pathology spot exams Flashcards
Epithelial neoplasms
Epithelial tumors are cohesive and form clusters or sheets. They can show trabecular, circular to papilliform arrangements. Acini may be seen in cells that produce secretory product. Examples of epithelial tumors include perianal gland adenoma, transitional cell carcinoma, biliary carcinoma, squamous cell carcinoma. Epithelial cells generally have the following features:
Large, round to polygonal cells Distinct cell borders Tightly adherent to each other Round to oval nuclei that can be basilar in columnar cells or eccentric in other cell shapes
Epithelial tumors can be benign (adenoma) or malignant (carcinoma). Benign versions consist of well-differentiated cells that can be difficult to distinguish from normal tissue (unless in excess for the aspirated site) or hyperplastic lesions (which may require evaluation of tissue architecture, e.g. normal architecture and arrangement around ducts would indicate hyperplasia versus neoplasia for skin adnexal tumors). Malignant epithelial cells usually demonstrate cytologic criteria of malignancy particularly as they become more aggressive or advanced. However, some carcinomas (e.g. the rare perianal carcinomas) do not always show features of malignancy but behave in a malignant fashion.
Mesenchymal neoplasms
Mesenchymal neoplasms carry features of their embryonic tissue of origin, the mesenchyme. The cells are generally individualized and spindled in shape. They can be seen in aggregates (not clusters), often held together by extracellular matrix. They do not typically demonstrate cell-to-cell adhesion. Due to increased matrix production, there are some mesenchymal tumors (e.g. fibroma) that do not exfoliate well and aspirates may be of low cellularity making a definitive cytologic diagnosis difficult. Examples include myxoma, fibrosarcoma, osteosarcoma, melanoma and hemangiosarcoma. Mesenchymal tumors generally have the following features:
Spindle, oval or stellate-shaped cells Indistinct cell borders, that taper into the background Round to oval to elongate nuclei that are usually centrally located Cells are scattered individually or in aggregates, usually within matrix. Less cellular than the other tumors due to matrix Matrix can be present in the background as well as within aggregates
As for epithelial tumors, mesenchymal tumors can be benign (“..oma”) or malignant (“sarcoma”). Some types of mesenchymal tumors, e.g. soft tissue sarcomas, are called sarcomas, even though they do not metastasize quickly. They are, however, locally invasive. There are also certain types of mesenchymal tumors that mimic epithelial tumors
Discrete (round) cell neoplasms
Discrete or round cell tumors often are of hematopoietic origin (lymphoma, histiocytic, mast cell tumor) and as the term suggests, consists of individualized round cells. Cells tend to exfoliate readily and aspirates are often of high cellularity. We can use morphologic features of the cells, including the presence or absence of granules and cytoplasmic and nuclear features, to determine the type of round cell tumor.
Mast cell tumor
Histiocytoma
Plasmacytoma
Lymphoma
Transmissible venereal tumor
Mast cell tumor
These are readily recognized by the presence of purple cytoplasmic granules.
They also have round eccentric nuclei with smooth chromatin. The nuclei can be hard to see as the granules soak up the stain
The degree of granularity varies between tumors. Granules may be harder to discern with water-based stains, such as Rapid stains, particularly in the less well-granulated tumors.
Low grade tumors are typically well-granulated. Higher grade tumors can be poorly or well-granulated and nuclear criteria of malignancy (nuclear atypia, binucleation, large nuclei, mitotic figures) are more reliable than granularity for determining the grade of mast cell tumors on cytology. Tumor grading for dermal (not subcutaneous) mast cell tumors in dogs is best done by histopathology.
Histiocytoma
Round to oval with variably distinct cytoplasmic borders.
Moderate to abundant amounts of clear to light blue cytoplasm
Nuclei are eccentric and round to oval to indented
Nuclei have finely stippled chromatin and nucleoli are not apparent
Cells are often found dispersed within a moderately blue background
Minimal cellular atypia, uniform cell size and morphology – they have a bland appearance
Regressing tumors are associated with increased numbers of small lymphocytes (tumor infiltrating cytotoxic T-cells)
Note: Histiocytomas generally consist of very bland, minimally atypical cells. If a high degree of cellular atypia (numerous criteria of malignancy) are found and a histiocytic lineage is still suspected, histiocytic sarcoma should be considered a differential diagnosis.
The main differential diagnosis is an extramedullary plasmacytoma. Lightly stippled chromatin, abundant light blue cytoplasm, indented nuclei and the blue background are used to distinguish between these lesions (not all features may be present in every tumor).
Plasmacytoma:
These arise from plasma cells, which form tumors (usually solitary) in extramedullary sites, such as the skin (digit, ears, mouth) in dogs.
Round to slightly oval cells Distinct cell borders Variable amounts of blue cytoplasm (often deep blue), some have perinuclear clear zones Nuclei are round, occasionally oval, and eccentric Nuclei have clumped chromatin and nucleoli are not apparent More atypia (anisocytosis and anisokaryosis) than histiocytic tumors Binucleation and, occasional, multinucleation is common. Multinucleated cells may show marked intracellular anisokaryosis Amyloid may be present in skin tumors. The main differential diagnoses are a histiocytoma or plasmacytoid variants of lymphoma. Compared to a histiocytoma, the cells have more distinct boundaries, darker cytoplasm, rounder nuclei (even in multinucleated cells) and clumpier chromatin. They may have perinuclear clear zones. With plasmacytoid variants of lymphoma, cells with higher nuclear to cytoplasmic ratios resembling lymphocytes are expected to be present.
Endocrine/neuroendocrine tumors
These tumors have a characteristic appearance, forming packets of cells. Cells often exfoliate in large numbers but are fragile and aspirates contain many bare nuclei from ruptured cells, hence some people call them “naked nuclei” neoplasms. They are of secretory epithelial (producing hormones, e.g. thyroid tumors) or neuroectodermal origin, with the latter secreting neurotransmitters, such as epinephrine in phaechromocytomas. Many of these tumors have quite uniform or bland cytologic features, but show aggressive malignant behavior (e.g. thyroid carcinomas in dogs), therefore cytologic criteria of malignancy are unreliable and we go by the known biologic behavior of the tumors. The type of endocrine or neuroendocrine tumor is generally determined by site, e.g. a cervical neck mass could be thyroid or parathyroid in origin, with the former being more common. In some types of tumors, we can be more definitive, for example thyroid follicular tumors can contain tyrosine granules (blue green pigment) in the cytoplasm.
Round to polygonal cells found in cohesive packets or small sheets Nuclei are round to oval and central to eccentric Nuclear chromatin is fine to smooth Indistinct cell borders
mitotic figures
A mitotic figure is a cell that is in the process of dividing to create two new cells.
Mitotic figures are easy to see because the genetic material inside the nucleus changes colour and shape before the cell divides.
Counting mitotic figures (MF) in tumors is one of the most widely used methods
of predicting tumor behavior. The mitotic count (MC)* is a rapid, inexpensive test that can be
performed by any pathologist, is part of many grading schemes, and aids in clinical prognostic
decisions.
Prometaphase
Central dark aggregate
Spikes/projections
Metaphase-
Linear or ring shaped
Spikes/projections
Anaphase -
2 separated aggregates
Distances variable
Telophase (1 MF)-
Separated aggregates
Cleavage furrow
atypical mf:
Multipolar-
More than 2 spindle
poles in any phase
Asymmetrical bipolar-
Unequal size of
chromosome clusters
Chromosome Bridging-
Chromosomes
stretching from one
cluster to opposite pole
Chromosome Lagging-
Fragments not in
contact with cluster
haematoxylin and eosin stain
Hematoxylin shows the ribosomes, chromatin (genetic material) within the nucleus, and other structures as a deep blue-purple color. Eosin shows the cytoplasm, collagen, connective tissue, and other structures that surround and support the cell as an orange-pink-red color.
Livor mortis
Livor mortis, also known as hypostatic congestion, is a post mortem change that occurs when blood pools on the dependent side of a dead animal due to gravity.
Psuedomelanosis
is a post mortem change
Green-blue staining by FeS.
FeS formed by H2S from putrefactive bacteria and iron from Hb from lysed RBC’s.
Will progress to appear back under the right circumstances
Colour is due to the development of blackish particles of ferrous sulphide in the tissues.
The sulphide part is due to the development of hydrogen sulphide in the putrefying tissues.
The iron part comes from the haemoglobin of the blood.
Haemoglobin is acted on in putrefaction by bacteria, which split off the iron at the same time as they produce hydrogen sulphide.
Components combine to form ferrous sulphide.
Discolouration therefore depends on the presence of both blood and bacteria.
Putrefaction
Putrefaction is the action of bacteria on tissues after death.
bacteria can produced gas bubbles. The tissue will likely feel soft and smell.
post mortem evidence of
Barbiturate euthanasia
enlarged spleen-
Congestion is due to passive engorgement of a vascular bed.
Occurs by decreased outflow or increased inflow of blood.
Can be acute or chronic.
Acute occurs with barbiturate euthanasia due to smooth muscle relaxation, resulting in blood pooling in the vessels or typically the spleen, liver and lungs.
Chilling artefact
Chilling a carcass will often result in opacity of the cornea and/or lens. Warming the carcass back to room temperature will return this to normal
Diffuse red discolouration of the intima of the base of the thoracic aorta is evidence of which post mortem change?
Haemoglobin imbibition
Haemoglobin imbibition is due to the red discolouration of tissue due to the release of haem from lysed erythrocytes.
Whilst this is most commonly seen as a freeze-thaw artefact as freezing expands erythrocytes and bursts them en masse, this can also happen if a carcass is left long enough post mortem for the ertyrhocytes to lyse “naturally” in organs that contain a lot of blood, such as the right side of the heart and large vessels.
The post mortem change negatively affected by cachexia is
Rigor mortis
cachexia-weakness and wasting of the body due to severe chronic illness.
Rigor mortis
a post mortem change
Appearance is that of hyperextended limbs and neck
Onset is 1-6 hours after death, lasts 1-2 days.
It is due to depletion of ATP and glycogen which are required to RELAX muscles and is reversed by autolysis
Rigor mortis also occurs in the heart, so will typically see the left ventricle devoid of blood as it contracts
May not see rigor mortis at all if an animal is cachexic
Post mortem cooling of the carcass is known as
Algor mortis
Melanosis in the pig is …
incidental
Known as congenital macular melanosis
agonal changes
take place immediately before death and are due to circulatory failure.
Desiccation
The postmortem drying of mucous membranes and delicate skin surfaces may result in artifactual changes in color or texture. This desiccation process begins immediately upon death and may progress quite rapidly in normally moist mucous membranes. This effect is often most prominent in the eye in humans, resulting in a horizontal band of red to brown-black discoloration of the sclera where the eyelids fail to close; this is commonly referred to as tache noire
Skin surfaces most commonly affected are thin, delicate areas such as the lips and genitalia. The gross appearance is dark red to black with a variably irregular surface.
Decomposition- autolysis
The most definitive and distinctive postmortem change is the decomposition of the soft tissues. Immediately upon death, decomposition begins on a molecular level because of the failure to maintain ion gradients and cell membrane integrity. As cell membranes begin to degrade and eventually rupture, they spill their contents into the interstitium, exposing the cell membranes of surrounding cells and connective tissue fibers to cytosolic proteolytic enzymes that further degrade exposed cell surfaces. This chain reaction of decomposition that results from the digestion of tissues by intrinsic enzymes is autolysis
Macroscopically
early autolysis may not be obvious
with time tissue becomes paler, soft, friable and may exude fluid
mucosal linings may slough off easily e.g. intestine
Microscopically
early autolysis cells will swell
cytoplasmic and nuclear detail are lost
cells lose their cohesion to each other
no inflammatory response
Decomposition- autolysis
The most definitive and distinctive postmortem change is the decomposition of the soft tissues. Immediately upon death, decomposition begins on a molecular level because of the failure to maintain ion gradients and cell membrane integrity. As cell membranes begin to degrade and eventually rupture, they spill their contents into the interstitium, exposing the cell membranes of surrounding cells and connective tissue fibers to cytosolic proteolytic enzymes that further degrade exposed cell surfaces. This chain reaction of decomposition that results from the digestion of tissues by intrinsic enzymes is autolysis
decomposition-putrefaction
Bacterial putrefaction typically begins slightly after autolysis, which creates ideal conditions for bacterial growth.
Macroscopically -
Carcass blown up
Gas bubbling
Psuedomelanosis
A blue-green to block post mortem discoloration due to bacterial breakdown of haemoglobin produces hydrogen sulphide.
Microscopically-
Bacteria
typically rods in farm animals (Clostridia)
No inflammation
what are the two types of decomposition
autolysis
putrefaction
phases of decomposition
Fresh stage: death until bloating begins (4–36 days)
*
Bloated stage: onset of bloating until resolution of bloating (3–19 additional days)
*
Decay stage: resolution of bloating until drying of carcass (6–183 additional days)
*
Dry stage: drying of carcass until no evidence of carrion insects (13–27 additional days)
Mummification
Under dry environmental conditions, either cool or warm, with low humidity and sufficient ventilation, the body may become desiccated rather than undergoing the more typical process of decomposition.48 The skin becomes tight and yellow-brown to black and takes on a leathery or parchment paper consistency.61 As a result of exposure to such dry conditions, the processes of autolysis and putrefaction are retarded or completely inhibited, and the tissues become dehydrated. The resulting desiccation produces changes in the body such as contraction or wrinkling of skin, retraction of the nailbeds and finger tips, and contraction of the erector pili muscles
inhibition of bile pigment after death
One of the earliest local colour changes.
Bile salts diffuse out of the gall bladder.
Stain nearby tissue like the liver, gut, stomach and omentum.
