
Pulmonic regurgitation-
Uncommon
Left base
Pulmonary hypertension
Mitral stenosis-
Rare
Left apex
Mid-diastole
Continuous (machinery) murmer
Patent ductus arteriosus (PDA)
Loudest heart base
Radiate to thoracic inlet
MISS THEM if only auscultate apical region
Other causes very rare
Differentials for heart disease
Depends on clinical sign/ physical examination finding
Respiratory disease – cyanosis, dyspnoea/ tachypnoea, muffled heart sounds, crackles, dull percussion, cough, syncope
Neoplasia – ascites, muffled heart sounds, dyspnoea/tachypnoea, dull percussion, weakness
Hypoproteinaemia – ascites, muffled heart sounds
Neurological disease – paresis, paralysis, weakness, syncope
Metabolic disease – syncope, weakness, tachypnoea
when should you be concerned with heart murmers in dogs
Symptomatic – tachypnoeic/ dyspnoeic, syncopal, exercise intolerant, cyanotic etc.
Pyrexic and new murmur
Small breed dog with grade 3/6 or louder left apical murmur (MMVD)
Breed predisposed to DCM with an apical murmur (remember Cocker spaniels too)
Intended for breeding/ athletic activity
Grade 3/6 or louder murmurs over heart base
ANY diastolic murmur
ANY continuous murmur
when should you be concerned with heart murmers in cats
Symptomatic – tachypnoeic/ dyspnoeic, syncopal, exercise intolerant, cyanotic etc.
Pyrexic and new murmur
Breeds predisposed to HCM e.g. Sphynx, Ragdoll, Toyger, Persian, Maine Coon, Bengal etc.
ANY diastolic murmur (too fast to tell?)
ANY continuous murmur
Arrhythmia
Gallop sounds
Murmur intensity less useful in cats!
tests for heart murmer
Blood pressure
Blood tests-
CBC, biochem, electrolytes
NT-proBNP- released form heart when it streaches
Cardiac troponin I- use for hf hd and arhythmias, released when cardiomyocyes break down
ECG-
(Holter monitor)
(Loop recorder)
Thoracic imaging-
Radiography
Echocardiography
(CT)
When you’re suspicious of heart disease…..
Gold standard approach:
Bloods (NT-proBNP, cardiac troponin I in certain situations)
Blood pressure
ECG
Echocardiography (in-house vs referral)
Thoracic radiography?
If financial concerns:
ECG if arrhythmia
Echocardiography (likely in-house)
Just NT-proBNP?
Just thoracic radiography?
Pre-analytical error checklist
Is the sample haemolysed/lipaemic/icteric?
Has my sample been taken/handled properly?-
Clotted?
Artefacts can occur if not stored properly
Serum should be separated/spun soon after collection
Too much or too little sample?
Contamination by anti-coagulant
Delay
Pre-analytical error – the right test?
Antigen versus antibody
Most useful test?- FNA in canine mammary tumours vs FNA in canine diffuse large B-cell lymphoma in a lymph node
More than one test:
e.g
Cushing’s:
CBC and biochem
Urine cortisol:creatinine ratio
Basal cortisol
ACTH-stim test
Low-dose dex suppression test
High-dose dex suppression tes
Imaging
Oedema
Changes in the distribution of fluid between the plasma and interstitium are most commonly manifested as oedema, which is defined as accumulation of excess interstitial fluid.
Occurs by:
increased microvascular permeability
increased intravascular hydrostatic pressure
decreased intravascular osmotic pressure
decreased lymphatic drainage
Permeability of endothelium will increase as a physiological response to…
inflammation
Inflammatory mediators will result in the release of cytokines:
interleukin 1 (IL-1)
tumour necrosis factor (TNF)
interferon-γ
Resulting in endothelial cell retraction and widening of interendothelial gaps.
Microvasculature permeability
Increased intravascular hydrostatic pressure as a cause of odema
Increased flow or volume of blood
hyperaemia (active)
congestion (passive)
Decreased Intravascular Osmotic Pressure
as a cause of odema
Osmotic (colloidal) pressure- Maintained by plasma proteins, particularly albumin
Albumin can be decreased due to:
Reduced production- Severe liver disease, Protein starvation
Increased losses-
GI disease-protein losing enteropathy, parasitism
Renal disease- protein losing nephropathy
Decreased lymphatic drainage asa cause of odema
Lymphatic obstruction or compression
Congenital lymphatic aplasia or hypoplasia
Intestinal lymphangiectasia
Lymphangitis- jhones disease
Effusions
Same mechanisms as oedema but into a larger space or cavity
Tap it!
Blood, bile, urine or GIT content?
Transudate, modified transudate or exudate
Cytology
Transudate
extravascular fluid with low protein content (Total protein (g/dL)- <2.5) and a low specific gravity (< 1.012)
It has low nucleated cell counts (Nucleated cells (x103/ul)- <1.5)
Transudates develop most commonly as a result of decreased plasma osmotic pressure.
can be caused by Hypoalbuminaemia.
Modified transudate
an effusion that occurs by transudative mechanisms where vascular fluids leak out of “normal” or “noninflamed” vessels (e.g. via increased capillary hydrostatic pressure or lymphatic obstruction).
Total protein (g/dL)- >2.5
Nucleated cells (x103/ul)- <5
Develops in response to increased hydrostatic pressure or permeability of capillaries and/or lymphatic vessels.
Causes- Cardiac disease, neoplasms, hepatic disease, and feline infectious peritonitis.
