Ocular Diagnostics

Question 1

When is running haematology/biochemistry/urinalysis valuable in ocular cases?

Answer 1

Suspicion of systemic disease (e.g lymphoma), bilateral eye disease, uveitis etc
Pre-operatively before anaesthesia

Question 2

What types of errors may occur as part of taking a sample?

Answer 2

Pre-analytical error - wrong collection tube, animal not starved prior to sample collection
Analytical error - instrument malfunction, insufficient aspiration of sample
Post analytical error - incorrect interpretation of significance of results.

Question 3

How do EDTA tubes works - what are they used for?

Answer 3

EDTA - chelates calcium and prevents clotting
Sample of choice for haematology
Optimum for cell preservation so good for fluid sample collection also
Cannot be used for culture as effects bacterial growth

Question 4

How do heparin tubes work - what are they used for?

Answer 4

Heparin activates antithrombin III which in turns inhibits bloods clotting factors, principally thrombin.
Can be useful for biochem/haem but EDTA preferred for haem
Sample of choice if very short sample, submit with blood smear and most tests can be run.

Question 5

How do plain tubes work - what are they used for?

Answer 5

Blood will clot in plain tube leaving you with serum and a clot of cells
Serum separated off and decanted into plain tube before sending away.
Serum = choice for biochemistry

Question 6

What are serum tubes used for/how do they work?

Answer 6

Serum = biochemistry
Must be spun
Gel like material will sit between cells and plasma/serum preventing issues with haemolysis

Question 7

What are fluoride oxalate tubes used for?

Answer 7

Glucose - falls by 5-10% an hour in whole blood
Can be measured in serum/plasma if red cells have been removed within hour of collection.

Question 8

What are citrate tubes used for?

Answer 8

Clotting times - D dimer evaluation
Underfilled - artificially prolong clotting time
Overfilled - sample will clot

Question 9

Which ocular conditions can be associated with changes in the haematology?

Answer 9

Non regenerative - chronic disease, inflammation, neoplasia, endocrine disease
Regenerative (RBC loss/haemolysis) - clotting issues (hyphema etc) - widespread systemic signs, coagulopathy
Erythrocytosis - PCV >70%, associated with hyperviscosity, engorged torturous retinal vessels

Question 10

What ocular changes may we see with thrombocytopaenia and what value does it have to be at for spontaneous haemorrhage? What other test would you do alongside?

Answer 10

Spontaneous haemorrhage may = intraocular haemorrhage
Platelet counts <50 for spontaneous bleeding
May see petechiation, ecchymoses etc as well as intraocular signs
Haemorrhages may be seen on retina, iris, conjunctiva or as hyphema
Platelet count <30-50 often associated with immune mediated IMHA< DIC or bone marrow dysfunction

D dimer for clotting times alongside platelet count = test of choice
A.Vasorum = common parasitic cause of coagulopathy in dogs

Question 11

What is secondary haemostasis and how can we assess this?

Answer 11

Secondary haemostasis = formation of fibrin clot
Primary = platelet plug

Measurement of clotting times - PT (prothrombin) and PTT (activated partial thromboplastin)
>25% prolongation = abnormal

Causes = vitamin K ingestion, severe liver disease, DIC

Question 12

Biochemistry - what can raised globulins indicate and which ocular condition can those things be linked to?

Answer 12

Increased globulin = inflammation, antigenic stimulation, neoplasia
All can lead to uveitis

Question 13

Biochemistry - when is assessment of triglycerides indicated in ocular disease, how is it performed?

Answer 13

Performed after 12-16hr fast
EDTA plasma = sample collection method of choice
Diabetes mellitus and pancreatitis can see elevation

May want to test if see corneal lipidosis (lipid deposition in middle of cornea) - can be associated with hypertriglyceridaemia

Question 14

Biochemistry - what diseases may we see elevations of cholesterol with and what ocular presentations

Answer 14

May see elevations with diabetes mellitus and hypothyroidism
Assess with corneal lipidosis but condition is not always dependent and even if is elevated may not be direct association.

Question 15

What syndrome may be associated with hypothyroidism? What tests would we do to confirm this diagnosis?

Answer 15

Horner’s syndrome can be associated with hypothyroidism

TSH and T4 (freeT4) evaluation

Question 16

What biochemical changes would we expect to see with diabetes mellitus and what ocular presentation do we see associated with diabetes?

Answer 16

Persistent Hyperglycaemia on bloods
Glucosuria - urine (ketones may also be present)
Elevations of hepatic enzymes, lipids and electrolytes

Associated with cataract formation

Question 17

How can we differentiate between persistent hyperglycaemia and transient stress related hyperglycaemia?

Answer 17

Cats especially prone to stress hyperglycaemia
Fructosamine
When persistently high glucose body tries to reduce by glycating proteins - adding glucose molecule to protein (protein usually albumin)
Fructosamine gives indication of glycaemic state for 1-3 weeks prior to sample collection

Question 18

What are the main ketones seen in diabetic ketoacidosis?
How can they be measured?