NOT the same thing as jaundice- more generalised
Generalised discoloration of tissues due to bile pigments seen in the living animal.
external exam of a post mortem
Overview and BCS
Crown rump length
Pelage and skin
Eyes
Feet
Faecal or urine staining
Blood
Mucous membranes
Dentition
The pluck comprises….
Tongue
Thyroid glands
Trachea
Oesophagus
Lungs
Heart
Thymus
summerise the steps of a post mortem
External exam
Stabilize carcass
Skin the ventral aspect of the carcass
Open the abdomen
Check for negative pressure
Open the thoracic cavity
Remove the pluck
Check for gall bladder patency
Remove the adrenal glands
Remove the spleen and liver
Remove GIT
Remove the urogenital tract
Open and examine at least seven limb joints
Remove and bisect a femur for assessment of bone marrow
If indicated in the history, for example in cases of trauma or neglect, skin entirely
A technician will remove the brain and eyes
Arrange all removed organs neatly on a chopping board
GIT should be placed on a separate board
Examine organs and take samples
what bosy systems may be involved in sudden death
nervous
cardiovascular
respiritory
How can the autonomic system be damaged?
Trauma
Hypoxia/anoxia
Oedema
Toxins
Seizures
neurogenic shock results in widespread and massive vasodilation
Explain the pathophysiology of death due to cardiovascular failure
Pathophysiology of death due to cardiac failure:
Structural
Electrical
Ischaemic
Pathophysiology of death due to vascular failure:
Ischaemic
Shock
Pathology of the vessels
Evidence of haemorrhage?
Evidence of disease of organs involved in fluid and electrolyte haemostasis?-
GI
Urinary
Endocrine
Open pulmonary arteries and aortic bifurcation
Take sections of kidney - End arteries therefore good place to look for small thromboemboli
Examining the heart-
Pericardial effusion?
Weight
Measurements
Gross lesions
Sections for histopathology
Explain the pathophysiology of death due to respiratory failure
“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
Heat stroke
Peri-anaesthetic
Sudden death – respiratory – pulmonary haemorrhage in horses
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
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
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
Seen frequently in young ruminants
Incidental
endocardiosis
the development and accumulation of fibrous tissue within the heart valves which in turn alters the physical structure of the valves
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 dogs
incidental
Ascarid migration
incidental
Multifocally affecting all lobes of the liver are poorly-demarcated, flat, vaguely round, white to pale tan foci
Various species
Hepatocellular adenoma/carcinoma
incidental
Description: Entirely obliterating the left lateral lobe of the lever is a large nodular vaguely round pink to red firm mass
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
incidental
a benign liver lesion that is composed of a proliferation of hyperplastic hepatocytes surrounding a central stellate scar.
There are however lots of malignant neoplasms that can form masses in the spleen:
Haemangiosarcoma
Lymphoma
Histiocytic sarcoma
Focally expanding the parenchyma and raising the contour of the spleen is a focal, well-demarcated, black to red to pink mass
siderotic plaques
benign golden brown or black patches that are frequently seen on the surface of the spleen. They result from focal accumulations of stored iron (hemosiderosis) derived from erythrophagocytosis and subsequent hemoglobin breakdown
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 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
Can be incidental
Can also be pathological
Quantifying and contextualising is important
Describe and explain the process of PCR
Veterinary molecular diagnostics
Uses oligonucleotide (hort single strands of synthetic DNA or RNA that serve as the starting point) primers to amplify region of interest (gene)
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
occurs in steps-
denaturation-High temperature breaks hydrogen bonds holding base pairs together
‘Melts’ double-stranded DNA revealing bases in specific order
annelaing-
At cooler temperatures, complementary bases can bind
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
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
Specificity is very high
Sensitivity is very high
Rapid turnaround time
Overcomes culturing restrictions
PPV can be low
Not for all assays/samples
Requires specialist equipment
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
qPCR – SYBR assays
SYBR Green is one of the most commonly used fluorescent dyes in qPCR. It binds to double-stranded DNA molecules by intercalating between the DNA bases. Once intercalated to DNA, SYBR Green becomes less mobile, causing its energy to be released as fluorescence. Therefore, the fluorescence intensity is directly associated with the concentration of double-stranded DNA, which can be measured at the end of each amplification cycle to determine the PCR progress.
qPCR – TaqMan assays
Each TaqMan Assay employs a TaqMan probe that specifically anneals to a complementary sequence between the forward and reverse primer sites. When the probe is intact, the proximity of the reporter dye to the nonfluorescent quencher (NFQ) results in suppression of reporter fluorescence. Probe cleavage by DNA polymerase during primer extension separates the reporter dye from the NFQ, resulting in increased fluorescence of the reporter.
enable the detection of a specific PCR product as it accumulates during PCR cycles.
qPCR
stands for quantitative polymerase chain reaction and is a technology used for measuring DNA using PCR
The main difference between the two is that qPCR is a real-time method, while PCR is not. This means that with qPCR, you can monitor the amplification of your target DNA in real-time as it is happening
RT-PCR
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
commonly used in the diagnosis and quantification of RNA virus infections (e.g., human immunodeficiency virus and hepatitis C virus) and the analysis of mRNA transcripts such as those produced by translocations commonly associated with non-Hodgkin’s lymphomas, leukemias, and sarcomas.
PCR for viral infections
Serological assays are not always feasible when detecting viral infections
Lack of species-specific secondary antibodies
Suitable cells for growth and titration are not available
daignostics for bacterial infections
Culture
Stain
Test
Helicobacter diagnostics
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
diagnostics 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
non invasive molecular tests
Better animal welfare
Can be performed more frequently
Multiple samples over short period (sequential samples)
Detect agents shed for short periods or intermittently
faeces
Skin swabs
Fur swabs
Environmental tests-
Soiled bedding
Environmental swabs
Air filters
Cage tops
Does not require handling
Culturing not always possible
PCR can detect presence of infectious agents
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
can result in false negatives
Natural inhibitors include:
Bile salts
Polysaccharides in faeces
Haem from blood
Glycogen and fats in tissues
Proteinases in milk
Urea in urine
Co-purified with DNA/RNA
Extraction kits designed to remove them
summerie the use of 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
ELISA
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,
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.
Direct ELISA
Detection of 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 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
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
Epithelial tissue
All endoderm and some ectoderm origin
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 tissue
Mesoderm origin
Supporting cells
Fibroblasts -> collagen
Endothelia
Bone
Round cell tissue
Mesoderm origin
Cells of the haemo-lympho system-
Erythrocytes
Leukocytes
histological appearence of epithelial tissue types
Polygonal
Poorly-defined cell borders
Abundant cytoplasm
Polar to central round nuclei
Basement membrane
histological appearence of mesenchymal tissue types
Histological appearance
Fusiform/spindloid
Poorly-defined cell borders
Variable cytoplasm
Fusiform/spindloid nuclei
Extra cellular matrix
histological appearence of round cell tissue types
Round
Well-defined cell borders
Individualised
Variable cytoplasm
Round to variable nuclei
Two types of antibodies for immunohistochemistry
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
in immunohistochemistry, 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
what can cytokeratin and vientin be used to differentaite betweeen in immunohystochemistry
Cytokeratin- proteins found in the intracytoplasmic cytoskeleton of epithelial tissue.
Vimentin- a type III intermediate filament (IF) protein that is expressed in mesenchymal cells.
therefore the presence of eeach can differantaite between epithelial and mesenchymal cell
Immunohistochemistry
A laboratory method that uses antibodies to check for certain antigens (markers) in a sample of tissue. The antibodies are usually linked to an enzyme or a fluorescent dye.
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 is, once a urinary tract infection is ruled out, almost always due to
disease of the glomerulus
low urine specific gravity, which would indicate reduced ability to concentrate urine, is typically due to
disorder of the tubule.
Podocytes
highly specialized cells of the kidney glomerulus that wrap around capillaries and that neighbor cells of the Bowman’s capsule.
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
PAS and Trichrome are ….
special histochemical stains that can be used to highlight immune complex deposition in glomeruli
IM-glomerulonephritis
due to immune complex deposition within the glomerular tuft
Results from the deposition of immune complexes in glomeruli
formation of antibodies against the glomerular basement membrane
activation of inflammatory cascade
PAS and Trichrome are special histochemical stains that can be used to highlight immune complex deposition in glomeruli
Immunofluorescence and transmission electron microscopy can be used to confirm immune complex deposition in glomeruli
Any condition that stimulates the immune system for long periods of time can cause IM-glomerulonephritis
what are the three types of error
Pre-analytical
Specimen collection
Analytical
The test
Post analytical
Interpreting
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
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
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
Analytical error
Sensitivity-
ability of a test to detect diseased animalscorrectly
the proportion of diseased animals testing positive
SnOUT
Specificity-
ability of a test to give the correct answer if notdiseased
i.e.proportion of non-diseased animals testing negative.
SpIN
i.e. a highly specific test will have few false positive
Eg: bTB skin test
Sensitivity = 57%
If 100 infected cattle are tested, it will potentially fail to detect 43 infected animals
= Lots of false negatives
Specificity = 99.5%
If 100 uninfected cattle are tested, it wrongly diagnose <1% as positive
= Few false positives
This is important as it is a screening test
post-analytical error
dependent on history and location
karyolysis
he complete dissolution of nuclear components of a dying cell.
karyorrhexis
the destructive fragmentation of the nucleus of a dying cell whereby its chromatin is distributed irregularly throughout the cytoplasm.
combination of increased AST and TBil, alongside the low haematocrit indicates what process?
haemolysis
poikilocytosi
an increase in abnormal red blood cells of any shape that makes up 10% or more of the total population. Poikilocytes can be flat, elongated, teardrop-shaped, crescent-shaped, sickle-shaped, or can have pointy or thorn-like projections, or may have other abnormal features.
e.g heinz bodies
oxidative damage
Increased creatinine with low urine specific gravity indicates what type of azotaemia?
Renal – free haemoglobin at high enough levels is toxic to the kidney, also this animal is anemic so has reduced O2 carrying capacity which will further damage the kidney
azotaemia
elevation, or buildup of, nitrogenous products (BUN-usually ranging 7 to 21 mg/dL), creatinine in the blood, and other secondary waste products within the body
Brown mucous membrane is also called what?
Methaemaglobinaemia
Define haemostasis and platelet role in haemostasis
Haemostasis
Complex physiological processactivated vascular injury.
Imbalance in the haemostasis pendulum results in haemostatic disorder characterised by either thrombosis or haemorrhage
Cellular component: platelets, especially but also fibroblasts
Soluble proteins (coagulation factors and inhibitors)
Insoluble proteins (extracellular matrix proteins).
Haemostatic component interplay
Primary haemostasis (platelet plug formation)
Secondary haemostasis (fibrin clot crosslinking)
Fibrinolysis
Platelet count
Usually perform as part of complete blood count and haemostatic disorder screening
Rule out/rule in quantitative platelet level disorder
thrombocytopenia/thrombocytosis?
Methods
Platelet count estimation (blood smear)
Manual count using haemocytometer
Automated counter
Diagnostic considerations
Appropriate venipuncture to reduce endothelial damage and platelet activation/clumping
Use of EDTA tubes prevent platelet clumping and clot formation
Proper sample handling and storage
Test sample ASAP!!!
Direct blood tube contact with ice-pack could trigger platelet clumping(false-positivethrombocytopenia)
protiens that contribute to oncotic pressure
Most abundant molecule: ALBUMIN
Main driver of oncotic pressure
Lots of larger molecules each present in small numbers: GLOBULINS
Subdivided into: Alpha, beta, and gamma globulins
Includes: Inflammatory cytokines, immunoglobulins
Albumin
Produced in the liver at a constant rate
Major contributor to plasma oncotic pressure
Carries ion molecules (calcium, magnesium)
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
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
High albumin =
dehydration
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
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)
Panhypoproteinaemia
When both albumin and globulin are lost together.
Two main categories:
Protein-losing enteropathy (common)- Differentials:
Lymphoma
IBD
Lymphangiectasia
Parasitism
Protein-losing dermatopathy (rare → severe burns)
enzymes are 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.
There can be multiple different versions of one enzyme: “isoenzyme”
SDH (sorbitol dehydrogenase)
Liver
Generally only used in large animals
ALT (alanine aminotransferase)
Liver, muscle
Much more specific to liver vs muscle. Not useful in large animals.
Cat ALT and ALP have much shorter half life compared to dogs → smaller elevations are more clinically significant
GLDH (glutamate dehydrogenase)
Liver
More stable than SDH
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
AST (aspartate aminotransferase)
Liver, muscle
Equally specific to liver vs muscle
Long half life
longer half life than ck
ALP (alkaline phosphatase)
Biliary, bone, intestines, steroid
Steroid isoenzyme only in dogs
Cat ALT and ALP have 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
Bone ALP can increase with growth in young animals as well as patients with high osteoblastic activity (e.g. hyperparathyroidism)
GGT (gamma glutamyl transferase)
Biliary
Small increases significant
hepatobiliary markers
Bile acids
Bilirubin
Cholesterol
Albumin
Glucose
Coagulation factors
alt
alp
sdh
gldh
ggt
ast
Bile acid stimulation
Tests the ability of the liver to re-uptake bile acids from the portal vein.
Patient is sampled after being starved for 8 hrs, 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
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 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
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”
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
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
Decreases due to:
Reduced intestinal absorption
GI disease, hypoadrenocorticism
Albumin & Glucose
If the liver is end-stage, then these can drop due to reduced production/storage.
Other causes of hypoalbuminaemia already discussed
Other causes of hypoglycaemia include:
Diabetic ketoacidosis
Starvation (puppies, working dogs)
Insulinoma → pancreatic neoplasm which produces insulin
Artefact → use fluoride oxalate tube
link between liver faliur and Coagulation factors
Liver synthesizes coagulation factors
Liver failure → prolonged coagulation times
Muscular enzymes
ck
ast
alt
CK (creatinine kinase)
Muscle
Short half life
ast has longer half life
Pancreatic lipase
Used to diagnose pancreatitis but can also go up when Glomelular Filtration Rate is reduced.