Exudate
fluid that leaks out of blood vessels into nearby tissues. The fluid is made of cells, proteins, and solid materials. Exudate may ooze from cuts or from areas of infection or inflammation. It is also called pus
Total protein (g/dL)- >4
Nucleated cells (x103/ul)- >5
Develop because of increased vascular permeability caused by inflammation.
Non-septic exudates are caused by irritants such as bile, urine, and pancreatic enzymes or because of tissue necrosis. Neutrophils in such effusions are nondegenerate.
Septic exudates are caused by microorganisms which may or may not be evident cytologically.
Rivalta test
a test for FIP using effusion
To perform this test, a transparent test tube (volume 10 mL) is filled with approximately 7–8 mL of distilled water to which any one of the following is added:
one drop (20–30 μL) of acetic acid (98–100%)
OR 2-3 drops 25% white vinegar
OR 10 drops of 5% white vinegar
and mixed thoroughly.
On the surface of this solution, 1 drop of the effusion fluid is carefully layered. If the drop disappears and the solution remains clear, the Rivalta’s test is defined as negative. If the drop retains its shape, stays attached to the surface or slowly floats down to the bottom of the tube (drop- or jelly-fish-like), the Rivalta’s test is defined as positive.
Vasculopathy
vascular abnormalities caused by degenerative, metabolic and inflammatory conditions, embolic diseases, coagulative disorders, and functional disorders
Vasculitis
inflammation of the blood vessels/vasculature
Arteritis
inflammation of arteries
Phlebitis
inflammation of veins
Lymphangitis
inflammation of lymphatics
Arteriosclerosis
narrowing of vessels
Atherosclerosis
narrowing of vessels due to deposition of fatty material in vessel walls
Different types of shock
Hypovolaemic
Cardiogenic
Distributive
Hypoxic
(Obstructive)- rare, and falling out of fashion
what does shock mean
HYPOPERFUSION
AND/OR
CELLULAR ANAEROBIC CONDITIONS resulting in HYPERLACTERAEMIA
Hypoxic shock
results from Impaired oxygen delivery to the cells
e.g
Anaemia
Decreased haemoglobin saturation – carbon monoxide. carbon monoxide has greater affinity for haemoglobin than oxogen so takes up the haemoglobin instead of the oxygen
Respiratory disease
Hypovolaemic shock
most common form of shock
fluid lost form intravascular space
comon caues-
acute blood loss
sever dehydration
third spacing- fluid comes form intravascular space into the intrasticial space due to loss of protien and oncotic pressure- e.g protien loosing enteropathy, parvo
Cardiogenic Shock
heart doesnt function properly- weak contractions
could be caused by damage to the myocardium- dilated cardiomyopathy
pulses may not match the heart rate
Distributive Shock
inflamitory markers cuase vessels to become less receptive to constrictive elements and then reamian dilated in situations in which they’d normally constrict
Systemic inflammatory responses (SIRs)-
Heat stroke
Sepsis
may cause cold extremities- freezing mucus membranes
shock symproms
cold extremities
cold mucus membranes
Increased lactate
Metabolic acidosis
Mucous membrane colour change- pale exept for distributive shock
Weak pulses
Increased heart rate (Dogs) or decreased heart rate (cats)
obstructive shock
could be caused by GDV
ondition that prevents blood and oxygen from getting to your organs
types of shcok a GDV can cause
Hypovolaemic shock- third spacing
Cardiogenic shock
Obstructive shock
Distributive
How can we use lactate as a prognostic indicator?
lactate faling with treatment = good prognosis
Haematopoiesis
the formation of:
Erythrocytes
Leukocytes
Platelets
Typically occurs in the bone marrow but extramedullary haematopoiesis also occurs in the spleen and liver
The thymus is responsible for T-lymphocyte maturation- Compare the Bursa in birds
Lymph nodes:
Not present in birds
Filter lymph for antigen presentation and response
Clinical signs associated with disorders of the haemolymphoid system:
Enlarged lymph nodes
Anaemia
Coagulopathies
Oedema
lymphomegaly
Enlarged lymph nodes
Causes of lymphomegaly:
Reactive- Reactive lymphoid hyperplasia. reacting to infection in other areas. the lymphnode affected depends on the sites that drain into it
Inflammation/infection- Lymphadenitis
Neoplastic
Diagnostic tools:
FNA- Avoid those lymphnodes draining the mouth
Biopsy
Questions for the pathologist:
One cell predominates or mixed?
Which cells are there more of?
Regular or irregular?
Maintenance of tissue architecture?
Necrosis?
Lymphoid atrophy
Congenital immunodeficiencies
Viral infections
Cachexia/malnutrition
Aging
Radiation and chemotherapy
Toxins
Bone marrow histology
Hematopoietic tissue is highly proliferative.
Pluripotent hematopoietic stem cells (HSCs) are a self-renewing population, giving rise to cells with committed differentiation programs, and are common ancestors of all blood cells.
Control of haematopoiesis is complex
The dominant regulator of erythropoiesis is erythropoietin (Epo) produced by the kidney
Iron is essential to haemoglobin formation and function
Typically the bone marrow only releases mature cells, however in times of increased need, immature cells will be released into the blood stream
Hence looking for polychromasia/reticulocytes in cases of anaemia to assess for regeneration
And band neutrophils/left shift in inflammation/infection
Infectious agents of circulating cells
Others are essentially non-pathogenic under most circumstances
Do not confuse with artefacts/mimics – Heinz bodies, etc
Anaplasma-
A. phagocytophilum – Tick-Borne Fever in ruminants
Leukocytes, erythrocytes, platelets
Babesia-
In cattle in UK – Babesia bovis
Erythrocytes
Theileria-
Not in UK – East Coast Fever in cattle
Reproduction by schizogony in lymphocytes (tissue/leukocytic phase) then fission in erythrocytes
Ehrlichia-
Not in UK?
