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.