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, herparin
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
amylase
Used to diagnose pancreatitis but can also go up when Glomelular Filtration Rate is reduced.
Poorly sensitive in cats
TLI
Usually used to diagnose Exocrine Pancreatic Inefficency (whereby levels are decreased)
Can go up with pancreatitis or with incomplete starvation
Renal physiology: Proximal tubule
Resorb most electrolytes
Activate Vitamin D
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
Renal physiology: Glomerulus
Electrolytes filtered out.
Proteins should remain in blood.
Small amount of protein present in canine urine
Location of juxtaglomerular apparatus (RAAS)
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
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
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 or diabetes insipidus → loss of electrolytes = “medullary washout”
Liver failure → loss of urea production
Oncotic pressure-
Amount of albumin within the peripheral blood
Hypoalbuminaemia
Loss via:
Kidneys → glomerular injury
Gut → IBD, lymphoma, lymphangiectasia
Skin → burns
Decreased production → Liver disease
Hyperalbuminaemia
Dehydration
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)
Requires damage to 75% of nephrons
Urea
Produced by the liver during protein metabolism
Excreted by the kidneys - small amount of reuptake
Provides the concentration gradient for loop of Henle
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)
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
NB: Greyhounds have naturally high SDMA
Azotaemia
Increase in urea, creatinine, and/or SDMA
levation, or buildup of, nitrogenous products
Classification:
Pre-renal → renal blood supply, increased urea production
Renal → problem with the kidney itself
Post-renal → obstruction of urine outflow
USG of a dog
1.020-1.045
USG of a cat
1.020-1.050
Uraemia
a buildup of toxins in your blood. It occurs when the kidneys stop filtering toxins out through your urine.
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
Electrolyte disturbances in kidney disease
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 suplimentation
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
Magnesium
Increases supplementation
Metabolic acidosis
Occurs when acids start building up in the tissues/blood or when bases are lost
If you retain acids → measurable with Anion Gap
Ketones → DIABETES MELLITUS
Lactate → INJURED OR HYPOXIC TISSUES
Uraemic acids → RENAL INJURY
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 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…
excess H+
H+ taken into tissue and exchanged for K+ → rise in blood K+
Respiratory alkalosis
Occurs when CO2 is exhaled excessively
Respiratory alkalosis causes:
Tachypnoea
CO2 is an acid → increased exhalation → alkalosis
= H+ deficit
H+ taken out tissue and exchanged for K+ → drop in blood K+
causes of Mixed acid/base disorders
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
causes of Mixed acid/base disorders
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
Calcium & 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
Calcium
Total calcium = bound to albumin and uraemic acids
Total can increase or decrease with fluctuations in these negatively charged molecules → especially albumin
Free (aka ionised) calcium = unbound calcium
Levels very tightly controlled by PTH, vitamin D and calcitonin
causes 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
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
Phosphorous
Increases:
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
Decreases:
Increased excretion-
Hyperparathyroidism
Fanconi’s syndrome (dogs)
Renal failure in horses
Reduced intake
Hypovitaminosis D
Magnesium
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)
Can I trust my results checklist
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
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
Haematopoiesis
formation of:
Erythrocytes
Leukocytes
Platelets
Typically occurs in the bone marrow but extramedullary haematopoiesis also occurs in the spleen and liver
Clinical signs associated with disorders of the haemolymphoid system
Enlarged lymph nodes
Anaemia
Coagulopathies
Oedema
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
polychromasia/reticulocytes
Polychromasia occurs on a lab test when some of your red blood cells show up as bluish-gray when they are stained with a particular type of dye. This happens when red blood cells are immature because they were released too early from your bone marrow. These immature cells are called reticulocytes.
Typically the bone marrow only releases mature cells, however in times of increased need, immature cells will be released into the blood stream
neutrophils/left shift
when immature neutrophils are released from the bone marrow due to an outpouring of cells, typically due to infection. In any acute inflammation, an increase in neutrophils is often seen.
Pancytopenia
defined as a complete lack of production of all lineages of haematopoiesis (bone marrow aplasia)
diffusely enlarged spleen
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
Benign spelnic masses
Nodular hyperplasia
Haemangioma
Haematoma
Indolent splenic masses
Marginal zone lymphoma
Malignant splenic masses
Haemangiosarcoma
Histiocytic sarcoma
Histiocytic neoplasia
Histiocytes are a subset of leukocytes
Originate from CD34 stem cells
Types:
Macrophages
Dendritic cells (histiocytic sarcoma)
Langerhan’s cells (histiocytoma)
Blood monocytes
Benign -
Histiocytoma
Skin
Young dogs and Boxers
Spontaneously regress
Malignant
Histiocytic sarcoma
Burnese Mountain Dogs, Flat Coated Retrievers, Rottweilers
Can be localised, often around joint, or systemic involving the spleen
Also, several slightly bizarre reactive histiocytic syndromes
Malignant histiocytosis (Burnese mountain dogs)
Pulmonary Langerhans cell histiocytosis (cats)
The urinary filtration barrier comprises
Fenestrated endothelium of glomerular capillaries
Glomerular basement membrane
Foot processes of the podocytes
The filtration barrier is selectively permeable
Under normal circumstances, all cellular components and plasma proteins the size of albumin or larger are retained in the bloodstream.
Water and solutes are freely filtered.
Molecular charge is also important.
Renin-angiotensin-aldosterone system
Renin
Released by juxtaglomerular apparatus in response to low blood pressure and flow to the kidney
Transforms angiotensinogen (made by the liver) to angiotensin I
Angiotensin I is converted to angiotensin II by angiotensin-converting enzyme (ACE – made by the lung)
Angiotensin II
Acts directly on vessels to result in vasoconstriction-> increase blood pressure
Acts on the adrenal gland to produce aldosterone-> kidney tubules resorb salt and water
Hydronephrosis
refers to dilation of the renal pelvis, which fills within urine.
Typically due to downstream blockage
Increased pressure in the renal pelvis results in no where for urine to go and glomeruli continue to “make” urine-> urine is forced into renal interstitium -> collapse of interstitial vessels -> hypoxia -> repair by fibrosis
pathophysiology
the study of structural and functional changes in tissue ans organs in the disease state
Uraemia
The systemic changes associated with severe azotaemia
Uraemia is a clinical syndrome
Whilst in clinical pathology we associate elevated levels of urea and creatinine with renal failure, in reality, over 90 toxins that would otherwise be filtered by the kidney build up in the blood
Uraemic toxins damage tissue by
Endothelial damage
Also, some of these toxins are leached into saliva and gastric secretions-> metabolised to ammonia -> caustic ulceration of mucosa (tongue, stomach, colon)
Animals with chronic renal failure are azotaemic and will eventually become uraemic
Chronic renal failure
When one part of the nephron is damaged, eventually the rest of the nephron follows
Replaced by fibrosis
Animals with chronic renal failure are azotaemic and will eventually become uraemic
Other findings:
Hypertension
Non-regenerative anaemia
Hypokalaemia
Calcium deposition in soft tissues
role of the kidney with calcium
The role of the kidney is three-fold
Resorption of calcium
Phosphate excretion
Activation of vitamin D
Failing kidneys result in -
Reduced calcium
Increased phosphate
Inactivated vitamin D
Less calcium absorbed from intestine
Chronic renal failure ultimately results in renal secondary hyperparathyroidism
Due to low calcium, the parathyroid glands become hyperplastic and produce more parathyroid hormone
As there is no other way in the animal of increasing plasma calcium, calcium is resorbed from bone
This results in weak bones, replaced by fibrosis = rubber jaw
Also the combination of high phosphate and acidosis means calcium is deposited in soft tissues, particularly stomach, kidney and pleura
Renal dysplasia
Progressive juvenile nephropathies
Polycystic kidney disease
Neoplasia of the kidney
From within – typically unilateral and singular
Renal cell carcinoma
Urothelial cell carcinoma
From without – typically bilateral and multiple
Lymphoma
Endocrinopathies are due to
An under or overproduction of hormones
An inability to respond to hormone production.
Exogenous hormone administration.
Production of hormone-like substances from certain cancers
Underproduction of hormones is due to
Immune-mediated destruction of the endocrine organ.
Upstream endocrine organ destruction.
The inability to produce a hormone due to a nutritional deficiency.
Typically, overproduction is due to hyperplasia or neoplasia.
Neurohypophysis (posterior pituitary, pars nervosa):
Oxytocin
ADH (antidiuretic hormone/vasopressin)
Adenohypophysis (anterior pituitary)
Pars distalis:
Lactotrophs (acidophils): PRL (prolactin)
Somatotrophs (acidophils): GH (growth hormone),
Thyrotrophs (basophils): TSH (thyroid-stimulating hormone)
Gonadotrophs (basophils): FSH (follicle-stimulating hormone, LH (luteinizing hormone)
Corticotrophs (chromophobes): ACTH (adrenocorticotrophic hormone)
Pars tuberalis: Scaffold for the capillary network of the hypophyseal portal system; has secretory granules, stellate cells, and receptors for melatonin
Pars intermedia:
Melanotrophs: MSH (melanocyte-stimulating hormone), b-endorphin, and corticotropin-like intermediate lobe peptide (CLIP)
Clinical Categories of Laminitis
Multifactorial disease process, with common end-point of laminar degeneration
Endocrinopathic-
Insulin dysregulation (Equine Metabolic Disease)
Equine Pituitary Pars Intermedia Dysfunction (PPID)
80% of cases of laminitis have underlying endocrinopathic disorders
Inflammatory
Severe infection (sepsis, colitis, endometritis)
Traumatic
Excess weight bearing on one limb (contralateral limb laminitis)
Pituitary pars intermedia dysfunction
Age related degenerative condition
Loss of dopaminergic inhibition
Hypothalamus unable to regulate pars intermedia of pituitary gland
Hypertrophy / hyperplasia of PI
Increase production of many hormones from PI which have wide array of effects on body
Increase gluconeogenesis
Decrease glucose utilisation
Increase glycogen deposition in liver
Decrease protein synthesis in muscles
Increase fat breakdown and redistribution
Decrease production and function of WBCs (immunosuppression)
Decrease cell division
Increase gluconeogenesis
Decrease glucose utilisation
Increase glycogen deposition in liver
Decrease protein synthesis in muscles
Increase fat breakdown and redistribution
Decrease production and function of WBCs (immunosuppression)
Decrease cell division
SMEDI
Still birth, mummification, embryonic death and infertility
In the pig is classic for parvovirus
Maceration of the foetus
When a fetus dies in utero, there are changes in the skin and tissues—termed fetal maceration. This process takes place entirely in the womb and stops once the fetus is delivered
Foetus is liquified
Foetid smell
Bones will remain
Bacterial cause
Endometritis
Open cervix
Emphysema in the foetus
A disorder affecting the alveoli (tiny air sacs) of the lungs. The transfer of oxygen and carbon dioxide in the lungs takes place in the walls of the alveoli. In emphysema, the alveoli become abnormally inflated, damaging their walls and making it harder to breathe.
Associated with
Protracted dystocia
Late expulsion of dead foetus
Putrefactive ascending bacteria
e.g. clostridial organisms)
Foul smell and gas under skin (crepitant)
Advanced uterin lesions and dam may die due to toxaemia
how can lepto be diagnosed from the foetus
Foetal kidney PCR for lepto
how can chlamydia be diagnosed form the foetus
MZN stain of foetal stomach content or PCR for chlamydia
what can be cultured from foetal stomach content
Salmonella and other bacterial and fungal contents
what is tested for in occult instanses of abortion
Salmonella and other bacterial and fungal contents can be cultured from foetal stomach content
Chlamydia and lepto difficult to culture so
MZN stain of foetal stomach content or PCR for chlamydia
Foetal kidney PCR for lepto
Coxiella also tested for with MZN before proceeding with sheep abortions due to zoonotic risk
Brucella testing to maintain Brucella-free testing also MZN
Histopathological +/- IHC of foetal tissues- Brain, liver, lung AND PLACENTA
Serology – dam blood and foetal fluid
Perinatal mortality
may be defined as death of the foetus or perinate before, during or within 48 h of calving at full term (> 260 days in cattle)
Includes both stillbirth and early neonatal mortality
As well as the previously discussed samples, examination of the foetal thyroid gland for
absolute goitre (thyroid enlarged relative to a criterion-referenced threshold thyroid weight, e.g. > 30 g)
or relative goitre (thyroid enlarged relative to a criterion-referenced threshold thyroid g: kg ratio with body weight, e.g. > 0.80)
and submission of a fresh (I2 content) and formalinised lobe (histopathology) will detect dietary iodine imbalance.
Where selenium deficiency is suspected a fresh sample of the foetal liver preferably or kidneys should be submitted.
what samples do you take from a still birth
Fresh tissues: lung, liver, kidney, thymus (viral PCR), stomach contents,other foetal fluid, placenta
Fixed tissues: lung, liver, kidney, thymus, placenta, brain, spleen, heart, thyroid, adrenal, skeletal muscle
Cysts that occur within the ovarian parenchyma include cysts derived from
anovulatory Graafian follicles (luteal and follicular cysts)
cystic corpora lutea
cystic rete ovarii- The rete ovarii, the homolog of the rete testis, is present in the hilus of all ovaries.
cysts of the subsurface epithelial structures
Luteal and follicular cysts both are derived from anovulatory Graafian follicles and differ only in the degree of luteinisation- During normal proestrus, regression of the CL coincides with development of a selected follicle, while the growth of any additional follicles is inhibited. In animals developing COD, ovulation fails to occur and the dominant follicle continues to enlarge.