Leukocytes
Mycoplasma-
Cats
Formerly Haemobartonella
Erythrocytes
The hemolytic anemia caused by M. haemofelis is called Feline Infectious Anemia (FIA)
Cytauxzoon-
Not UK
Cats
Ertyrhocytes plus macrophages in tissues
Birds-
Hemoproteus, Leukocytozoon, Plasmodium
Infectious agents within macrophages in tissues
Leishmania-
Protozoa
Spread by sandfly
Amastigote stage found in macrophages
Granulomatous dermatitis in cutaneous form
Granulomatous splenitis, lymphadenitis and hepatitis in visceral form
Histoplasmosis-
Fungus
Soil-borne
Typically seen in the lungs (ddx: Blastomycosis)
Will spread to lymph nodes
Acute lymphadenitis
Typically due to pyogenic bacteria- Acute suppurative lymphadenitis
Streptococcus equissp.equi (Strangles)
Rhodococcus equi
Trueperella pyogenes
Chronic lymphadenitis
Typically due to intracellular and/or persistent bacteria
Chronic (pyo)granulomatous lymphadenitis
Mycobacterium bovis
Mycobacterium aviumsubspeciesparatuberculosis- Johne’s
Corynebacterium pseudotuberculosis
Also parasites and fungi – see previous slide
Canine sterile granulomatous lymphadenitis
Puppy strangles
Infection and inflammation of the spleen
Primary isolated splenitis is generally uncommon in dogs and cats-
Often secondary to another disease process, such as peritonitis or neoplasia
Or in cattle may see granulomas caused by Mycobacterium bovis in addition to other organs
Leishmania and Hepatozoon (not in UK) should be consider as primary causes of splenitis in dogs however often other organs affected
In cats consider Tularaemia - Francisella tularensis
Zoonotic and potential to be weaponised
Not in UK
Lymphadenitis also
Severe lymphoid depletion in the spleen and thymus is a feature of numerous viral diseases, including rinderpest, canine distemper, equine herpes virus, canine parvoviral enteritis, feline panleukopenia, and bovine viral diarrhoea
Necrotizing lymphadenitis, splenitis, and hepatitis in laboratory animals due to various infectious agents:
Tyzzer’s disease (Clostridium piliformis):
Mice, gerbils, and other laboratory and wild rodents
Multifocal foci of hepatic necrosis (older mice), catarrhal enterocolitis with mesenteric lymph node abscesses (younger mice); myocardial lesions occasionally (especially gerbils)
Yersiniosis (Yersinia pseudotuberculosis):
Cats, Guinea pigs, rabbits, rats, other rodents
Acute: Enteritis (ileum) with mucosal ulceration
Subacute to chronic: Discrete miliary to caseous lesions in lymph nodes, spleen, liver and lungs
Bacteria are easy to visualize, in contrast to F. tularensis
Salmonellosis (Salmonella sp.):
Guinea pigs, mice, rats, other rodents
Acutely–focal areas of necrosis in liver, spleen, and lymphoid tissue and intestine
Bubonic plague (Yersinia pestis):
Rats, squirrels, and other rodents
Viral causes of immunodeficiency
Parvoviruses-
Replication depends on host-cell DNA polymerases produced in the S phase of the cell cycle. Therefore, has a predilection for cells that are rapidly dividing- Foetal tissues – heart (puppies), cerebellum (kittens), haematopoietic and lymphoid tissues, intestinal crypt epithelium
Bovine Viral Diarrhoea Virus (BVDV)-
Subclinical infection usually by non-cytopathic BVD followed by seroconversion is the most common form- Transient leukopenia may be seen with onset of signs of disease.
Infection of the dam- 50-100 days gestation: Foetal death, abortion, mummification
100-150 days gestation: Congenital brain defects, cataracts microphthalmia
Persistent infection is an important sequela of foetal infection with noncytopathic BVDV occurring with infection prior to 125 days gestation- Persistently infected calves may appear healthy and normal in size, or may be stunted and prone to respiratory or enteric infections. Viraemic but do not seroconvert
Superinfection of the PI calf with cytopathic form = mucosal disease- Fever, leukopenia, dysenteric diarrhoea, inappetence, dehydration, ulcers of the nares and mouth. Death within a few days of onset.
PM -> Erosions and ulcerations throughout the GI tract., mucosa over Peyer’s patches haemorrhagic and necrotic, extensive necrosis of lymphoid tissues
Feline immunodeficiency virus (FIV)
Three phases of infection - the acute phase, the asymptomatic (or latent) phase, and the progressive phase.
Acute phase-
generally occurs 1-3 months after infection
Replicates in T-cells of within lymph nodes
Spreads to other lymph nodes, resulting intransient lymphomegaly and fever
Asymptomatic phase-
Months to years.
Replicates very slowly
Cyclical lymphopaenia and/or increased globulins
Some cats will remain in this stage and never progress to more severe disease.