Answer 18

B-hydroxybutyrate
Acetoacetic acid
Acetone

Can measure in the urine - do not detect BHB just acetoaectic acid and acetone
BHB’s can be measured in serum

Question 19

What ocular changes may we see in a dog with hyperadrenocorticisim?

Answer 19

No specific ocular changes pathognomonic for HAC

Often hypertensive so can get secondary retinal changes
Low dose dexamethasone/ACTH stim testing - both will be significantly affected by concurrent non adrenal disease such as diabetes mellitus

Question 20

What ocular changes might we see in a cat with hyperthyroidism?

Answer 20

No specific ocular changes for hyperT4

Often hypertensive so can get secondary retinal changes associated with this.

Question 21

How should conjunctival/corneal swabs be taken?

Answer 21

Can use local anaesthesia but may reduce usefulness for culture
Samples taken for investigation of corneal/conjunctival disease should not contact the lid margin or skin of the face as this will cause contamination of the sample and may lead to incorrect diagnosis/treatment.
Mini tip swabs often the best for sample collection.

Amies transport medium - helps prevent loss of the organism or overgrowth
Survival of fastidious bacteria can be prolonged by the addition of charcoal to Amies medium.

Mucopurulent discharge adhered to globe, lid or conjunctiva - sampled prior to cleaning site for ocular examination.

Question 22

What is the test of choice for both Chlamydia and Mycoplasma?

Answer 22

PCR

Question 23

What things will affect the recovery of organisms for culture?

Answer 23

Recent antimicrobial therapy
Insufficient material collected - greater amount of material submitted, the greater chances of culture
Storing swab samples in fridge -increase possibility of false negative

Question 24

How does virus isolation work? How does it compare in terms of sensitivity/specificity?

Answer 24

Collection of sufficient viable organisms to cause a cytopathic effect in cell culture
Highly specific but poorly sensitive - false negatives a problem with this type of test (may not have collected sufficient numbers of micro-organisms)
Swabs for VI must be submitted in viral transport medium.

Rarely used now as has been surpassed by PCR in a lot of cases.

Question 25

How should samples for fungal culture be submitted?

Answer 25

Collection of sufficient material = limiting factor
Material should be collected from periphery of lesions where more likely to recover viable fungi.
Submitted in standard Amies transport medium or whole tissue samples in plain pot.

Question 26

What are the most commonly performed serological tests in ophthalmology? What are the used for?

Answer 26

Immunofluorescent assay (IFA) and enzyme-linked immunosorbent assays (ELISA)
Used to detect pathogen-specific antibodies or detect antigen

Question 27

How do IFA assays work?

Answer 27

IFA - antibody detection
Test serum incubated with plates or slides containing immobilised antigen.
If antibodies to antigen present within the serum they will bind and then are detected by incubation with a secondary antibody that targets the antigen-bound antibody.
This secondary antibody is labelled with a fluorescent marker that can then be visualised - results are given as an antibody titre and represent the greatest dilution of the sample that still shows fluorescence.

Question 28

How does PCR work?

Answer 28

Exponential amplification of very small amounts of DNA to detectable levels.
Very sensitive for infectious and inherited disease
In case of infectious disease sensitivity can also be downfall as may amplify small amounts of non clinically significant DNA - risk positive results of uncertain significance (can be inferred by CT value provided by lab)
Herpesvirus = prime example where significance on PCR can be difficult to ascertain.

Question 29

What are the different types of PCR methods?

Answer 29

Conventional - heated and cooled cycles, temperature dependent DNA polymerase with code specific primers is used to amplify and therefore detect tiny quantities of DNA. Con = DNA does not need to be intact/organism does not need to be vital, unexpected DNA may be amplified if primer sticks to another part of the target DNA than expected)

Nested (2 sets of primers to reduce amplification of inappropriate DNA segment)

Real time PCR (qRT-PCR) - after every PCR cycle the amount of product formed is measured and displayed in fluorescent units, greater the target DNA the more quickly product and therefore fluorescence is formed. Once certain threshold reached of fluorescence in a sample it is deemed to be positive and the number of cycles taken to achieve this allows quantitative measurement of DNA in the original sample.
Pro = allows estimate of DNA present and thus interpretation of clinical significance

Reverse transcriptase PCR (RT-PCR) - reverse transcriptase added to produce complimentary DNA from an RNA sequence.
Used for the detection of RNA viruses such as FIV

Question 30

What cytological preparations can be made from ocular lesions?

Answer 30

Impression smear, scrapes, aspirates

Scrapes and smears = more superficial lesions than aspiration.

Question 31

How are direct impression smears performed on the conjunctiva/cornea?