Cysts derived from anovulatory Graafian follicles are most common in the cow and sow, but also occur sporadically in the bitch and queen. They often cause altered reproductive activity through secretion of steroid hormones. Affected animals, especially bitches, may show marked manifestations of hyperestrinism, such as altered reproductive behavior, anemia, and hemorrhagic diathesis. In the cow and sow they may be associated with anestrus, persistent estrus, or nymphomania
Cystic corpora lutea are essentially a variation of a normal luteal structure and the animal may be pregnant.
Taylorella equigenitalis
CEM, caused byTaylorella equigenitalis, a microaerophilic gram negative coccobacillus, is ahighly contagiousvenereal diseaseofmaresthat is characterized by endometritis, transient infertility, and rarely abortion
Stallions do not develop clinical disease, but can transmit the organism; recovered mares can harbour the organism for several months and are an important reservoir
Infectious endometritis – similar clinical, gross, and histologic changes; bacteriologic culture to differentiate
Endometrial hyperplasia
is common in domestic canines, often involves cystic distension of endometrial glands (cystic endometrial hyperplasia, CEH).
Exogenous sources of progesterone can be found in megestrol acetate, etc.
Chronically hyperplastic endometrial glands lead to the gross accumulation of mucoid fluid = mucometra and hydrometra
There is debate among authors whether cystic endometrial hyperplasia (CEH) and pyometra are linked, it is currently thought that prolonged exposure to high levels of progesterone cause endometrial gland proliferation which may increase susceptibility of the uterus to infection.
E.coli
Uterine endometrial adenocarcinoma
The most common neoplasia of the rabbit reproductive tract and probably the most common neoplasia of any body system of female rabbits
Carcinoma = malignant
80% will metastasize
lungs
Leiomyoma/leiomyosarcoma
Tumour of smooth muscle
Leiomyomas of genital origin are among the most frequently encountered neoplasms of the female reproductive system in almost all domestic animals, including elephants
Severe necrotizing/gangrenous Mastitis
Gram –ve bacteria
Endotoxin production
->Massive cytokine release
-> necrosis
-> increase in vascular perm
Wet gangrene
Quarter may slough
Sick cow
Granulomatous Mastitis
Contamination of teat-administered drugs are contaminated
Nocardia
Cryptococcus
Atypical Mycobacteria
Candida
Viral mastitis in goats
Caprine arthritis and encephalitis (CAEV)
Retrovirus
Indurative mastitis
Hard udder
Pathogenesis
Not fully understood.
Virus-infected macrophages in colostrum and milk are absorbed intact through the gastrointestinal mucosa.
Spread throughout the body via infected mononuclear cells.
Periodic viral replication and macrophage maturation induce massive lymphoproliferative lesions in target tissues and organs such as the lungs, synovium, choroid plexus, and udder.
Persists by residing as provirus within host cells
Fibroadenomatous hyperplasia
Cats
Young intact females
Progesterone
a non-neoplastic, benign condition that is seen most often in young, intact female cats. This lesion is associated with prolonged progesterone or other hormonal exposure, via endogenous or exogenous sources
Dogs – herpes virus
More commonly causes death of puppies <2weeks old.
Herpes virus is latent in the bitch causing no to minimal clinical signs but recrudesces during late pregnancy.
Some adults will have ocular and/or vaginal discharge.
Puppies are infected during birth.
Viraemia as virus spread via macrophages.
Incubation period 6-10 days.
Puppies are anorexic, hypothermic, vocal, disorientated. Fading puppies? Most if not all of litter will die.
Optimum temperature for replication of the virus is 34-36c, hence (partly) why neonates are worse affected, and active warming can improve prognosis.
Gross lesions include bilateral, multifocal, petechial and ecchymotic haemorrhages of the kidneys and liver.
Necrosis in kidneys, liver and lungs. Typical intranuclear viral inclusion bodies.
Virus, like a lot of herpesviruses, is endotheliotropic so most lesions are typically due to death of endothelial cells resulting in damage to blood vessels.
Herpes virus is not a core vaccine, but can be given to breeding bitches at request BEFORE and during pregnancy
Dogs – Brucella canis
Zoonotic
Transmitted primarily through mating to the bitch then to puppies in utero and milk.
Abortion mid pregnancy due to placentitis and endometritis, and aborted material source of infection. Female may continue to shed bacteria intermittently for weeks to months.
Aborted foetuses may have renal haemorrhages but bronchopneumonia most typical.
If mated again, subsequent pregnancies may reach full term but neonates may be weak or die.
In the male, it causes infertility and epididymitis.
Dogs with no clinical signs can still be infectious.
Discospondylitis common clinical presentation.
Testing methods include bacterial culture of any fluid from an adult/foetus and some practices are now offering serology for any imported animal prior to surgery.
Cats – feline panleukopenia virus
Parvovirus
Faeco-oral spread
Requires infection of rapidly dividing cells to replicate-
GIT
Bone marrow
Early in utero infection:
Foetal death and resorption
Perinatal infection
2 wks prenatal - 2 wks postnatal
Infection of cerebellum at crucial developmental period
cerebellar hypoplasia -> ataxia
2-4 months postnatal
Infection of bone marrow, thymus, GIT and mucosal lymphoid tissue
Leukopenia and enteritis
4-12months
Enteritis
what is desribed in descriptive pathology of the bovine placenta
Chorioallantois not amnion
*Size and number of cotyledons (range 72-125)
*Freshness: smell, colour, texture
*Cotyledons: red, pale, necrotic, exudate; fibrin/pus
*Intercotyledonaryareas:thickened/’plaques’, exudate; fibrin/pus
what is desribed in descriptive pathology of the aborted calf
Freshness: smell, colour of viscera (all similar after 24-48hr death in utero)
*Size, weight, crown rump length, features of development
*Skin lesions, covered with exudate, meconium
*Excess fluid in body cavities: clear, pink, red, fibrin
*Stomach content: meconium
what is desribed in descriptive pathology of the bovine organs
*Lungs –inflated (pink, spongey) or not (atelectic; dark pink/red)
Liver –haemorrhages, multifocal lesions, abnormal size/shape/structuresIntestine –atresia (colon, rectum, etc), inflammation/necrosis
*Kidney –number, structure
*Heart–size,shape,anatomy
*Musculoskeletal and others–‘describe whatyousee’
what is desribed in descriptive pathology of the bovine brain
Size and structure, abnormalities?
*Cavitation of cerebral hemispheres: hydranencephaly, porencephaly
*Cerebellar hypoplasia
what samples are taken from an abortion in the calf
*Aborted calf (if suitable)
*Placenta
*Maternal blood?
*Bulk milk?
*Cohort bloods?
*Faeces, environmental samples, etc?
Calf samples
*Fresh-
*Stomach content (liver or lung if unavailable)- Bacteriology, fungal examination
*Spleen or thymus- PCR pestivirus
*Kidney-PCR Leptospires
*Foetal fluid-Antibody tests
*Brain-Weigh, iodine
*Thyroid (stillborn)
Adrenal or liver- PCR BoHV-1
*Fixed: Lung, brain, liver, heart, + stomach wall, eyelid
Placental samples*Fixed and fresh cotyledon
pathology of Chlamydia abortus: Enzootic abortion on sheep placenta
inflamed cotyledons/ intercotyledonary
*Thick necrotic exudate
pathology of Toxoplasma gondii: toxoplasmosis abortion on sheep placenta
*Inflamed cotyledons with necrotic foci → totally necrotic cotyledons- straberry cotelydons
*Little/no intercotyledonaryreaction
*Non specificfoetalpathology
*Fresh →mummies
pathology of Campylobacteriosis on sheep placenta
Lambs: non specific → multifocal hepatitis
*Placenta –pale/small necrotic cotyledons
Campylobacter fetus fetus
*Campylobacter coli/jejuni
equipmetn for horse abortion PM
Scales (up to ~100kg, and grams -10kg)
*Measuring tape
*PM knife or scalpel
*(Sterile scalpel)
*Rat tooth forceps
*(Sterile forceps)
*Universal containers –2
*Formalin and suitable pots
*Charcoal swabs –3
*Gas burner
*SpatulaOr
*1/2 swabs and 1/2 extra universal containers/60ml pots
CRL
generally used to assess gestational age
crown-rump length
Samples for virology (PCR)
thymus
lung
liver
spleen
kidney
all in one container
4 small pieces of chorioalatous in another pot
samples for bacteriology
*Liver(or heart blood if liver too soft)-
this is done by heating a scalple, searing the flesh, cutting into the steralised area andusing a charcole swab to collect a sample from the cut
*Lung (or stomach content)
*Chorioallantois- the swab from here shoul be taken from the cervical pole, which is an area aound the cervical scar
samples for histology
*Liver–4 small samples ~1-2 cm cubed
*Lungs–4 small samples from different lobes
*Spleen
*Adrenal gland
*Kidney–wedge from centre of each kidney (cortex to pelvis)
*Thymus
*Conjunctiva
*Thyroid
*Heart
*Chorioallantois –5 samples –see later
*Amnion –2 samples
Umbilical cord –2 cm sectionAny abnormal findings should be sampled
Amniotic plaques
Amniotic plaques are commonly observed in the placenta of cattle as well as other species. These plaques are 2–4 mm in diameter [28]. They typically appear as a slightly raised annular lesion that histologically appears as a proliferation of hyperplastic epithelium that often shows squamous metaplasia.
normal
what preservative should samples for histopathology be routinely collected?
Formalin
Which additional histochemical stain is used to highlight mycobacteria in histopathological sections?
Ziehl-Neelsen / ZN
What process visible via light microscopy will differentiate between autolysis and necrosis?
Inflammation or lack of inflammation
Blood samples for performing haematology should be collected into tubes containing which anticoagulant?
EDTA
Which benign round cell tumour is most common in the skin of young dogs and regresses spontaneously following T-cell infiltration?
Histiocytoma
embolic nephritis
inflammation of a specific segment of glomeruli, which is associated with subacute bacterial endocarditis, and frequently produces microscopic hematuria without azotemia
Which baterial agent is the most common cause of cystitis in dogs?
Escherichia coli or E. coli
Darkfield microscopy is typically used to diagnose which infectious disease of the kidney?
Leptospirosis
Which basal endocrinology test can be used to diagnose canine hypoadrenocorticism?
Cortisol
arthrogryposis.
term used to describe a variety of conditions involving multiple joint contractures (or stiffness). A contracture is a condition where the range of motion of a joint is limited
in the fetus its a sign of schmallenberg
The combination of still birth, mummification, embryonic death and infertility primarily in primiparous sows is most indicative of which infectious agent?
Parvovirus
What is the most common non-odontogenic oral neoplasm in dogs?
Melanoma/malignant melanoma
Mucosal mineralisation of the stomach is typically due to failure of which organ?
Kidney/renal/urinary
Which equine parasite undergoes hypobiosis in the large intestine, resulting in severe disease upon emergence?
cyathostomins/small strongyles
Define pre-patent period in the context of gastrointestinal parastiology.
Time between infection and oocyst/egg shedding in the faeces
Cirrhosis
Cirrhosis is scarring (fibrosis) of the liver caused by long-term liver damage. The scar tissue prevents the liver working properly. Cirrhosis is sometimes called end-stage liver disease because it happens after other stages of damage from conditions that affect the liver, such as hepatitis.
Nutmeg liver is typically secondary to failure of which other organ or tissue?
Heart/cardiac
EHV abortion
Markedly increased clear yellow fluid, thorax and pericardium
Jaundice- multifocal hepatic necrosis
Thymic necrosis –colourchange from pink to cream-tan
liquefaction
Pulmonary consolidationIntranuclear inclusion bodies in hepatocytes
EHV-4
▪Usually sporadic cases▪Usually have excess of body fluids
▪Liver / lung lesions –sparse or absent
▪Spleen –best site for virus isolation
▪Many lesions secondary to hypoxia (alive foals may survive)
Hypospadia
Due to failure of the urogenital groove to close in the male
Tumours of the testicle
Interstitial/Leydig cell tumour
Seminoma
Sertoli cell tumour
Tumours of the scrotum
Vascular hamartoma, haemangioma and haemangiosarcoma
Melanoma
Mast cell tumour
– Sertoli cell tumours
Grossly:
The tumours are white
Irregularly ovoid
Lobulated
Bulge when cut
May be cystic
Abundant fibrous stroma makes then firm to hard
May cause marked distortion of the testicle
Most are benign
The incidence of Sertoli cell tumours is 20 times higher in cryptorchid dogs
Up to 30% of affected dogs produce excessive oestrogen, resulting in:
Feminization, including attraction of male dogs
Reduced libido
Testicular and penile atrophy
Preputial swelling
Perineal hernia
Gynecomastia
Redistribution of fat
Symmetrical, often ventral, alopecia.
Squamous metaplasia of the prostate gland (see later slide)
Oestrogenic depression of bone marrow can result in-
Anaemia
Thrombocytopenia
Granulocytopenia
Any defect, injury or infection, that results in leakage of spermatozoa or spermatozoal antigens into the extra tubular compartment results in…
a foreign body or granulomatous response, fibrosis, continued disruption of tubules, spermiostasis, or spermatocele
Innate and acquired immune function is actively suppressed in testicular parenchyma, as spermatocytes, spermatids, and spermatozoa are highly antigenic and outside the blood-testis barrier
Infections of the testicle and epididymis
Orchitis and epididymitis
Sheep
Brucella ovis
Orchitis: Sheep/Goat pox virus, Visna/maedi virus, Trueperella pyogenes, Corynebacterium pseudotuberculosis, Brucella melitensis, Histophilus ovis
Epididmytis: Actinobacillus seminis (most important); Histophilus somni, Mannheimia haemolytica, E.coli, Trueperella pyogenes
Pigs-
Brucella suis
Cattle-
Brucella abortus
Orchitis: Mycobacterium bovis, M. tuberculosis, E. coli, Proteus vulgaris, Corynebacterium ovis, Streptococcus , Staphylococcus sp., Trueperella pyogenes, Actinobacillus spp, Nocardia farcinica, Chlamydia spp,, and Mycoplasma sp
Epididymitis: bovine herpesvirus 4 (cytomegalovirus), Actinobacillus seminis, Mycoplasma bovigenitalium, Trypanosoma brucei
Dogs-
Brucella canis
Cats-
Naturally resistant to Brucella
Orchitis: FIP
Epididymitis is rare
Scrotal inflammation
Chorioptes bovis-> scrotal mange in SHEEP
Diseases of the prostate - prostatitis
Typically ascending
E.coli common
Abscessation and sepsis
Benign prostatic hyperplasia is the most common disorder of the prostate in intact males
Under the influence of testosterone the prostate will become hyperplastic
Is symmetrical
May not cause issue but is palpable on rectal exam
Clinical signs include haematuria and preputial discharge
May be cystic
Resolves post castration
prostatic squamous metaplasia-
due to excess oestrogen via unknown pathogenesis
The excess oestrogen in this case would be from a Sertoli cell tumour
Metaplasia
the change from one differentiated cell type to another of the same germ layer.