NEVER euthanise a cat on a snap test alone
FIV (Feline Immunodeficiency Virus) (cats.org.uk)
Progressive phase-
Progressive immuno-compromised state
Secondary infections may occur – gingivitis very common
Ongogenic (see next slide)
Snap test tests for the antibody
Feline leukaemia virus (FeLV)-
Subgroups A,B,C & T; A, B, and C are most important
FeLV-A is the only one that is contagious, found in almost 100% infected cats and is predominately immunosuppressive; FeLV-B and C results from recombination or mutation
FeLV-B is found in ~50% of infected cats; commonly associated with neoplastic transformation and lymphoma (see next slide)
FeLV-C arises from a mutation of the envelope gene of FeLV-A; it is present in about 1% of infections and is associated with anaemia following bone marrow infection (non-regenerative)
Snap test tests for the antigen
Oncogenic viruses of the lymphoid system
Retroviruses
Insert copies of their RNA into host DNA
Bovine leukaemia virus - Notifiable
Avian leukosis
Lentiviruses-
FIV
FeLV
Maedi-Visna
FIV- The risk for developing lymphoma in FIV-infected cats is fivefold to sixfold higher than in uninfected cats
Intestinal B-cell lymphoma
FeLV- Lymphoma is increased 60-fold in infected cats
Commonly results in T-cell mediastinal/thymic lymphoma
Vaccination has markedly reduced this
diffusely enlarged spleen causes
Congested/bloody-
Torsion
Barbiturate euthanasia
Acute haemolytic crisis
African swine fever
Septicaemia- Salmonella, Anthrax
Non-congested/firm/meaty
Neoplasia- Lymphoma, Mast cell tumour (cats)
Chronic immune-mediated haemolytic anaemia
Chronic infection- Mycoplasma, etc
Chronic inflammation
Anthrax
Anthrax is a cause of outbreaks of sudden death, particularly in ruminants, however can affect other grazing animals and humans.
Clinical signs in animals that do not die suddenly include high fever, tachycardia and tachypnoea, and congested and terminally cyanotic mucosae with haemorrhage. Animals that survive longer than a day may have dysentery, abortion, oedematous swellings of the perineum, throat and abdominal wall, and blood-stained milk.
The characteristic sign in pigs is swelling of the pharyngeal region and neck.
Anthrax is notifiable and zoonotic, so if you suspect it you must call APHA
Due to the high fever, animals decompose rapidly resulting in typical saw-horse bloated appearance
DO NOT open up the animal, it is spread by spores which can become aerosolised
If the animal has been accidentally opened up, immediate gross post mortem findings include blood filled cavities and marked splenomegaly
Diagnosed in field by taking a blood smear from the ear
Rod-shaped bacteria with capsule
African Swine Fever
Forms and clinical signs-
Peracute, acute, subacute, and chronic forms
High fever
Cutaneous erythema in all forms
Dyspnoea, vomiting
Chronic form – abortion, lameness, pneumonia, wasting
Pathology-
Enlarged friable spleen
Haemorrhages in lymph nodes, particularly gastrohepatic
Petechial haemorrhage in kidneys and peri-renal oedema
Tri-cavity effusions
Marked pulmonary oedema
Gall bladder oedema
Classical swine fever
Forms and clinical signs-
Peracute, acute, subacute, and chronic forms
High fever
Generalised vasculitis results in haemorrhages, infarction and cyanosis
Infarction of ear tips will result in necrosis
Chronic form – marked weight loss, poor reproductive performance, abortion or weak tremoring piglets
Pathology-
Splenic infarctions
Haemorrhages in lymph nodes and necrosis of tonsils
Chronic - mucosal ulcers at ileocaecal junction, lymphoid atrophy in lymph nodes and thymus
Splenic masses
Discrete single or multiple splenic masses are common in the aged dog
Bloody versus firm
Irrespective of the diagnosis, they will commonly rupture resulting in a haemoabdomen
Rarer in cats, where mast cell tumours are the most common splenic tumour and tend to cause diffuse enlargement
Benign- Nodular hyperplasia
Haemangioma
Haematoma
Indolent- Marginal zone lymphoma
Malignant- Haemangiosarcoma
Histiocytic sarcoma
petechia
is a pinpoint (1 to 2 mm) haemorrhage that occurs mainly because of diapedesis associated with minor vascular damage
ecchymosis
larger than petechia
up to 2 to 3 cm in diameter haemorrhage that occurs with more extensive vascular damage
Suffusive haemorrhage
affects larger contiguous areas of tissue than petechia and ecchymosis
haematoma
Haemorrhage that occurs in a focal, confined space forms a haematoma, most common in the ears of long-eared dogs or pigs and in the spleen after trauma to the vasculature
Haemoperitoneum
blood in the peritoneal cavity
Haemothorax
blood in the thoracic cavity
Haemopericardium
blood in the pericardial sac
Aneurysm
bulge in an artery due to defect in the wall
Arterial aneurysms
Arterial aneurysms and their subsequent ruptures are rare in animals
Rupture of the aorta, pulmonary artery and coronary artery has been reported in swine and turkeys fed a diet deficient in copper
Dissecting aneurysms in the coronary and renal arteries in young male racing greyhounds, probably due to arteriosclerosis and haemodynamic stress
In horses, aortic sinus of Valsalva aneurysms or tears in the aortic root are well‐recognised conditions in breeding stallions, often leading to sudden death.