Answer 31

Can either apply asymmetric strips of millipore filter onto the ocular surface or with aid of cytology brush/swab
Cytological samples using swab improved with being moistened with local anaesthetic, transport medium or saline.
Roll onto conjunctival/corneal surface then roll opposite direction on slide to maintain cell preservation.

Question 32

How are scraping techniques performed for the cornea/conjunctiva?

Answer 32

Used when larger sample required for submission

Local anaesthesia required
Handle end of no 11 or 15 Bard Parker scalpel blade
Scalpel handle held at an acute angle to tissue surface and gently dragged backwards to collect sample within acute angle
Gently place onto submission slide and if necessary smeared with another slide using digital pressure and drawing slides apart.

Question 33

What are the most important principles for cytological sampling?

Answer 33

Thin smear of any material collected
Appropriately labelled submission + relevant history
Material rapidly air dried to prevent deterioration of cells
Slides stored appropriately (room temp, out of light, not sent with formalin pots)

Question 34

What type of stains are used for cytological preparations?

Answer 34

Romanowsky, Wright-Giemsa, Diff Quik (in house)

Question 35

With aspirates what type of lesions will aspirate well and which not so well?

Answer 35

Eputhelial/round cell = excellent aspiration (adenomas, SCC, MCT)
Mesenchymal (connective) tissues = poor aspiration
Cystic masses - very acellular unless marked inflammation

Question 36

How is aqueocentesis conducted and which tubes should be used for collection of the aqueous humour?

Answer 36

Technique in which AH is aspirated from the anterior chamber allowing for cytological or serological evaluation of the fluid or to aspirate cells from masses in the anterior segment or iridial surface.

Conducted under GA or deep sedation with topical anaesthesia as incorrect placement of the needle of movement could cause significant intraocular damage.

Fluid for culture = plain (paediatric tube if possible)
Cytological evaluation = direct smear of fluid or submission in EDTA (paediatric tube if possible), EDTA helps preserve cellular morphology but in high concentration if use just normal EDTA tube it will cause cellular lysis.

Most common complications - acute hypotony, haemorrhage
Important aqueous is not removed too fast or subsequent severe hypotony can lead to choroidal effusion and retinal detachment.
Haemorrhage most likely with aspiration of masses or if iris stroma punctured with needle

Normal findings for AH analysis = no cells, total protein 21-37mg/dl, no microbes

Question 37

When may vitreocentesis be performed?

Answer 37

Under GA
Generally globes where prognosis for vision poor for diagnosis of potential infectious disease e.g cryptococssis, blastomycosis

Question 38

What is the definition of a true positive?

Answer 38

True positive = positive result in which the animal has the disease

Question 39

What is the definition of a true negative?

Answer 39

True negative = negative result in which the animal does not have the disease

Question 40

What is the sensitivity of a test?

Answer 40

True positives/number of animals with disease (true positives + false negatives) x 100
Highly sensitive test = good screening test
Will almost always detect animals with the disease but there may be a few false positives

Question 41

What is the specificity of a test?

Answer 41

True negatives/number of animals without disease (true negatives + false positives) x 100
Highly specific test = unlikely to give a false positive result, if positive = diagnostic
A highly specific test may lose some sensitivity so there may be false negatives.
If positive = has disease, if negative can’t fully rule out

Question 42

What is the diagnostic accuracy of a test?

Answer 42

Diagnostic accuracy = frequency with which correctly classifies an animal as having or not having the disease
Number correctly classified/number of animals in study x 100

Question 43

What is the predictive value of a test? What other factors affect the PV of a test?

Answer 43

Predictive value of a positive test = True positives/all positives x 100
Predictive value of a negative test = true negatives/all negatives x 100

PV affected by disease prevalence, where cut offs are drawn for positive or negative result, what is the gold standard and what if the new test is actually better than the gold standard?

Question 44

What is a ROC curve?

Answer 44

ROC curves plot diagnostic specificity (false negative rate) on the x axis against sensitivity (true positive rate).
ROC curve with 45 degree angle = test no better than tossing a coin
Best type of ROC curve = head directly to the top of graph then horizontal - suggests ability of the test to differentiate diseased from non diseased animals is good.

Question 45

Give some examples of wheres serology antibody testing (ELISA/IFA) may be used in ophthalmology.
What does detection of antibodies tell you?

Answer 45

Detection of antibodies = exposure and seroconversion - interpret in light of vaccination status and prevalence within the population.

Toxoplasma titre in dogs/cats (care with interpretation as high prevalence in general population - significance of titre levels?)

Feline coronavirus - again high normally in population so not synonymous with active infection. Negative titre does not fully exclude FIP - 10% of cats with FIP may have negative titre.
Pathology arises due to 2 main reasons - some forms of FCoV mutate and able to enter macrophages rather than being confined to enteroytes. 2 = cats own immune response to this virus is exaggerated- marked production of antibodies, humoural response and vasculitis (wet FIP) or inappropriate cell mediated response leading to granuloma formation (dry FIP)