In this case is from cuboidal epithelium in gland formation to stratified squamous
In the normal animal the change from specialised epithelia to strat. squamous is protective
neoplasia of the prostate
Carcinoma as epithelial and typically malignant
In the dog, arise more commonly from the urothelial tissue in the urethra= urothelial cell carcinoma (transitional cell carcinoma)
Less commonly arise from the glandular parenchyma= prostatic adenocarcinoma - more common in humans and a mouse model
Posthitis
inflammation of the prepuce
Balanoposthitis
inflammation of the glans penis
Phalitis
inflammation of the entire penis
Phalophosthitis
inflammation of both penis and prepuce
Pizzle rot
Posthitis in sheep
Corynebacterium renale
Bovine herpes virus 1
Phaloposthitis in the bull
Infectious pustular vulvovaginitis and abortion in the cow (see dry lab)
Respiratory disease in the calf
neoplasm of th epenis and prepuce
Papillomas
Genital papillomas, or warts, on the penis seen in horses and cattle
Viral cause
In horses can progress to squamous cell carcinoma
Melanoma
Volvulus
occurs when a loop of intestine twists around itself and the mesentery that supplies it, causing a bowel obstruction. Symptoms include abdominal distension, pain, vomiting, constipation, and bloody stools. The onset of symptoms may be insidious or sudden.
Hyperaemia
he process by which the body adjusts blood flow to meet the metabolic needs of its different tissues in health and disease
general pathological apearence of intestines with acute infection
Hyperaemia
Fluid filled lumen
Enterotoxaemia
bacterial toxins absorbed into bloodstream from intestines
Typically in veterinary species we are referring to various types of Clostridium perfringens
Types A to E
All produce a different of combination of toxins
Type D produces epsilon toxin
Typically seen in fat weaned lambs after sudden diet change/increase in grain = overeating disease
Pore forming
Enterocyte necrosis
Necrohaemorrhagic enteritis
Kidney tubular cell necrosis
“pulpy kidney”
glucosuria
Increases vascular permeability
Petechial haemorrhages
Pericardial effusion
Brain oedema -> neurological signs
Blindness, headpressing, ataxia
viruses in the GIT tract
Most viruses generally prefer to infect cells with rapid turnover as can use their replicative mechanisms to produce more virus
GIT has constant cell turnover
Most viruses generally prefer to infect cells with rapid turnover as can use their replicative mechanisms to produce more virus
GIT has constant cell turnover
Some viruses take this one step further and stimulate prolific growth
e.g. papilloma viruses
Stimulate the cell cycle
In some cases inhibit innate cell-mediated immunity
Hyperplastic lesions -> papilloma (and sarcoids)
Malignant transformation in some cases
Squamous cell carcinoma
Cervical carcinoma in humans
Due to purpose of the GIT, viral shedding and therefore infectivity is high
Faeco-oral transmission
Saliva
Many viruses of significant veterinary importance spend all if not most of their transmission cycle within the GIT
Erosion
Loss of the superficial surface epithelium but basement membrane/lamina propria intact
Ulceration
Loss of surface epithelium and extends into lamina propria/submucosa
Vesicle
Fluid filled space between layers of the epithelium
Blister
Also common in auto-immune diseases
Pustule
As per vesicle but contains necrotic material/pus
motility issues in the GIT
Ileus
= arrest of intestinal motility in the absence of an obstruction
Clinical signs
Colic
Distension
Reflux/regurgitation
Vomiting
Neuropathic
Myasthenia gravis (more later)
Inflammation
Ileus common post surgery and stress
rabbits
metabolic causes of pathology in the GIT
Non GI causes of vomiting
Uraemia
Neurological
Addison’s
Hypoadrenocorticism
Intermittent v+d+
Glucocorticoids maintain normal gastrointestinal mucosal integrity and function
Intestinal epithelial barrier disruption leads to permeability defects and the subsequent interaction of intestinal immune cells with the luminal contents. Activated immune cells release pro-inflammatory cytokines, such as TNF. In turn, TNF results in tight junction (TJ) disruption and intestinal epithelial cell (IEC) apoptosis and thereby exacerbates local inflammation. TNF also directly stimulates IECs to synthesize and release immunoregulatory glucocorticoids (GCs) to counter-balance excessive tissue damage. GCs act via the glucocorticoid receptor (GR) to inhibit TNF-mediated tissue damage in a negative feedback loop. The GR also inhibits pro-inflammatory transcription factors, including NF-κB, AP-1, and STATs leading to the resolution of the inflammation.
FELINE CHRONIC GINGIVOSTOMATITIS (FCGS)
Presents as severe inflammation of the oral
cavity
* Most commonly affects the caudal oral mucosa:
- palatoglossal arches (fauces),
- alveolar and buccal mucosa of the
caudal oral cavity,
- less commonly soft palate and dorsal
aspect of the caudal tongue
* The hard palate, labial mucosa and sublingual
mucosa are usually spared
- Not fully understood – probably several factors:
- Dental and periodontal disease,
- Altered immunological response,
- Infections:
- feline calici virus,
- FeLV, FIV, feline herpes virus-1,
- Pasteurella multocida, Bartonella spp., Mycoplasma felis …
- Usually affects adult cats
- Extremely painful
- Diminished food intake
- Weight loss
- Ptyalism
- Halitosis
- Unkempt appearance
May be bippsied to distinguish from squamous cell carcinoma (SCC)-
* Marked lymphoplasmacytic inflammation
with prominent numbers of plasma cells
* Localised to the mucosa, but can extend
into the submucosa (occasionally also
sialadenitis, myositis)
* Mott cells = plasma cells with numerous
globular cytoplasmic inclusions composed
of immunoglobulin (Russell bodies)
* Hyperplasia and erosion of overlying epithelium * Migration of neutrophils into the epithelium * Variable numbers of macrophages, mast cells
Ptyalism
a condition where you make too much saliva.
Mott cells
plasma cells with numerous
globular cytoplasmic inclusions composed
of immunoglobulin (Russell bodies)
ssociated with stress conditions occurring in a number of conditions including chronic inflammation, autoimmune diseases, lymphomas, multiple myeloma, and Wiskott–Aldrich syndrome
Russell bodies
eosinophilic spherical or globular cytoplasmic inclusions that accumulate in the rough endoplasmic reticulum of mature plasma cells. These plasma cells containing Russell bodies are also known as Mott cells
EOSINOPHILIC GRANULOMA COMPLEX (EGC)
- In cats, it affects a broad age and breed
range with common sites being: - dorsal surface of the tongue,
- palate,
- mucocutaneous junction of the
rostral lips
In dogs, it is over-represented in Siberian
huskies and Cavalier King Charles
spaniels with common sites being:
- palate,
- less commonly tongue, lips, other
mucosal sites
A complex of inflammatory diseases
that represents a group of similar
hypersensitivity reactions to various
antigens (environmental, ingested
materials, parasitic)
* Several clinical presentations/terms:
- linear granuloma,
- eosinophilic plaque/granuloma,
- collagenolytic granuloma,
- indolent ulcer
histopathology-
* Variable numbers of eosinophils
(can be a mixed inflammatory
population with eosinophils)
* ↑ neutrophils in ulcerated areas
* Collagenolysis- The proteolytic processing of collagen
* Cytology can often provide a
diagnosis, so histology is not
necessarily needed
The pathologist provides a diagnosis of
eosinophilic or mixed inflammation with
eosinophils
- In highly indicative cases (based on
histology and clinical history), the
pathologist can mention EGC in the
commen
OSTEOMYELITIS of the mouth
- The source of infection can be:
- hematogenous (bacteraemia),
- implantation (open jaw
fractures, contamination of
surgical sites, bite wounds,
gunshot wounds), - local extension (from an
infected tooth or periodontal
tissues) - Common infectious agents are:
- Staphylococcus spp.,
- Streptococcus spp.,
- Truperella pyogenes,
- Nocardia spp.,
- Coccidioides immitis,
- Cryptococcus neoformans
Osteomyelitis and destructive
malignant neoplasms can have a
similar appearance!
* Several things need to be taken
into consideration:
- clinical examination,
- diagnostic imaging,
- histology,
- microbiology
SQUAMOUS CELL CARCINOMA (SCC) in the oral cavity
- The most common non-odontogenic
oral neoplasm in cats - Can present as a proliferative, ulcerated
lesion or a non-healing wound - Often invades underlying bone
- Can metastasize, but usually later on
- Can mimic osteomyelitis on X-ray!
The 2 nd most common nonodontogenic oral neoplasm in dog
histopathology-
Keratin pearls- neoplastic keratinised
squamous cells forming
concentric layers
* Neoplastic cells in SCC extend past
the basement membrane into the
underlying stroma
* The majority of them is either well
or moderately differentiated
* Dysplastic changes of the epithelium
may possibly evolve into SCC
CANINE CHRONIC ULCERATIVE (GINGIVO)STOMATITIS
(CCUS)
- Lesions most often occur on the
buccal mucosa and lateral lingual
mucosa opposite to larger tooth
surfaces - Affected mucosa is often
depigmented and mirrors the shape
of the associated tooth → contact
stomatitis - Predisposed breeds: greyhound
- Poorly understood, most likely due to
inflammatory reaction to persistent
plaque bacterial biofilm that damages
the mucosa - Clinical signs:
- drooling,
- halitosis,
- reluctance to eat
histopathology
- Lichenoid and perivascular infiltrate
of B and T lymphocytes and plasma
cells - Often ulcerated epithelium,
intercellular oedema, transmigration
of neutrophils and T lymphocytes and
sparse necrotic/apoptotic epithelial
cells - Granulation tissue formation beneath
ulceratio
Lichenoid infiltration
a bandlike infiltrate of inflammatory cells in the superficial dermis, parallel to the epidermis
perivascular infiltrate
Inflammatory cells are clustered around blood vessels
MALIGNANT MELANOMA
- The most common non-odontogenic
oral neoplasm in dogs - Mean age: 10.5 – 12 years
- Early and high metastatic rate
- Often invades underlying bone (50%)
- Very rare in cats
- Melanocytoma, benign neoplasm of
melanocytic origin, is very rare in the
oral cavit
histopathology-
* MI > 4/10 HPF
* ↑ nuclear atypia
* Ki67 > 19.5
* ↓ pigmentation
Amelanotic MALIGNANT MELANOMA-
* Immunohistochemistry:
- Melan A
- PNL2 (slightly ↑ sensitive than Melan A)
may metastisise
FIBROSARCOMA
- The 3rd most common non-odontogenic oral neoplasm in dogs
- Mean age: 8 years
- Maxillary and palatal lesions are
more common than mandibular - Locally aggressive, invasive and
destructive - Low metastatic rate
- The 2nd most common malignant oral
neoplasm in cats (after SCC)
histology to distinguishe Fibrosarcoma from
amelanotic malignant melanoma
- Final diagnosis with IHC:
- vimentin +
- Melan A -
- PNL2 -
Canine biologically high-grade/histologically low-grade
FIBROSARCOMA (H/L FSA)
Most often
arises from the
maxillary gingiva
* Most frequent in
large breed dogs,
especially
Golden retrievers
* Biologically very
aggressive
histopathology-
* Very bland histology reminiscent of fibrous
connective tissue
* Histopathology cannot distinguish between
H/L FSA and fibrous gingival hyperplasia or
fibroma
* Clinicopathological correlation is VITAL!
FIBROMATOUS EPULIS OF PERIODONTAL LIGAMENT ORIGIN
(FEPLO)/PERIPHERAL ODONTOGENIC FIBROMA (POF
- A common most likely reactive
gingival lesion - Mean age: 8.5 years
- Rostral maxilla is the most common
site - Locally invasive
- Very good prognosis
histopathology-
* Three main components:
- proliferative mesenchymal cells
embedded in a collagenous
stroma reminiscent of
periodontal/gingival ligament,
- cemento-osseous matrix,
- odontogenic epithelium
CANINE ACANTHOMATOUS AMELOBLASTOMA (CAA)
- The most common odontogenic
neoplasm in dogs - Mean age: 8.8 years
- Rostral mandible is the most common
site - Local invasion of underlying bone
- Does not metastasise
histopathology-
Cardinal histologic features of odontogenic epithelium:
- palisading of the basilar epithelium,
- palisading epithelial cells have antibasilar nuclei,
- palisading epithelial cells have a basilar clear zone within
the cytoplasm,
- odontogenic islands have central areas reminiscent of
stellate reticulum (not present in CAA!)
- Can morphologically mimic SCC
- No reliable IHC markers for odontogenic
epithelium in veterinary medicin
Diseases of the oesophagus - inflammatory
Oesophagitis
Inflammation of the mucosa
Often due to acid reflux
Weak sphincter?