Developmental collagen disorders, such as the Ehlers-Danlos and Marfan syndrome
Smaller haemorrhages in vitamin C deficiency (scurvy) in guinea pigs and primates
Minor endothelial injury
->small numbers of erythrocytes escape by diapedesis
caused by:
Endotoxemia- toxins produced by baceria in the gut
Infectious agents- canine adenovirus-1, adeno viruses are endothelialtrophic
Chemicals- uremic toxins
Immune complexes can become entrapped between endothelial cells and activate complement and neutrophil influx to result in damage to the endothelium and vessel wall-
Feline infectious peritonitis- why cats get swollen belly. damaged endothelium causes leakage
Recap - see glomerulonephritis section in kidney biopsy lecture
the 4 phases of Haemostasis
haemostasis- stopping bleeding
Arteriolar vasoconstriction-
Transient
Mediated by reflex neurogenic mechanisms
Primary haemostasis-
Formation of platelet plug
Secondary haemostasis-
Clotting factors and production of fibrin
Thrombus and antithrombotic events (resolution)
Primary haemostasis
Adhesion-
vWF (von willebrand factor) is released by local activated endothelium and coats the exposed collagen
vWF acts as a bridge between platelet surface receptor and collagen
Activation-
Caused by thrombin and ADP
Results in shape change and release of thromboxane- go from round to flattened to bridge gap
further release of prothrombotic agents
Aggregation-
Glycoprotein IIb/IIIa allows binding of fibrinogen
Fibrin (formed by 2ry haemostasis) stabilises
chediak-higashi syndrome
assosiated with chnge in coat colour
aleutian mink, cattle, persian cata
Congenital disorders of 1ry haemostasis
defective storage of ADP
what can negativly effect platlet function
NSAIDs
Aspirin inhibits cox
Resulting in less thromboxane and thus no platelet aggregation
Uraemia
Idiopathic immune-mediated thrombocytopaenia- imunne system destroys platlets
Buccal Mucosal Bleeding Time
The buccal mucosal bleeding time (BMBT) is a test of platelet function. It is indicated in patients suspected of having a primary hemostatic defect despite adequate platelet numbers. Its use and accuracy for the assessment of platelet function are controversial, but it is still widely used in clinical practice for lack of a more accurate or specific test.
Method:For a BMBT, the patient is restrained in lateral recumbency and may require light sedation. A strip of gauze is tied around the maxilla to fold up the upper lip sufficiently to cause moderate mucosal engorgement. It is important not to place the gauze too tightly so that blood vessels are not constricted. The BMBT should be performed with a commercial spring-loaded device that creates a small incision of uniform width and depth into the inside of the upper lip. An area devoid of visible blood vessels should be chosen. Any shed blood must be carefully blotted with filter paper, with extreme care taken not to blot the incision itself; doing so may disturb the fragile primary platelet plug (Figure 3). The time taken from incision to cessation of bleeding by first clot formation is recorded (normal = 2–4 minutes in dogs, 1–2.5 minutes in cats).
Secondary haemostasis
Coagulation factors are-
Plasma proteins, Produced mainly by the liver.
Subdivided into :
Intrinsic pathway-
factors XI and XII
Extrinsic pathway-
VII
both lead into common pathway
Common pathway
Starts with X
Results in formation of thrombin which cleaves fibrinogen to fibrin
Vitamin K– dependent factors
Span all three parts
II, VII, IX, and X
Congenital disorders of 2ry haemostasis
Factor IX deficiency- Haemophilia B: dogs, cats and rarely horses. generally mild in cats and small dogs. more severe in large dogs
Factor VIII deficiency- Hemophilia A:
dogs, horses, cattle sheep , cats. vleeding can be severe in large dogs and horses
Liver disease
VitK deficiency-
factors II, VII, IX, X
warfarin containing/ like rodenticides
Testing 2ry haemostasis
Prothrombin Time-
Purpose. The prothrombin time (PT), also known as one-stage PT, is the principal test of the extrinsic pathway, but it also tests the common pathway. Because of the short half-life of factor VII, PT is a very sensitive test for early vitamin K deficiency or antagonism (eg, anticoagulant rodenticides). This test result is not affected by the presence of primary hemostatic disorders.
Activated Partial Thromboplastin Time-
Purpose. The activated partial thromboplastin time (aPTT) tests the intrinsic and common pathways. It is typically prolonged in patients with heritable factor deficiencies (eg, hemophilia A) or hepatic synthetic dysfunction, or in patients with disseminated intravascular coagulation that have consumed clotting factors.
Prothrombin Time
Purpose. The prothrombin time (PT), also known as one-stage PT, is the principal test of the extrinsic pathway, but it also tests the common pathway. Because of the short half-life of factor VII, PT is a very sensitive test for early vitamin K deficiency or antagonism (eg, anticoagulant rodenticides). This test result is not affected by the presence of primary hemostatic disorders.
Activated Partial Thromboplastin Time
Purpose. The activated partial thromboplastin time (aPTT) tests the intrinsic and common pathways. It is typically prolonged in patients with heritable factor deficiencies (eg, hemophilia A) or hepatic synthetic dysfunction, or in patients with disseminated intravascular coagulation that have consumed clotting factors.
thrombus
A thrombus forms antemortem within a vessel
thromboembolism
a section of a thrombus that has broken off and lodged elsewhere
antemortem clot
An antemortem clot is a coagulum of blood and/or plasma that forms when bleeding into a cavity
Post mortem clots
are found within vessels and the heart and are a product of blood stasis and clotting post mortem
virchows triad
need one of these three things to form a thrombus
Abnormal blood flow-
Valvular disease
Heart disease
Shunts
Aneurysms
Hypovolaemia
Torsions
Hypercoagulability-
Glomerular disease- Loss of antithrombin 3
Metabolic disease
Inflammation
Platelet activation- Neoplasia
Endothelial injury-
Infectious diseases
Free-radicals - vit e/selenium deficiency
Toxins
Trauma
severe cases will have all three
Thrombosis factor – abnormal flow
Local stasis or reduced flow (e.g., gastric dilation and volvulus, intestinal torsion and volvulus, varicocoele, external compression of vessel)
Cardiac disease (e.g., cardiomyopathy, cardiac hypertrophy)
Aneurysm (e.g., copper deficiency in pigs, Strongylus vulgaris)
Hypovolemia (e.g., shock, diarrhoea, and burns)
Normal blood flow is laminar such that the platelets (and other blood cellular elements) flow centrally in the vessel lumen, separated from endothelium by a slower moving layer of plasma.