Hernia – most common
Oesophageal mucosal metaplasia- Strat sq to columnar
Can be iatrogenic
Doxycycline in cats
Regurge during GA
May result in stricture (narroeing)
Diseases of the oesophagus - trauma
Choke
Diseases of the oesophagus - anomalous
Persistent right aortic arch
Vascular ring anomaly
German Shepherd dogs
Results in dilated oesophagus cranial to constriction
Myasthenia gravis-
Another important cause of megaoesophagus
Congenital and idiopathic forms
Idiopathic due to antibodies against acetylcholine receptor
This can be secondary tumours of the thymus
Aspiration pneumonia
abomasitis
disease of the stomach
Abomasitis (abomasal bloat) is a relatively rare ruminant disease characterized by inflammation of abomasum in young calves, lambs, and goat kids.
Bacterial-
Clostridium septicum
Braxy
Clostridium sordelli
Viral-
Rarely just the abomasum
BVD
Malignant catarrhal fever
Parasitic
gastritis
Gastritis is when the lining of your stomach becomes irritated (inflamed)
Gastritis as a single entity is rare in veterinary species
Inflammatory bowel disease
The stomach has a unique microbial flora due to the pH
Helicobacter spp are important in humans and ferrets (H. mustelae)
Associated with ulceration
Low numbers are consider unremarkable on gastric biopsies from dogs and cats
stomach ulcers
Primarily an issue in horses and pigs
Complicated and unclear pathogenesis-
Inappropriate feed
Stress
NSAIDs
Pigs-
Non-glandular oesophageal portion
Ruminal ulcers in cows typically associated with ruminal acidosis
Caudal vena cava syndrome
Fungal overgrowth
Abomasal ulceration cause unclear
Traumatic reticuloperitonitis
Hardware disease
Wire or similar penetrates wall of reticulum
Pathophysiology varies from localised transmural inflammation of the wall of the reticulum to peritonitis to pericarditis
Clinical signs initially include-
ruminoreticular atony
moderate ruminal tympany,
decrease in milk production
pyrexia
abdominal pain -
arched back, erect hairs at the withers, anxious expression, reluctance to move, and an uneasy, careful gait
pain going down hill
pole test
Chronically ill to sudden death from heart failure
Uraemic gastropathy
Diseases of the stomach – metabolic
In renal failure, nitrogenous toxins build up in the blood
Ulceration and mineralisation
Diseases of the stomach – neoplastic
Stomach tumours are generally rare in veterinary species
Squamous cell carcinoma in horses ->
Adenocarcinoma ->
Leiomyoma/sarcoma
Gastrointestinal stromal tumours
Lymphoma
Carcinoids
abomasal displacement
Left or right displaced abomasum
High yielding dairy cows
Multifactorial-
Hypomotility
Hypocalcaemia
High concentrate diet
RDA more commonly seen within 1 month of calving
LDA more common than RDA
Metabolic alkalosis with hypochloremia and hypokalemia
bloat
Bloat is a clinical syndrome relating to the clinically appreciable distension of the abdomen due to distension of the abdomen
Typically due to the inability of liquid or gas to exit the stomach
In cows referred to as ruminal tympany
In the cow there are two forms-
Frothy (primary)
Gas (secondary)
Frothy-
Typically due to consumption of legumes
Lower ruminal ph (normal is 6.5 to 7.5)
Blocks eructation
Acute and deadly
Gas-
Due to physical or physiological inability to eructate
Choke
Vagal indigestion
More chronic
Difficult to detect post mortem-
Bloat line
in dogs-
Gastric dilation and gastric dilation/dilatation and volvulus (GDV)
Gastric dilation/distension can occur if gorge on kibble
GDV is typically gaseous with some liquid and food
Pathogenesis incompletely understood
Volvulus occurs first?
The pylorus and duodenum first migrate ventrally and cranially.
A volvulus of >180° causes occlusion of the distal oesophagus.
Compress vasculature
Caudal vena cava
Stomach and splenic -> necrosis
Fluid “lost” into the stomach
Metabolic acidosis
Hypovolaemia shock
Severely ill, clinical emergency
DIC
gastric dilation in horses
Gastric dilation as a primary disease rare in horses
Will quickly fill with fluid in cases of obstructive colic
Also will occur in motility disorders
Most well known would be grass sickness
Clostridium botulinum
Autonomic neurones lost
No peristalsis
Diseases of the stomach – perforation
Gaseous distension of the GIT is a common post mortem finding, especially farm animals
May perforate if left long enough
Did this perforation occur before or after death?
INFLAMMATION CANNOT OCCUR AFTER DEATH
Gross- Fibrin
Histo- No cellular response
Volvulus
twisting of the gut on mesenteric axis
Torsion
twisting of the gut on long axis
Intussusception
Intussusception (in-tuh-suh-SEP-shun) is a serious condition in which part of the intestine slides into an adjacent part of the intestine. This telescoping action often blocks food or fluid from passing through. Intussusception also cuts off the blood supply to the part of the intestine that’s affected
Volvulus, Torsion, and Intussusception can cause
- Thin walled veins get compressed
- Venous congestion
- Ischaemic infarction
- Necrosis
- lowered Gut barrier function (bacterial translocation/endotoxaemia)
- Obstruction proximal
- Perforation
- Acute fibrinous/suppurative peritonitis
Pathogenesis of diarrhoea
1 Altered structure / permeability (malabsorption)
2 Altered epithelial cell transport (secretory diarrhoea)
3 Osmotic effects (e.g. maldigestion)
4 Altered motility
Loss of water
Dehydration
Haemoconcentration
Hypovolaemic shock
Loss of ions (principally sodium, potassium and
bicarbonate)
Metabolic acidosis
Hypokalaemia
Protein-losing enteropath
(i) Increased permeability to plasma proteins -
lost to intestinal lumen
(ii) Chronic inflammation - lymphatic blockage
Main protein lost is albumin. Loss exceeds liver
synthesis ➔hypoalbuminaemia ➔plasma osmotic
pressure➔oedema and ascites
(wasting + emaciation may also be present
Oedema disease of pigs
Enterotoxaemic colibacillosis
E. coli F18
Pathogenesis:
Associated with dietary changes at weaning
Bacterial overgrowth in small intestine
Produce verotoxin (Shiga-like)
Necrosis of enterocytes and endothelial cells
Leakage from vessels results in oedema, which in the brain results in swelling and neurological signs
Classical gross post mortem presentation is marked oedema of the spiral colon
Swine dysentery
Necrohaemorrhagic enterocolitis
Brachyspira hyodysenteriae
Salmonellosis
Infectious colitis
All salmonella species are enteroinvasive
Zoonotic and reportable
enterica species serovar Typhimurium
Second most important cause of food poisoning in humans
Pathogenicity factors include ability to neutralise NO in phagocyte
Are phagocytosed but able to survive within phagolysosome
Pathophysiology-
Septicaemia-
Fibrinoid necrosis of vessels and DIC
Hepatitis and pneumonia
S. choleraesuis
Acute enteric -
Necrotising ileotyphlocolitis
S. typhimurium
Chronic enteric-
Button ulcers – ddx classical swine fever
S. typhimurium
Rectal strictures
Trichuris
Whipworm
Carnivores, ruminants, pigs people
Direct life cycle
Clinical signs
Transient, recurring large bowel diarrhoea with or without blood
Rarely, severe infestations result in pseudo-Addison’s
Histiocytic ulcerative colitis
Histiocytic ulcerative colitis
Boxer dogs and their kin
E. coli
Histiocytes - granulomatous
Anal furunculosis
GSDs
Immune-mediated
Peri-anal fistulation
A fistula is an opening between areas of the body that are not usually connected.
In this case between the anus and skin
Furunculosis is generally a term used to imply a deep infection of the dermis, typically with ruptured hair follicles and free hair shafts which themselves add to the immune reaction
Epithelial tumours of the intestine
tend to be upper GIT
In the cat, more commonly lower GIT
Papilloma and
polyp
Adenoma
Carcinoma
Anal and peri-anal glands
Near the anus there are the anal sac glands and the perianal/circumanal (hepatoid glands)
Anal sac glands
Apocrine
Perianal glands
Sebaceous
vascular pathology of the liver
Due to the unique blood supply, type of endothelia and architecture of the liver, oedema does not occur
Congestion however is very common
Infarction is very rare in the liver and tends to occur at the tips
Centrilobular (zone 3) hepatocytes are the furthest away from oxygenated blood and are also metabolically the most active, therefore are sensitive to cardiogenic failure
Chronic passive congestion due to chronic heart failure will result in chronic low oxygen delivery to the centrilobular hepatocytes
Grossly this appears as a zonal pattern with congestion of the central veins and pallor of the centrilobular hepatocytes
The centrilobular hepatocytes are pale due to hydropic degeneration (cell swelling – reversible)
Chronically these centrilobular hepatocytes may undergo necrosis and may be replaced by fibrosis which grossly appears as a “nutmeg” liver
Chronic anaemia will also cause loss of centrilobular hepatocytes
Liver lobe torsion is generally rare in veterinary medicine, except in rabbits
Telangiectasia is benign distension of sinusoids by blood seen most frequently in the cow.
Blood flow may also be compromised secondarily to diaphragmatic hernias
Portosystemic shunts
Blood from the portal system bypasses the liver
Due to anomalous vessel(s)
Can be congenital (Yorkshire Terrier) or acquired (Spaniels due to chronic liver disease)
Extra-hepatic (small breed) or intra-hepatic (large breed)
Clinically
Stunted growth
Hepatic encephalopathy
Biochemistry: Elevated serum bile acids, hypoalbuminemia, hyperammonaemia, hypoglobulinaemia, hypoglycaemia, decreased BUN, hypocholesterolemia
Urinalysis: Ammonium biurate crystals in alkaline urine
Haematology: mild to moderate microcytic, normochromic, nonregenerative anaemia
Grossly the liver is atrophic as lacking growth stimuli
viral pathologies of the liver
The liver can be exposed to infectious agents through three main routes
Haematogenous
Biliary (ascending)
Direct extension
Neonates also have a direct connection between umbilicus and liver
The liver receives 100% of the blood flow from the GIT
Defence
Kupffer cells
Resident macrophages
IgA secreted into bile
Multifocal random necrosis
Herpes
Typically affects foetus and neonates
Characteristic intranuclear viral inclusion bodies
Adenoviruses
Canine infectious hepatitis
Chickens
bacterial pathologies of the iver
Innumerable types of bacteria may infect the liver
Bacteria from the GIT
May initially be peri-portal
Tyzzer’s disease
Clostridium pilliforme
Foals and laboratory species (gerbils)
Abscessation common
Ruminal acidosis damages the rumen mucosa, resulting in translocation of Fusobacterium necrophorum into the portal circulation-> hepatic abcesses -> caudal vena cava syndrome
Haematogenous bacteria
Typically random necrosis
Salmonella, Listeria, Clostridia, Yersinia
Mycobacteria – pyogranulomas, Ziehl-Neelsen
Leptospirosis
Campylobacter (aborted lambs)
Clinical signs associated with leptospirosis vary and depend on the serovar and the host.
See Adewole’s lecture
In maintenance hosts, leptospirosis generally is characterized by a low serological response, relatively mild acute clinical signs, and a prolonged renal carrier state which may be associated with chronic renal disease.
In incidental hosts, leptospirosis can cause severe disease.
Dogs
Young > old
Serovars icterohemmorrhagiae and canicola were believed to be responsible for most clinical cases of canine leptospirosis and after a bivalent serovar-specific vaccine against canicola and icterohemmorrhagiae came into widespread use, the incidence of “classic” leptospirosis in dogs decreased.
Clinical signs: fever, inappetence, vomiting, abdominal pain, diarrhoea, PUPD.
Depending on strain and host response, pathogenesis can be primarily due to hepatic or renal dysfunction or a combination.
Whilst the renal consequences of lepto were covered in a previous lecture, the liver is another major organ damaged during leptospirosis.
The degree of icterus in canine disease usually corresponds to the severity of hepatic necrosis.
Cattle
The icterus and haemoglobinuria that develop in cattle with leptospirosis results from a specific haemolytic toxin produced by serovar pomona.
Chronic canine hepatitis
pathogenesis
Spaniels, Dobermans and Labradors all over-represented, therefore genetic factors implicated
May be secondary to chronic infection, such as lepto, or copper toxicosis (see later)
Clinically the animal will have raised liver enzymes
Grossly characterized by a small liver with nodules of hepatocyte regeneration and hyperplasia, separated by bands of fibrosis
Proxy for cirrhosis
Histopathology reveals periportal inflammation predominantly with portal areas bridged by fibrosis
Feline triaditis
In dogs, the common bile duct joins the duodenum at the major duodenal papilla, separately from the pancreatic duct. An accessory pancreatic duct joins the duodenum at the minor papilla in most dogs though anatomic variation exists.
In cats, the common bile duct fuses with the pancreatic duct before entering the major papilla. Only 20% of cats are estimated to have an accessory pancreatic duct
The canine CBD is 3mm in diameter and the feline CBD is 4mm in diameter
Probably due to their unique anatomical features of cats, whenever the intestine is inflamed, so too will be the pancreas and/or biliary tree
Cats are typically impressively jaundice
Likely post-hepatic
Histopath reveals portal and peri-portal inflammation, indicative of ascending infection
Infectious - helminths of the liver
The liver can be affected by helminths in two ways
Target organ
Trematodes
Cestodes
Visceral migrans
Nematodes
Fascioloides hepatica
Common liver fluke of ruminants
Indirect life cycle
Three syndromes
Acute fasciolosis, normally seen in sheep, is caused by large numbers of juvenile fluke migrating through the liver. These cause extensive haemorrhage and damage to the liver parenchyma. Animals are typically weak, and anaemic, often with palpably large livers, abdominal pain, ascites and sudden death is common.
Chronic fasciolosisoccurs in both sheep and cattle and occurs several months after moderate intake of infective cysts. Chronic disease is associated with adult fluke in the bile ducts. Anaemia, loss of appetite and gradual weight loss are common clinical signs. Infection also has an impact on fertility, growth rates and milk production.
Black disease (Clostridium novyi) in sheep and bacillary hemoglobinuria (C. hemolyticum) in cattle (and sheep). Migration of immature flukes through the hepatic parenchyma may result in the generation of necessary ischemic conditions for the proliferation of clostridial spores, already within the liver, to proliferate. Once activated the clostridial bacteria produce toxins, resulting in necrosis in the liver and death in sheep with black disease or intravascular haemolysis with associated anaemia and haemoglobinuria in cattle
toxic pathology of the liver
Because of its function and exposure to portal blood flow, the liver is a major organ affected by toxins.
Centrilobular hepatocytes are most commonly affected, as most toxins are not truly toxic until metabolised by cytochrome P450.
Few toxins are toxic without metabolism and will thus cause periportal necrosis.
An inexhaustive list of hepatotoxins include
Plants and similar
Blue-green algae
Ragwort (pyrrollizidine alkaloids)
Amanita mushrooms
Mycotoxins
Chemical/drug
Xylitol
Carprofen
Acetaminophen/paracetamol
Copper
Whilst copper is an essential cofactor of many cellular processes, it is also a toxicant at high enough levels resulting in free radical formation
Acute
death primarily in ruminants due to haemolytic crisis and acute liver necrosis
sheep fed cattle feed and/or deficient in molybdenum
Gross – icterus, multifocal pan-lobular hepatic necrosis and gunmetal blue kidneys
Chronic
Bedlington Terriers have a defect in a copper transport gene, may be other breeds too
All dogs with CCH should have copper levels tested
Copper levels in dog food too high?
metabolic pathology of the liver
Fatty liver disease/ketosis/twin lamb disease/hepatic lipidosis/steatosis
Terms used for lipid deposition in the liver
Physiology of lipid metabolism - recap
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.
Whilst hypoxia and toxins such as aflatoxins that affect protein synthesis in the liver can cause this, one of the main times to see this is when an animal can no longer rely on glucose as an energy source and free fatty acids are thus mobilised from adipose tissue as an alternative energy source
This is an example of reversible injury
idiopathic pathology of the liver
Gall bladder mucocele
Border Terriers
neaoplastic pathology of the liver
The liver comprises predominantly
Hepatocytes
Bile ducts
Hepatocellular adenoma/carcinoma
Cholangiocellularcarcinoma
Lactate
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
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- See huge elevation in Calcium to produce egg shell
This is predominantly Bound and not metabolically active
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
Renal
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 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
ERYTHROCYTOSIS (POLYCYTHAEMIA)
a high concentration of red blood cells in the 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
Dehydration (PCV up to 60%)
fluid loss with a stable red cell mass
Clinical signs may be present
Lab test abnormalities
serum total proteins
Often but not always increased serum sodium
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
Leukocyte disorders/patterns
Neutrophilia and neutropenia
Left shift and toxic change
Lymphocytosis and lymphopenia
Monocytosis
Eosinophilia/Basophila
“Stress leukogram”
Neutrophilia
Increased production
- TO MEET DEMAND
Infections
Immune mediated diseases
Inflammation
Neoplasia
(COMMON)
2.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
a low number of white blood cells called neutrophils in your blood.
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
Band neutrophils
released with increased demand
slightly less mature than segmented neutrophils and have indented, unsegmented “C” or “S” shaped nuclei. Band neutrophils normally account for approximately 5-10% of peripheral blood leukocytes. An increased proportion of band neutrophils can be seen in infectious and inflammatory conditions.
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.
under conditions that intensely stimulate neutrophil production and shorten the maturation time in marrow.
Signs of toxicity
Dohle bodies
Foamy cytoplasm
Bluish cytoplasm
Toxic granules - rare
Peripheral blood: Toxic verses non-toxic neutrophils
Tissues/fluids: Degenerate verses non-degenerate neutrophils
Dohle bodies
Döhle bodies are single or multiple blue cytoplasmic inclusions that are remnants of rough endoplasmic reticulum. They are associated with myeloid left shifts and are seen in conjunction with toxic granulation.
he presence of Döhle bodies, nuclear immaturity, “toxic” cytoplasmic granulation, and giant platelets may indicate, at least in some measure, a general metabolic disturbance of the hematopoietic system.
Foamy cytoplasm
have a bubbly (foamy) cytoplasm. Most are macrophages (1.76) that have phagocytized lipid material, but some are cells of another derivation that have a similar multivacuolated cytoplasm. Compare with clear cells
Foam cells form through dysregulated lipid metabolism in mammalian macrophages: lipid accumulation that exceeds the homeostatic capacity of macrophages triggers lipid droplet formation
Toxic granules
rare
he term used to describe an increase in staining density and possibly number of granules that occurs regularly with bacterial infection and often with other causes of inflammation
Toxic granulation is seen in cases of severe infection, as a result of denatured proteins in rheumatoid arthritis or, less frequently, as a result of autophagocytosis. Infection is the most frequent cause of toxic granulatio
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
Important species differences in Red cell morphology
Regenerative response
Eosinophil morphology
and Leukocyte responses
red cells-
Dogs – have most obvious central pallor
Alpacas – oval shaped but no nucleus
Birds/reptiles - nucleated red cells
regenerative resonse-
Regeneration in most species noted by increased polychromatic red cells (reticulocytes) in circulation
Horses do not release polychromatic red cells into circulation so cannot assess regeneration this way
eukocyte response-
Cat leukocyte response to epinephrine more “extreme” than other species
Classic complete”stress” glucocorticoid response most commonly seen in dogs
Dogs typically display neutrophilia with inflammation (unless severe), cattle more commonly neutropenia, cats and horses somewhere in between.
Complete Blood Count (CBC)
EDTA BLOOD SAMPLE
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
Mean corpuscularhaemoglobin(MCH)
calculated (MCH =Hbx10 / RBC)
Mean corpuscularhaemoglobinconcentration (MCHC)
MCHC=Hb/HCT
Some analysers:
Red cell distribution width (RDW) – indication of variation in red cell size
Reticulocyte counts/percentage – measure of regeneration
Reticulocyte Hb – potential marker for iron deficiency
Plateletcrit (PCT) – equivalent of HCT for platelets
Packed cell volume
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
Regenerative anemia
Anisocytosis- red blood cells (RBCs) that are unequal in size
Polychromatophils- efers to how blood cells look under a microscope when the cells are stained with special dyes. It means there is more staining than normal with certain dyes. The extra staining is due to an increased number of immature red blood cells (RBCs) called reticulocytes
Reticulocytes- red blood cells that are still developing
Nucleated RBC- the body is so desperate for red blood cells that it has begun producing them outside of the bone marrow.
Howell-Jolly bodies-nuclear remnants that are found in the RBCs
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
Spherocytes
Spherocytes are often difficult to recognize – MUST LOOK FOR THEM IN THE MONOLAYER
They are smaller, darker and have no central pallor
Can be extremely difficult/impossible to see in species other than dogs
Heinz bodies
ggregates of denatured, precipitated hemoglobin within erythrocytes that form as hemoglobin with oxidative damage is metabolized.
look like blebs on outside of cell
sign of oxidative injury. Haemoglobin is oxidised and pushed to cell margin, no longer functional
Paracetamol in cats, onions, some toxins (eg Zinc), diabetic ketoacidosis are some common causes of Heinz bodies/eccentrocytes
eccentrocytes
appear in a peripheral blood smear to have their hemoglobin shifted to one side of the cell. This abnormality, which is confined to the RBC membrane and cytoskeleton, is induced by oxidative damage.
sign of oxidative injury. Haemoglobin is oxidised and pushed to cell margin, no longer functional
Paracetamol in cats, onions, some toxins (eg Zinc), diabetic ketoacidosis are some common causes of Heinz bodies/eccentrocytes
Acanthocytes
look like splats- long fingers come off cell body
Schistocytes
Red cell fragments forming secondary to being squeezed through abnormal blood vessels (eg haemangiosarcoma) or being fragile (eg iron deficiency)
PCR (EDTA sample) tests are available for:
the haemotropic Mycoplasmas in cats
Babesia species in dogs
Ehrlichia and Anaplasma in dogs
FIV/FeLV
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
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.
ANAEMIA
Reduction in red cell mass, evidenced in decreased:
Haemoglobin
Provides information on the total O2 carrying capacity
Packed cell volume (PCV)
Haematocrit (HCT)
Red blood cell count (RBC)
The effects of anaemia are due to reduced oxygenation of tissues.
Tissue hypoxia activates the following 4 main compensatory mechanisms, which serve to maintain tissue oxygen levels as near to normal as possible.
Increased oxygen delivery: The affinity of Hb for O2 is reduced 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).
Mucous membrane pallor
Lethargy
Exercise intolerance
Tachycardia
Tachypnoea
Collapse
Icterus
Melaena
Pica
Haemolysis
Extravascular
More common
Macrophages
Spleen and liver
± Icterus (jaundice)
Intravascular
Acute, severe
Haemoglobinaemia
Haemoglobinuria
Ghost cells
+/- icterus
Haemolytic anaemia
Immune-mediated (common – primary verses secondary)
Infections (less common)
Babesia
Mycoplasma
Inherited RBC metabolic defects (rare)
Toxins
Severe Hypophosphataemia (rare)
- IMHA
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
Pale mucous membranes +/- tachycardia, bounding pulses, systolic murmur
±
Tachypnoea
Jaundice
Hepatosplenomegaly
Pyrexia, mild lymphadenopathy
Thromboembolic
disease
Secondary IMHA
Infections
Neoplasia
Drugs
No single pathognomonic test
HAEMATOLOGY
Anaemia (usually regenerative) plus any of:
Agglutination(A)
Spherocytosis (S)
Positive Coombs’ test
Test detects the presence of antibodies on the RBC
. Haemolytic anaemia
Secondary to Infection
Parasites
Haemoplasmas in cats
Babesia in dogs/cattle
Bacteria
Leptospira
Clostridium
. Inherited RBC metabolic defects
These are RARE
PFK deficiency (Springers)
PK deficiency (Basenjis, Somalis, Abysinnians)
Genetic tests (PCR) available for diagnosis
Basophilic stippling
the presence of numerous basophilic granules that are dispersed through the cytoplasm
basophilic stippling is a frequent manifestation of hematologic disease in the peripheral blood, and it is also observable in bone marrow aspirates. It is implicated in cases of lead poisoning but can be an indicator of various heavy metal toxicities
Stain precipitate
Stain precipitate mimics bacteria, including cocci and Mycoplasma species. Generally, the stain precipitate is purple, whereas bacteria are blue (with a Wright’s stain).
usually results from the use of aged staining solutions and/or inadequate rinsing of slides following application of stain.
B. divergens apearence on Fresh blood smears
Single or paired round oval or pear shaped structures
Low sensitivity
pcr better- edta
Anaplasma phagocytophilium apperence on Blood smear
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 appearence on blood smear
B. caballi - Single or paired
pear shaped
meet at posterior end in erythrocytes
T. equi - Smaller
Round or ovoid
Tetra shapes
‘maltese cross’ in erythrocytes
Mycoplasma haemolamae appearene on blood smear
RBC small and elliptoid
Lots of small blue dots on surface of RBC
Identify on blood smear (make fresh as organisms will fall off)
mammal red blood cells
Anucleated (No nucleus)
Anisocytosis/Polychromasia common in some species
Shorter half life c/f dogs and cats
Red Blood Cell Appearance - Birds
Erythrocytes
Nucleated
Larger than mammals’ smaller than reptiles’
Elliptical cells
Elliptical, central nucleus
Red Blood Cell Appearance - Reptiles
Erythrocytes
Nucleated
Larger cells than birds and mammals
Blunt ended ellipse
Central round/ovoid nucleus with Irregular margin
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
Aspergillosis, Chlamydiosis, TB, circovirus, chronic bacterial infection
Heterophils
in birds and reptiles
Stain differently but comparable function to neutrophils
heterophils have diffrent shaped nucleus and granules
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
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/Neutrophilia
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
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
basophils in exotics
Round to oval nucleus, lobed in mammals
Frequent round purple cytoplasmic granules
Nucleus may be obscured by granules
Some species variation in appearance of granules/pattern
Higher numbers c/f eosinophils
Rabbits 5% (but can be <30%)
Birds <6%
Reptiles can have very high numbers
Eg. Up to 40% in aquatic turtles normal
Differentiate from mast cells (rare)
Lymphocytes in exotics
Similar function in all exotics species
Immunologic function
T lymphocytes - cell mediated immunity
B lymphocytes - humoral immunity
Scant weakly basophilic cytoplasm
High nucleus:cytoplasm ratio
No granules
Round, central or slightly eccentric nucleus
Easily confused with thrombocytes and monocytes
Predominant leukocyte in some species
Rodents, rabbits, waterfowl, some snakes and other reptiles
Neutro/Heterophil:Lymphocyte ratio often more important than absolute numbers
Lymphopaenia
Relative lymphopaenia in lymphocytic species with a heterophilia/neutrophilia
Severe stress
Corticosteroids (endogenous or exogenous)
Toxins
Acute infection
Lymphocytosis
Young animals have higher baseline, as do some breeds and species
Excitement
Lymphoma/Neoplasia
Monocytes in exotics
Large cells (generally largest leykocyte in peripheral blood – foten twice the size of RBCS)
Irregularly shaped nucleus
Lacy chromatin
Abundant cytoplasm
Reptiles
Variable number of fine red cytoplasmic granules
In snakes even more prominent granules and referred to as azurophils
Elevated in chronic inflammation
Granulomatous disease
Azurophils
– snakes only
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
Monocytosis/Monocytopenia
Function
Monocytosis
Acute and Chronic Inflammation
Corticosteroid response
Viral Infection
Granulomatous disease
Necrosis
Neoplasia
Foreign body
Suppuration
Monocytopenia
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
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
Hypoxic shock
Impaired oxygen delivery to the cells
Anaemia
Decreased haemoglobin saturation – carbon monoxide
Respiratory disease
Hypovolaemic shock
Hypovolaemia occurs when fluid is lost primarily from the intravascular compartment - a relatively small total loss of fluid has profound physiological consequences with the development of hypovolaemic shock. In this circumstance, treatment with fluids centers around rapid replacement of the lost volume to restore tissue perfusion. Conversely dehydration represents fluid loss from all three body fluid compartments. It commonly occurs with more gradual fluid losses where there is time for water to move between body fluid compartments. Total body fluid losses may be much larger than with hypovolaemia but as the fluid losses are borne between all the compartments, it has much less profound and immediate physiological effects.