Thrombosis factors– hypercoagulability
Anti-thrombin 3-
an important anti-coagulant
it is a small molecule thus one of the first to be lost with glomerulonephritis
Aortic trifurcation thrombosis is an important but relatively uncommon sequel of hyperadrenocorticism in dogs-
Pathogenesis not completely understood:
Thrombocytosis
Decreased antithrombin concentrations secondary to glomerular changes and proteinuria
Decreased fibrinolysis
Relative increases in the concentrations of circulating procoagulant molecules, such as factor VIII and fibrinogen
In humans, 80% of patients with diabetes mellitus die a thrombotic death- The plasma levels of many clotting factors including fibrinogen, factor VII, factor VIII, factor XI, factor XII, kallikrein, and von Willebrand factor are elevated in diabetes
DM a potential sequel of HAC
Thrombosis factors – the endothelial cell
Roles of the endothelial cell-
Solute/metabolite diffusion/transport
Local vasoconstriction and vasodilation
Coagulation
Inflammation
Wound healing and angiogenesis
In normal circumstances the endothelial cells are slightly anti-coagulant, but become pro-coagulant when needed
vonWillebrand’s factor plays a crucial role in primary coagulation-
Synthesized in endothelial cells (and megakaryocytes)
Stored in Weibel-Palade bodies
vWF enables adhesion of platelets to subendothelial extracellular matrix
Thrombosis factors – Disseminated Intravascular Coagulation
Diffuse or severe endothelial damage and/or platelet activation with or without severe inflammation can result in disseminated intravascular coagulation (DIC)
DIC results in widespread consumption of clotting factors and platelets = consumptive coagulopathy
The outcome is microthrombi within small vessels which can contribute to multi organ failure (MOD) and widespread haemorrhage
Thrombosis – morphology
Thrombi can develop anywhere within the CVS
Arterial thrombi
Venous thrombi-
Slower flowing blood so will typically completely occlude vessel and take the form of the vessel
Post mortem clotting-
Not adherent to the wall of the vessel
Separates into cells and plasma = chicken fat clot
Thrombosis – infarction
An infarct is a local area of very acute (peracute) ischemia (complete lack of oxygen) that undergoes coagulative necrosis.
Infarction is caused by the same events that result in ischemia and is most common secondary to thrombosis or thromboembolism.
The characteristics of an infarct are variable based on the type and size of vessel that was occluded (artery or vein), the duration of the occlusion, the tissue in which it occurs, and the prior perfusion and vitality of the tissue.
Disorders of Primary Haemostasis Clinical presentation
Epistaxis- nose bleed
Spontaneous haemorrhage
Gastrointestinal bleeding
Prolonged bleeding after surgery
Excessive menstruation in intact females
Ecchymosis, petechiae, bruising
Primary Haemostasis Investigation
Buccal mucosal bleeding time (BMBT)
Mucosal membrane of the lip
Standardised cut made in lip, e.g.: 5 x 1 mm
End point = bleeding stops & crescent of blood no longer forms on filter paper
Normal <4min (dogs) <3mins (cats)
Prolonged BMBT causes
Thrombocytopenia-
Consumption, haemorrhage
Immune mediated thrombocytopaenia
Disseminated intravascular haemolysis (DIC)
Decreased platelet function-
Rare inherited disorders
Platelet function inhibited by uraemia of renal failure
Therapy, e.g.: Aspirin
Von Willebrand’s Disease-
Lack of or reduced amounts of Von Willebrand’s Factor
Dobermans
Platelet concentration determined by
haematology analyser
Units: # x10^9/L
Counted in the same channel as erythrocytes
However platelets are smaller
pseudo thrombocytopenia
Platelet clumps-
Clotting during sampling
In vivo activation
Platelets counted as RBCs (and vice versa)-
Dogs:
Akitas: Small red blood cells
CKCS: Macroplatelets
Cats, Goats:
Small red blood cells
Platelets - Manual Assessment
Thrombocytopenia?
→ ALWAYS CHECK A BLOOD SMEAR!