Acute blood loss
Trauma
Severe dehydration
Third spacing = Third-spacingoccurs when too much fluid moves from the intravascular space (blood vessels) into the interstitial or “third” space—the nonfunctional area between cells. – edema, pleural or peritoneal cavity
Bilirubin
end product of haemoglobin metabolism
Unconjugated
Conjugated
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
Conjugated is water soluble and can be excreted 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)
Conjugation renders bilirubin water soluble. Only very small amounts in blood because it is normally excreted into bile.
What will cause Janudice – it is an increase in the pigmentation in the blood stream and tissues
Disease processes have to be caused by a breakdown in part of the pathway
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
pre-hepatic jandice
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 jandice
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
post hepatic jaundice
Disruption of bile flow through the extrahepatic biliary system
Megakaryocytes
Reside in the bone marrow
Large multinucleated cells with abundant cytoplasm
Platelets
Sheared off fragments of megakaryocyte cytoplasm
Smaller than a RBC
Anucleate, irregular, granular
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
D-Dimers
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
Localised or disseminated intravascular coagulation (DIC)
Sepsis, systemic inflammation, haemorrhage, neoplasia, surgery, immune-mediated disease
Decreased hepatic or renal clearance
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
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)
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
Haemophilia A =
Factor VIII
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).
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 help 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
Flaming plasma cell
Flame cells are plasma cells with a distinctive pinkish hue in the outer rim of the cytoplasm. This appearance is thought to be the result of precipitated immunoglobulin. These cells can be found in plasma cell dyscrasias as well as cases of chronic infection or inflammation
Reactive lymphoid hyperplasia
a benign nodular lesion, histopathologically characterized by marked proliferation of non-neoplastic, polyclonal lymphocytes forming follicles with an active germinal center
-Mott cells
-Mixed small, intermediate, large lymphocytes
types of inflamation
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
Degenerate neutrophils
Degeneration of neutrophils is indicated by swelling of the nucleus, with the nucleus appearing lighter staining and smudged- kind of like it is swollen. The cells may also lyse. When degenerate neutrophils are seen, you should look carefully for microorganisms – especially bacteria.
Hyperplasia
Hyperplasia = increase in the number of cells in a tissue
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
Dysplasia
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.
Neoplasia
Neoplasia = abnormal and excessive growth oftissue.
Growth of a neoplasm is uncoordinated with that of the normal surrounding tissue, and persists in growing abnormally, even if the original trigger is removed
Hyperplasia, metaplasia, and dysplasia are reversible because they are results of a stimulus.
Neoplasia is irreversible because it is autonomous
epithelial neoplasia
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 neoplasia
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 neoplasia
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
Round cells
Central round nuclei
Lots of magenta granules in the cytoplasm
Fibroblasts, eosinophils and ribbons of pink matrix
Histiocytoma
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
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
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 neoplasia
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- Size and shape
Chromatin
Nucleoli
Nuclear membrane
Relationship / nuclear moulding
Mitotic figures
Cytoplasmic features- Vacuolation
Basophilia
Product
N:C ratio
Nuclear position
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
Criteria of Malignancy
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
Anisokaryosis
one of the main features of cancer cells: variation of nuclear size and shape from cell to cell. Referred to as nuclear pleomorphism and anisonucleosis.
Nuclear moulding
conformity of adjacent cell nuclei to one another. It is a feature of small cell carcinomas and particularly useful for differentiation of small cell and non-small cell carcinomas, i.e. adenocarcinoma and squamous carcinoma.
Emperipolesis
the presence of an intact cell within the cytoplasm of another cell. It is derived from Greek (en is inside, peripoleomai is go round). Emperipolesis is an uncommon biological process, and can be physiological or pathological.
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
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
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
Morphology of cell injury
The common appearance of an injured cell is swelling
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)
Hydropic degeneration
extensive fluid accumulation
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
Senescence
Death
Dysplasia -> 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 homeostasis within the cell
cell adaptations
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.
Senescence
Senescent cells are somatic cells that stop dividing
Senescent cells remain metabolically active
Signals that induce senescence are typically due to DNA damage and/or tumour suppressor genes
p53-p21
p16
Morphological appearance of cell ageing
Long-lived post mitotic cells
Neurones and muscle
Lipofuscin
Senescent cells
Heterochromatin accumulations
Transmission electron microscopy
Not clinically relevant
Biochemical markers of senescence
Lack proliferation markers
Senescence-associated secretory phenotype (SASP)
Research orientated
Lipofuscin
Wear-and-tear pigment
Normal accumulation over time of lipoprotein in secondary lysosomes
May accumulate excessively in certain circumstances
Phalaris poisoning in ruminants
Intracellular, golden-brown, globular
Ceroid
Very similar to lipofuscin but only accumulates in disease states
Brown gut in dogs with vitamin E deficiency
Intracellular and extracellular
Endogenous pigmentation – non-haematogenous
Lipofuscin and ceroid
Melanin
Incidental colouration
Leptomeninges
Pig lungs - melanosis
Hyperpigmentation
Endocrine skin disease
Melanoma
Hypopigmentation
Vitiligo
Melanin incontinence
Some skin diseases of dermo-epidermal junction (pemphigus)
Endogenous pigmentation - haematogenous
Blood
Haemoglobin
Haemosiderin- brown iron-containing pigment usually derived from the disintegration of extravasated red blood cells. It tends to be golden brown, more refractile, and more clumped than melanin
Haematoidin- an orange-yellow pigment in the bile that forms as a product of hemoglobin; excess amounts in the blood produce the yellow appearance observed in jaundice
Porphyria-
Heme synthesis disorder
Deposition of porphyrin pigments in tissues
Methaemoglobinaemia
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/Acer
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
Carbon monoxide
CO bind haemoglobin, forming carboxyhaemoglobin
Much stronger binding than oxygen and slow to revert
Cherry red mucous membranes, muscle and brain
Carotenoids
Exogenous pigmentation
found in leafy vegetables and in horses and Jersey cattle impart a yellow colour to adipose tissue and lipid-laden organs.
Calcification
Pathological calcification = deposition of calcium salts in soft tissues - typically as phosphates and carbonates
Metastatic calcification is due to increased circulating calcium levels
Dystrophic calcification is secondary to necrosis
Morphology of necrosis
Continued swelling and hypereosinophilia
Nuclear changes
Pyknosis = shrinkage
Karyorrhexis = fragmentation
Karyolysis = dissolution
Inflammation
Causes of necrosis - anoxia
Reduction or cessation of ATP production due to hypoxia or anoxia respectively will result in loss of function on energy-dependent cell pumps
Na+/K+ pumps
Discussed in POP lecture 1
Results in cell swelling due to osmotic pressure
ONCOTIC NECROSIS
Cell swelling is the typical feature and distinguishes it from apoptosis
Calcium efflux pumps
are also affected
resulting in accumulation of intracellular calcium
Causes of necrosis – membrane damage
Membranes can be directly damaged by
Pore-forming infectious agents/toxins
Reactive oxygen species (ROS)
Phospholipase activation
Protease activation
One of the best examples of membrane damage by pore-forming toxins are those produced by Clostridium perfringens
Causes of necrosis – free radicals
Free radicals are any molecule with a free electron
Reactive oxygen species (ROS) and reactive nitrogen species (NO)
Produced by oxidative metabolism, therefore most frequently made by mitochondria, but will also damage the mitochondria if cannot be removed.
Vitamin E and selenium are important co-factors in the neutralisation of free radicals
apoptosis
Apoptosis is normal
Embryological
Physiological
May be due to a pathological process
Organ not receiving stimulus
portosystemic shunt
Cell contains infectious agent
Cell is irreparably damaged
DNA is irreparably damaged
Cell is cancerous
Two main mechanisms of apoptosis
Intrinsic
Extrinsic
Morphology differs to necrosis
Cell is shrunken
No/minimal inflammation
Chromatin condensation around nuclear periphery (most characteristic)
Formation of cytoplasmic blebs = apoptotic bodies
There are different types of programmed cell death
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
Due to hypoxia
Large amount of cells and cell membranes with little connective tissue
Gross
Soft, viscous focus, often with cavity containing creamy-yellow material (pus).
Histo:
Cell debris, eosinophilic fluid
Very messy
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.
Gross:
Friable, granular, white appearance
Typically encapsulated
Abscess/granuloma/pyogranuloma
Histo:
Loss of architecture
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
Gangrenous necrosis
Often a sequel of coagulative 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.
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.
Fibrinoid necrosis
Blood vessels
associated with inflammation = vasculitis
Antigen and antibody complexes deposition in arterial walls and fibrin leakage.
Histo:
Bright pink, amorphous hyaline (glassy)
Thrombosis
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
Increased intravascular hydrostatic pressure results in
Increased flow or volume of blood
hyperaemia (active)
congestion (passive)
Acute inflammation – serous exudation
Exudation of cell-poor fluid into spaces created by cell injury or into body cavities (effusions)
Fluid is not infected & does not contain high numbers of leukocytes.
Seen with:
Thermal injury to skin - blisters
Acute allergic responses - watery eyes/runny nose
Acute inflammation – Catarrhal/mucoid
exudation
Secretion of thick gelatinous fluid
Contains mucus and mucins
Occurs most commonly in tissue with abundant goblet cells & mucous glands – runny nose
Acute inflammation – Fibrinous
exudation
Characteristic of inflammation of membranes of body cavities and organs
meninges, pericardium, joints
Most commonly caused by infectious microbes
Acute inflammation – Suppurative/purulent
exudation
Pus = an exudate consisting of neutrophils, the liquefied debris of necrotic cells, and oedema fluid.
The most frequent cause = infection with bacteria that cause liquefactive tissue necrosis
e.g. Staphylococci - aka pyogenic (pus-producing) bacteria.
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
Thrombus
A thrombus forms antemortem within a vessel
A thromboembolism is a section of a thrombus that has broken off and lodged elsewhere
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
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
infarction
An infarct is a local area of very acute (peracute) ischemia 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.
Abscess and granulomatous inflammation
Abscesses form due to
Production of myeloperoxidase by neutrophils which themselves also undergo necrosis, resulting in liquefaction and pus
Rabbits and birds lack myeloperoxidase, resulting in an inability to form pus
Chronically, fibroblasts produce collagen and extracellular matrix proteins to wall off the area = fibrous capsule
Granulomatous inflammation
occurs when the inciting agent cannot be removed and is largely
mediated by macrophages which may become multinucleated or epithelioid
Granulomatous inflammation can be
Nodular = (tuberculoid) granulomas
Mycobacterium bovis
Diffuse = lepromatous
Eosinophilic granuloma
Mycobacterium leprae
Healing
Repair and healing are defined loosely as restoration of tissue architecture and function after an injury
Repair (parenchymal and connective tissues)
Healing (surface epithelia)
Two mechanisms
REGENERATION: some tissues are able to replace damaged components and essentially return to a normal state
occurs by proliferation of cells that survive the injury and retain the capacity to proliferate
In some cases, tissue stem cells may contribute to restoration of damaged tissues (mammals have limited capacity to regenerate damaged tissues/organs)
i.e. in the rapidly dividing epithelia of the skin and intestines and in some parenchymal organs, notably the liver.
Requires an intact basement membrane
CONNECTIVE (FIBROUS) TISSUE DEPOSITION
Occurs when injured tissue incapable of complete resolution or if supporting tissue structure is severely damaged
May result in scar formation – not normal but provides enough structural stability that the injured tissue is usually able to function
Fibrosis: describes the extensive deposition of collagen that occurs in the lungs, liver, kidney, etc. as a consequence of chronic inflammation or in the myocardium after extensive ischemic necrosis (infarction)
Macrophages play a central role in repair by
clearing offending agents and dead tissue,
providing growth factors for proliferation of cells
secreting cytokines that stimulate fibroblast proliferation and connective tissue development
Repair begins within 24 hours after injury with emigration of fibroblasts and induction of fibroblast and endothelial cell proliferation.
Angiogenesis (formation of new blood vessels) – supplies nutrients/oxygen for repair; vessels are leaky because VEGF (GF that drives angiogenesis) increases vascular permeability; also due to incomplete interendothelial junctions
Formation of granulation tissue – consists mainly of fibroblasts and new capillaries in a loose ECM often admixed with inflammatory cells (mainly macrophages); progressively invades site of injury; amount formed depends on size of tissue deficit and intensity of inflammation
Remodelling of connective tissue (produces stable fibrous scar) – process of maturation of the connective tissue. The amount increases gradually leading to reorganisation and formation of a scar.
Ethylene Diamine Taetra-acetic Acid (EDTA) is usedfor
Haematology
Lithium Heparin is used for
biochemistry
blood tubes without an addiive are use for
All serum tests
Sodium Fluoride/Potassium Oxalate blood tubes are used for
Blood glucose
Trisodium Citrate (Sodium Citrate) blood tubes are used for
Coagulation studies
Prothrombin test