Check for platelet clumps (feathered edge)
Manual count
Body of the smear
count how many platets you can see in each hightly magnified field of view
Average of 10 hpf x 15
count how many platets you can see in each hightly magnified field of view
over 150-200 is good
Check for Macroplatelets
May also effect [RBC]
lower count of macroplatlets ok
Immune Mediated Thrombocytopenia (IMTP
Thrombocytopenia (<10 x10^9/L, normal: >150)-
Antibodies bind directly or indirectly to platelets
Platelets are destroyed / phagocytosed by macrophages
Primary-
Idiopathic
Most common in dogs
IMHA + IMTP = Evan’s Syndrome
Secondary-
Drugs (e.g.: Sulphonamides in dogs, Methimazole in cats)
Infections (e.g.: Ehrlichia, Leishmania)
Neoplasia
Diagnosis-
Spontaneous haemorrhage
Thrombocytopenia <10 x10^9/L
Confirmed on blood film examination
Diagnosis of exclusion
Therapy-
Immunosuppression: Steroids, Azothioprine with supportive care
Disseminated intravascular coagulation
DIC-
Widespread uncontrolled activation of coagulation cascade
Platelets are consumed → thrombocytopenia
Clots are dissolved → Excessive fibrinolysis = ↑ D-Dimers
must have all 3
FDPs- no comercially availabe
Causes-
Envenomation
Systemic inflammation / infection (e.g.: Septicaemia, vasculitis)
Massive necrosis
Heat stroke
D-Dimers
Measured on citrated plasma
Form when cross linked fibrin is cleaved by plasmin
Used to assess ↑ fibrinolysis associated with coagulation
Clots are dissolved → excessive fibrinolysis = ↑ D-Dimers
Causes for high level
Localised or disseminated intravascular coagulation (DIC)
Sepsis, systemic inflammation, haemorrhage, neoplasia, surgery, immune-mediated disease
Decreased hepatic or renal clearance
Angiostrongylus vasorum
Angiostrongylus vasorum = Lungworm
Becoming more common in the UK
Infected by ingestion of slugs and snail (intermediate host)
Clinical presentation
Coughing / Difficulty breathing
Inappetent
Vomiting or diarrhoea
Weight loss
Depression / Lethargy
Bleeding: Cutaneous bruising and intra-cavitatory haemorrhage
Bleeding pathogenesis: Excessive intravascular coagulation
Thrombocytopenia (consumption)
Consumption of coagulation factors
Diagnosis
Broncho-alveolar Lavage (BAL) & Cytology
Faecal analysis: Baermann technique
PCR: Faeces, blood, BAL
ELISA test: Detects antigen
Therapy
Moxidectin & Imidacloprid, Milbemycin oxime & Praziquantel
Von Willebrand Disease
Von Willebrand factor (vWF)
Exposed when vessel wall is damaged
Binds platelets to endothelial wall
Von Willebrand Disease
Most common hereditary bleeding disease in dogs
Doberman most common breed
Different types = Reduced concentration or complete absence of vWF
Presentation-
Mucosal haemorrhage: Epistaxis, GI haemorrhage, prolonged oestrus
Prolonged bleeding after surgery
Cutaneous bruising
Diagnosis-
Prolonged buccal mucosal bleeding time
Measurement of vWF: <50% = Decreased
Genetic test for vWD type I (most common)
APTT and PT
Coagulation times’ = APTT and PT-
Stimulate the coagulation pathway in vivo
The it takes until a clot is detected
Activated partial thromboplastin time = APTT
Assesses intrinsic + common pathways
Prothrombin time = PT
Assesses extrinsic + common pathways
Differentials for prolonged APTT
Intrinsic + Common factors
XII, XI, IX, VIII, X, V, II, I
Hereditary-
Haemophilia A = deficiency in factor VIII
Haemophilia B = deficiency in factor IX
Factor XII deficiency = most common in cats, no clinical haemorrhage
Acquired-
Hepatic disease
Vitamin K malabsorption
Vitamin K antagonism (rodenticide toxicity)
DIC or local consumption of coagulation factors
Differentials for prolonged PT
Extrinsic + Common factors
VII, X, V, II, I
Hereditary
Factor VII - prolonged PT, normal aPTT
Affected dogs generally do not have a history of bleeding
Bruising / prolonged bleeding following surgery
Acquired
Hepatic disease
Vitamin K malabsorption / antagonism (rodenticide toxicity), this goes up first in this case
DIC or local consumption of coagulation factors
Vitamin K Deficiency / Antagonism
Vitamin K dependent factors-
Factors II, VII, IX & X
These factors require Vitamin K to be functional
Malabsorption of Vitamin K-
Gastrointestinal disease (e.g. inflammatory bowel disease)
Vitamin K antagonism-
Rodenticide toxicity (e.g. Warfarin) - dogs
Mouldy sweet clover - ruminants, horses, pigs
Rodenticide Toxicity
Very common in dogs
Clotting factors II, VII, IX and X affected
Diagnosis
Visible clinical signs can take 5-7 days to appear after ingestion
Monitor clotting times on a daily basis for at least 3 days
Prothrombin time (PT) will become prolonged first
Therapy
Vitamin K1 supplementation
Severe cases → plasma / blood transfusions
Replaces coagulation factors to allow rapid cessation of haemorrhage
Haemophilia A = Factor VIII
Diagnosis
<1% factor VIII activity: reference interval: 50-200%
Prolonged APTT only (not PT)
All animals
Severe in larger dogs, horses & japanese brown cattle
X-linked recessive mutation in Factor VIII gene
Males are either affected or not (never carriers)
Females can be free of the defect, carriers (heterozygous) or rarely affected (homozygous).
Haemophilia B = Factor IX
Diagnosis
<1% factor VIII activity: reference interval: 50-200%
Prolonged APTT only (not PT)
Dogs & cats
Mild in small dogs, severe in large dogs
X-linked recessive mutation in Factor VIII gene
Males are either affected or not (never carriers)
Females can be free of the defect, carriers (heterozygous) or rarely affected (homozygous).
What is jaundice?
Excessive accumulation of the yellow/orange pigment (bilirubin) in the tissues and blood (hyperbilirubinaemia)
Bilirubin ismade during the normal process of breaking down red blood cells.
Most often seen in the shclera , mm skin
Unconjudated bilirubin
direct breakdown product of haem – in soluble and can only travel in blood stream attached to albumin and unable to excrete directly from the body
Uncojudated bilirubin is formed by the breakdown of hemoglobin in the red blood cells. The liver converts this bilirubin into direct bilirubin, which can then be released into the intestine by the gallbladder for elimination. Total bilirubin levels are therefore indicative of both the destruction of red blood cells and the proper functioning of the liver, gallbladder, and bile ducts.
This is bound to albumin and is the dominant form of total bilirubin in blood. It is produced in macrophages from breakdown of heme groups (specifically the porphyrin ring of heme). The biggest source of heme is hemoglobin within red blood cells (RBC)
Unconjugated bilirubin is then released into plasma where it binds to albumin. Uptake of unconjugated bilirubin occurs in the liver and is carrier-mediated and passive.
Once within the hepatocyte mitochondria, unconjugated bilirubin is transported with ligandin (Y protein or glutathione-S-transferase A) or other proteins (e.g. fatty acid binding protein) and the majority is conjugated to glucuronic acid by UDP-glucuronyl transferase (from the UGT1A1 gene).
Under physiologic conditions, most of the unconjugated bilirubin in blood comes from normal RBC turnover, where RBCs that have completed their normal lifespan (effete) are removed by macrophages in the spleen, liver and bone marrow. Small amounts of bilirubin also come from other heme-containing proteins, such as myoglobin. As RBCs age, they accumulate oxidative injury, which causes exposure of an antigen on RBCs, which is recognized by macrophage receptors, resulting in phagocytosis (extravascular hemolysis) and conversion of the porphyrin ring of hemoglobin into biliverdin and then unconjugated bilirubin (indirect bilirubin on chemistry panels).Macrophages export the unconjugated bilirubin into blood, where it binds to albumin as it is water insoluble. The albumin-bound unconjugated bilirubin is take up by transporters in the hepatocytes on the sinusoidal (blood or lumen) side of the membrane. The unconjugated bilirubin is transported internally within the hepatocyte by ligandin or Z protein and then conjugated to glucuronic acid (glucose in horses) to form conjugated bilirubin (direct bilirubin on chemistry panels), which is water soluble.
conjudated bilirubin
water soluble and can be excreted from the body
Conjugation renders bilirubin water soluble. Only very small amounts in blood because it is normally excreted into bile.
The conjugated bilirubin is then excreted into the biliary system by transporters on the canalicular or biliary side of the membrane (different from those that take up unconjugated bilirubin from blood) along with bile salts and is either stored in the gall bladder (for those species with a gall bladder) or excreted directly into the small intestine. Within the small intestine, the conjugated bilirubin is degraded by bacterial proteases to urobilinogen, around 10% of which undergoes enterohepatic recirculation after uptake by the portal vein. The “reclaimed” urobilinogen is then freely filtered by the glomerulus and is found in urine (there is a pad for urobilinogen on a urinary dipstick but it is not used in veterinary medicine as a diagnostic test). The rest of the urobilinogen is reduced to stercobilin (either directly or via stercobilinogen), which is excreted in the feces. Stercobilin imparts the brown color to normal feces.The excretion of conjugated bilirubin into bile is the rate-limiting step of the entire pathway of bilirubin metabolism and is ATP- or energy-dependent.
Pre hepatic icterus
Accelerated red blood cell destruction and increased bilirubin production
Immune mediated haemolytic anaemia (IMHA)
If this bilirubinemia overwhelms the liver’s functional capacity for uptake, conjugation, and secretion, bilirubin is refluxed from the liver into the circulation resulting in hyperbilirubinemia and icterus.2
Hepatic icterus
Hepatocellular disease-
Reduced hepatocyte uptake
Reduced Conjugation
Reduced secretion of bilirubin
intrahepatic cholestasis associated with hepatocellular injury, necrosis, or dysfunction
Hyperbilirubinemia that occurs in most diseases of the liver is a mixture of conjugated and unconjugated bilirubin in varying proportions – reflux back into the intravascular system
Viruses heptatp toxic drugs
Post hepatic icterus
Disruption of bile flow through the extrahepatic biliary system- gall stones, tumours ect
often show the most yellowing of the skin
diagnostics for prehepatic jaundice
icteric plasma
Saline agglutination
Coombs test
Blood smear
diagnostics for Hepatic icterus
can detect whether the problem is the liver by Liver disease is more common cause of incterus in non=haemytic patients
can detect whether the problem is the liver by:
The blood test
ALT
ALP
GGT
AST
however most systemic diseases will affect liver and therefore affact these so instead you can do :
BAST= shows liver function
Bile acid stimulation test for liver function
liver disease include viral or bacterial infections, ingestion of toxic plants or chemicals, certain drugs and medications, cancer, autoimmune diseases, and certain breed-specific liver diseases.
diagnostics for post hepatic icterus
Gall bladder obstruction
Extremely icteric
Obstruction of the common bile duct is associated with a number of diverse primary conditions, including inflammation (eg, pancreatitis, duodenitis, duodenal foreign body, etc), cholelithiasis, gallbladder mucocele, choledochitis/cholecystitis, neoplasia, bile duct malformations, parasitic infection, extrinsic compression, fibrosis, and bile duct stricture.
can see some of these on ultrasound
ALP and GGT increase more
gallbladder mucocele- looks like kiwi fruit on ultrasound, common in older border terriers
steps of acute inflammation
Fluidic- Vasodilation
Increased vascular permeability
Cellular- Leukocyte recruitment
Causes of inflammation
infections- Bacterial, viral, fungal, parasitic
Microbial toxins
Different pathogens elicit varied inflammatory response
Tissue necrosis - Elicits inflammation by releasing molecules from necrotic cells, regardless of the cause of cell death
Ischaemia, trauma, and physical and chemical injury
Foreign bodies - May elicit inflammation themselves or because they cause traumatic tissue injury or carry microbes
Immune reactions = hypersensitivity - Reactions in which the normal protective immune system damages the individual’s own tissues.
